Highly water-soluble monoboronic acid probes that show optical sensitivity to glucose based on 4-Sulfo-1,8-naphthalic anhydride

Article abstract of DOI:10.1021/jo9002008

Two highly water-soluble monoboronic acid probes that display the more desirable off - on fluorescence response were synthesized based on 4-sulfo-l,8-naphthalic anhydride and a remarkable sensitivity for glucose rather than fructose and galactose was also

Full text of DOI:10.1021/jo9002008

Some key challenges in this field that continue to limit the
number of useful probes are the following: the design of
synthesis of reporters that have (1) excitation and emission
wavelength above 500 nm, (2) low molecular weight, (3)
photostability, and (4) perhaps most important water solubility.19,20
As a highly photostable and fluorescent probe, naphthalic
anhydrides and their derivatives have been widely used for
fluorescent tags and receptor antagonists.21-26 Of particular
interest, when appropriately substituted at both the naphthalic
and phenyl rings of N-aryl-1,8-naphthalimide, a clear dual-
fluorescence was observed.26,27 For instance, by introducing a
nitro group into the naphthalic anhydride ring, two emission
bands (430 nm/550 nm, respectively) of the dye molecule were
reported by our group.27
Highly Water-Soluble Monoboronic Acid Probes
That Show Optical Sensitivity to Glucose Based
on 4-Sulfo-1,8-naphthalic Anhydride
Zhi Cao, Premchendar Nandhikonda, and
Michael D. Heagy*
Department of Chemistry, New Mexico Institute of Mining &
Technology, Socorro, New Mexico 87801
mheagy@nmt.edu
ReceiVed January 29, 2009
A bis-boronic acid based probe was first synthesized by
Shinkai et al.28 when 3-aminophenylboronic acid was added
into a protoporphyrin system. At pH 10.5, in the presence of
fructose, the fluorescence signal of this probe could be increased
100-fold. In addition, other studies23,29 have shown that bis-
boronic acid probe designs exhibit higher binding affinity
specific for glucose than monoboronic acid probes by comparing
Kd values of both sensors, 10-5-10-4 M for bis-boronic acid
probe and 10-3-10-2 M for monoboronic acid probe, respec-
tively. Unlike changing distances between the boronic acids
through synthetic modifications of bis-boronic acid probes,
recent investigations on simple monoboronic acid probes showed
that fluorophores and substituents on fluorophores also could
contribute to saccharide selectivity.23,27,29 Therefore, more
complicated synthetic schemes of bis-boronic acid probes could
be avoided by employing appropriate substituents of fluorophore,
without losing saccharide binding efficiency.
Two highly water-soluble monoboronic acid probes that
display the more desirable off-on fluorescence response
were synthesized based on 4-sulfo-1,8-naphthalic anhydride
and a remarkable sensitivity for glucose rather than fructose
and galactose was also observed.
With cases of diabetes reaching epidemic proportions, there
continues to be a strong demand for methods of detecting
saccharide concentration in blood for those patients who are
suffering from this chronic disease.1-6 Since the ability of
recognition of saccharides, boronic acids with diol moiety have
been widely investigated for their huge potential biomedical
applications.1,3,7-9 Unfortunately, most of the carbohydrate
probes based on boronic acid moiety continue to have limited
water solubility and those based on enzymes exhibit poor
stability or consumption of substrate during the detection
procedure which also limit further biosensing applications.10,11
Because of their ability to bind to the diols of sugars,
phenylboronic acid and its derivatives have been developed for
saccharide sensing based on different measurements, such as
fluorescence,12,13 UV-vis absorption,14,15 and other methods.16-18
In this work, we synthesized two different N-phenylnaph-
thalimide-based monoboronic acid probes. Upon consideration
of the structurally related Lucifer yellow dye and its high water
solubility,30 we utilized the 4-sulfo potassium salt group of 1,8-
naphthalic anhydride. By changing substituted -B(OH)2 posi-
tions on the phenyl ring, we investigated the steric effect on
(15) Shinmori, H.; Takeuchi, M.; Shinkai, S. Tetrahedron 1995, 51, 1893–
1902.
(16) Gabai, R.; Sallacan, N.; Chegel, V.; Bourenko, T.; Katz, E.; Willner, I.
J. Phys. Chem. B 2001, 105, 8196–8202.
(17) Lee, M.; Kim, T.; Kim, K. H.; Kim, J. H.; Choi, M. S.; Choi, H. J.;
Koh, K. Anal. Biochem. 2002, 310, 163–170.
(18) Shoji, E.; Freund, M. S. J. Am. Chem. Soc. 2002, 124, 12486–12493.
(19) Norrild, J. C.; Eggert, H. J. Am. Chem. Soc. 1995, 117, 1479–1484.
(20) Eggert, H.; Frederiksen, J.; Morin, C.; Norrild, J. C. J. Org. Chem. 1999,
64, 3846–3852.
(21) Mader, H. S.; Wolfbeis, O. S. Microchim. Acta 2008, 162, 1–34.
(22) DiCesare, N.; Pinto, M. R.; Schanze, K. S.; Lakowicz, J. R. Langmuir
2002, 18, 7785–7787.
(23) Cao, H.; Diaz, D. I.; DiCesare, N. J.; Lakowicz, R.; Heagy, M. D. Org.
Lett. 2002, 4, 1503–1505.
(24) DiCesare, N.; Adhikari, D. P.; Heynekamp, J. J.; Heagy, M. D.;
Lakowicz, J. R. J. Fluoresc. 2002, 12, 147–154.
(1) James, T. D.; Shinkai, S. Top. Curr. Chem. 2002, 218, 159–200.
(2) Fang, H.; Kaur, G.; Wang, B. J. Fluoresc. 2004, 14, 481–489.
(3) Cao, H.; Heagy, M. D. J. Fluoresc. 2004, 14, 569–584.
(4) Finney, S. J.; Zekveld, C.; Elia, A.; Evans, T. W. J. Am. Med. Assoc.
2003, 290, 2041–2047.
(5) Wentholt, I. M.; Vollebregt, M. A.; Hart, A. A.; Hoekstra, J. B.; DcVrics,
J. H. Dibetes Care 2005, 28, 2871–2876.
(6) Pickup, J. C.; Hussain, F.; Evans, N. D.; Rolinski, O. J.; Birch, D. J. S.
Biosens. Bioelectron. 2005, 20, 2555–2565.
(7) James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. Nature (London)
1995, 374, 345–357.
(25) Adhikiri, D. P.; Heagy, M. D. Tetrahedron Lett. 1999, 40, 7893–7896.
(26) Cao, H.; Chang, V.; Hernandez, R.; Heagy, M. D. J. Org. Chem. 2005,
70, 4929–4934.
(8) Wang, W.; Gao, X.; Wang, B. Curr. Org. Chem. 2002, 6, 1285–1317.
(9) Striegler, S. Curr. Org. Chem. 2003, 7, 81–102.
(10) Yan, J.; Fang, H.; Wang, B. Med. Res. ReV. 2005, 25, 490–520.
(11) Heller, A. Annu. ReV. Biomed. Eng. 1999, 1, 153–175.
(12) Yoon, J. Y.; Czarnik, A. W. J. Am. Chem. Soc. 1992, 114, 5874–5875.
(13) James, T. D.; Linnane, P.; Shinkai, S. Chem. Commun. 1996, 281–288.
(14) Yamamoto, H.; Ori, A.; Ueda, K.; Dusemund, C.; Shinkai, S. Chem.
Commun. 1996, 407–408.
(27) Cao, H.; McGill, T.; Heagy, M. D. J. Org. Chem. 2004, 69, 2959–
2966.
(28) Murakami, H.; Nagasaki, T.; Hamachi, I.; Shinkai, S. Tetrahedron Lett.
1993, 34, 6273–6276.
(29) DiCesare, N.; Lakowicz, J. R. Org. Lett. 2001, 3, 3891–3893.
(30) Coskun, A.; Akkaya, E. U. Org. Lett. 2004, 6, 3107–3109.
3544 J. Org. Chem. 2009, 74, 3544–3546
10.1021/jo9002008 CCC: $40.75 2009 American Chemical Society
Published on Web 04/07/2009

SCHEME 1. Synthetic Route of o- and m-Phenyl
Monoboronic Acid Probes for Saccharides Based on
4-Sulfo-1,8-naphthalic Anhydride
saccharide binding of different probes and structural configu-
ration during this procedure.
To explore fluorescent properties through sugar binding, we
synthesized two probes, 1 and 2, by using a common fluorophore
4-sulfo-1,8-naphthalic anhydride as the starting material. 3-ami-
nophenylboronic acid and 2-aminophenylboronic pinacol ester
were selected for investigation of isomeric effects on different
boronic acid binding pathways with sugar. Sensor 1 was
prepared in a single-step through reaction of the commercial
potassium 4-sulfo-1,8-naphthalic anhydride and 3-aminophe-
nylboronic acid hemisulfate. The other probe was prepared
through a two-step reaction. After reaction between naphthalic
anhydride and aminophenylboronic pinacol ester, the product
was treated with 33% aqueous H2O2 for 1 h and probe 2 was
obtained.31 A synthetic scheme for construction of those isomers
is given in Scheme 1. High water solubility and significant
emission signal change are observed in our experiments.
Beyond our expectations, both saccharide probes displayed
a greater optical sensitivity to glucose than fructose. Numerous
papers indicate that most of the monoboronic saccharide
chemsensors favor fructose more than glucose. Some re-
ports3,21,23,29 indicated that the affinity of probe to fructose is
approximately 100 times greater than that of glucose for
monoboronic acid sensors. Probes 1 and 2 show large fluores-
cence intensity changes through chelation-enhanced fluorescence
(CHEF) via three most abundant monosaccharides in human
blood and receptor interactions, as shown in panels a, b and
cin Figure 1. Currently, we attribute the increased fluorescence
in the presence of analyte to rigidification of the biaryl probe.32
Next, we examined the selectivity of these probes to common
monosaccharides at pH 7.4. Figure 1d shows the relative
fluorescence of 1 at 400 and 474 nm as a function of
carbohydrate concentration. Photophysical properties of monobo-
ronic acid probes 1 and 2 are listed in Table 1. The increase in
fluorescence intensity ratios (I in the presence of saccharide, I0
in the absence of saccharide) for this series shows an increase
of about 2-5 times, respectively. Probe 1 displays an increase
in the ratio of intensities for three common monosaccharides,
showing the largest increase in fluorescence for glucose. Similar
observations were made by our group previously in which the
largest quenching effect took place on glucose.23 The dissocia-
tion constant Kd for fructose was found to be 6.5 mM, while a
higher Kd of 28.5 mM was obtained by calculation for glucose
FIGURE 1. Fluorescent spectral changes of boronic acid probe 1 upon
addition of different saccharides in phosphate buffer (0.1 M) at pH 7.4
(λex ) 360 nm, λ1em ) 400 nm, and λ2em ) 474 nm): (a) fructose,
(b) glucose, and (c) galactose. (d) Plots of fluorescent intensity changes
of 1 as a function of sugar concentration at 474 nm.
at pH 7.4. Though several bisboronic acid sensors have been
synthesized to favor glucose over fructose among saccharide
bindings, their limitations to complex glucose make them less
efficient as glucose probes at high blood glucose levels.
Consequently, probe 1 shows an advantage at pH 7.4 by
(31) Gypser, A.; Michel, D.; Nirschl, D. S.; Sharpless, K. B. J. Org. Chem.
1998, 63, 7322–7327.
(32) Takeuchi, M.; Mizuno, T.; Shinmori, H.; Nakashima, M.; Shinkai, S.
Tetrahedron 1996, 52, 1195–1204.
J. Org. Chem. Vol. 74, No. 9, 2009 3545

TABLE 1. Photophysical Properties of 1 and 2 Monoboronic Acid
Probes
TABLE 2. Dissociation Constants (Kd) of Probes 1 and 2 in the
Presence of Monosaccharides
Kd (mmol)
probe
D-fructose
D-galactose
D-glucose
1
2
6.5 ( 0.2
13.2 ( 0.4
15.2 ( 0.5
22 ( 0.6
28.5 ( 1.2
35.4 ( 1.5
and photophysical changes in comparison with the ortho
derivative. Plans are currently underway to extend the absorption
wavelength of theses dyes to longer wavelength.
Experimental Section
N-(1,8-Naphthaloyl)-3-aminophenyl Boronic Acid (1). 3-Ami-
nophenylboronic acid hemisulfate (0.100 g, 0.54 mmol), 4-sulfo-
1,8-naphthalic anhydride, potassium salt (0.143 g, 0.45 mmol), and
10 mL of pure water were placed in a 25 mL round-bottomed flask
equipped with a Dean-Stark receiver and condenser. The reaction
mixture was allowed to reflux for 24 h. Water was removed by
drying overnight, and excess 3-aminophenylboronic acid was
removed by crystallization in hot ethanol. The reaction offered a
displaying the largest fluorescence increase to glucose while
maintaining an affinity within physiological limits, similar to
our probe synthesized from 3-nitronaphthalic anhydride and
3-aminophenylboronic acid.21 The incorporation of the boronic
acid group in the meta position does not lead to significant
spectroscopic and photophysical changes in comparison with
the ortho derivative. A relatively smaller dissociation constant
was observed for the meta derivative (probe 1) in the presence
of saccharides, which could be explained by the effect of less
steric hindrance (Figure 2).24 Quantum yields for both probes
were within approximately the same order of magnitude. In
addition, the apparent dissociation constants for meta (probe 1)
and ortho (probe 2) derivatives are listed in Table 2. Fluores-
cence spectra as well as graphical analyses to detail Kd values
are also provided in the Supporting Information.
1
pink-white powder (0.162 g, 81%), mp 290-293 °C: H NMR
(D2O) δ 9.04 (d, J ) 9 Hz, 1H), 8.54 (d, J ) 6 Hz, 2H), 8.30 (d,
J ) 7.7 Hz, 1H), 7.93 (t, J ) 6.6 Hz, 1H), 7.65 (m, J ) 8.8 Hz,
2H), 7.50 (m, 2H); 13C NMR (DMSO) δ 164.5, 164.0, 150.5, 135.0,
134.8, 131.1, 131.0, 129.8, 128.3, 127.4, 125.6, 123.9, 123.0; IR
1217, 1366, 1730, 2970 cm-1; HRMS for C18H9KSBNO7, expected
m/z 396.0349, found m/z 396.0351.
N-(1,8-Naphthaloyl)-2-aminophenyl Boronic Acid (2). 2-Ami-
nophenylboronic acid pinacol ester (0.165 g, 0.75 mmol), 4-sulfo-
1,8-naphthalic anhydride, potassium salt (0.143 g, 0.45 mmol), and
10 mL of pure water were placed in a 25 mL round-bottomed flask
equipped with a Dean-Stark receiver and condenser. The reaction
mixture was allowed to reflux for 24 h. The mixture was allowed
to reflux by adding 2 mL of 30% aqueous hydrogen peroxide at
room temperature for 1 h. Water was removed by drying overnight,
and excess 3-aminophenylboronic acid was removed by crystal-
lization in hot ethanol. The reaction offered an orange-yellow
1
powder (0.174 g, 87%), mp 355-358 °C: H NMR (D2O) δ 8.98
(d, J ) 8.2 Hz, 2H), 8.50 (d, J ) 6.6 Hz, 2H), 8.27 (d, J ) 7.3 Hz,
1H), 7.89 (t, J ) 7.2 Hz, 1H), 7.61 (d, J ) 8.1 Hz, 1H), 7.35 (m,
J ) 7.2 Hz, 2H); 13C NMR (DMSO) δ 164.6, 164.0, 150.7, 135.1,
131.0, 130.8, 129.0, 128.3, 127.2, 125.4, 124.1, 123.1, 123.0; IR
1209, 1368, 1732, 3011 cm-1; HRMS for C18H9KSBNO7, expected
m/z 396.0349, found m/z 396.0348.
FIGURE 2. Prediction of apparent dissociation constants of probe 1
among different saccharides.
Acknowledgment. This work was supported by a grant from
the National Institutes of Health (GM R15-57855-03). M.D.H
wishes to thank Ms. Sherrel of the Department of Chemistry
and Chemical Biology, University of New Mexico for HRMS
analysis.
In conclusion, two highly water-soluble monoboronic acid
probes that exhibit large fluorescence increases in the presence
of monosaccharides show remarkable sensitivity for glucose
rather than fructose and galactose. To our knowledge, this is
the first highly water-soluble monoboronic acid probe to dis-
play the more desirable off-on fluorescence response. While
both the monoboronic acid probes displayed a greater change
in fluorescence intensity for glucose, it should be emphasized
that their binding affinities are still greater for fructose. By
changing the position of the boronic acid group from ortho to
meta on the phenyl ring, there are no significant spectroscopic
Supporting Information Available: Fluorescent spectral
study and apparent dissociation constants in the presence of
monosaccharides of sensor 2. This material is available free of
charge via the Internet at http://pubs.acs.org.
JO9002008
3546 J. Org. Chem. Vol. 74, No. 9, 2009