A highly fluorescent water-soluble boronic acid reporter for saccharide sensing that shows ratiometric UV changes and significant fluorescence changes

Article abstract of DOI:10.1016/j.tet.2005.07.035

One water-soluble naphthalene-based fluorescent boronic acid, 6-(dimethylamino)-naphthalene-2-boronic acid (6-DMANBA, 1), has been synthesized. 6-DMANBA shows significant ratiometric UV absorbance changes upon addition of a sugar. For example, addition of 50 mM fructose shifted the UV absorption wavelengths of 6-DMANBA from 306 and 251 to 280 and 244 nm, respectively. In addition, 6-DMANBA is highly fluorescent with a quantum yield of 89% in the absence of a sugar and shows significant fluorescence intensity changes with the addition of a saccharide in aqueous phosphate buffer at physiological pH. For example, with the addition of 50 mM fructose, 6-DMANBA shows an 80% fluorescent intensity decrease at 432 nm. All these spectroscopic properties make compound 1 unique and useful.

Full text of DOI:10.1016/j.tet.2005.07.035

Tetrahedron 61 (2005) 9111–9117
A highly fluorescent water-soluble boronic acid reporter for
saccharide sensing that shows ratiometric UV changes and
significant fluorescence changes
*
Xingming Gao, Yanling Zhang and Binghe Wang
Department of Chemistry and Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30302-4089, USA
Received 6 April 2005; revised 12 July 2005; accepted 12 July 2005
Available online 2 August 2005
Abstract—One water-soluble naphthalene-based fluorescent boronic acid, 6-(dimethylamino)-naphthalene-2-boronic acid (6-DMANBA,
1), has been synthesized. 6-DMANBA shows significant ratiometric UV absorbance changes upon addition of a sugar. For example, addition
of 50 mM fructose shifted the UV absorption wavelengths of 6-DMANBA from 306 and 251 to 280 and 244 nm, respectively. In addition,
6-DMANBA is highly fluorescent with a quantum yield of 89% in the absence of a sugar and shows significant fluorescence intensity changes
with the addition of a saccharide in aqueous phosphate buffer at physiological pH. For example, with the addition of 50 mM fructose,
6-DMANBA shows an 80% fluorescent intensity decrease at 432 nm. All these spectroscopic properties make compound 1 unique and
useful.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
of Hageman,8r Smith,9c,d and Houston,9e,f have also reported
using boronic acids as a way to enhance cell affinity and/or
fusion. We termed these boronic acid-based carbohydrate
sensors as fluorescent boronolectins because of their
similarity in functions with lectins.10 Because saccharide
biomarkers11 are implicated in various physiological and
pathological processes including cancer,12 immune
responses,13 and viral and bacterial infection,14 such
boronolectins have a wide variety of potential applications
in the diagnosis, detection, and therapeutic intervention of
human diseases.12,15 Critical to our effort in developing
fluorescent boronolectins is the availability of reporter
compounds that change fluorescent properties upon
binding.8a,b,e,f,16–17 In our continuing work in this area, we
are interested in developing a combinatorial approach for
the synthesis of fluorescent boronolectins selective for
various biologically important carbohydrates and for multi-
plex sensing. For this effort and increased diversity of the
combinatorial libraries, we are in need of a large number of
boronic acid-based fluorescent reporter compounds that are
(1) water soluble and (2) chemically and photochemically
stable.16,17
Fluorescent sensors that can selectively recognize various
biologically important ions and compounds are very useful
for a wide range of applications.1 This field typically has
two major directions: sensors for metal ions and for organic
compounds. Recent years have seen a major surge in interest
in developing sensors for biologically important organic
compounds such as neurotransmitters,2 amino acids,3
nucleic acids,4 and carbohydrates.5 Among these activities,
the carbohydrate sensing and recognition field is especially
active. In the design and synthesis of carbohydrate sensors
for application in aqueous solution, the boronic acid
functional group is quite often used as the key recognition
moiety because of its strong and reversible interactions with
1,2- or 1,3-diols to form five- or six-membered rings,
respectively.6 Our laboratory7 and other research groups8
have been actively pursuing fluorescent carbohydrate
sensors based on boronic acid.
Recently, our laboratory has demonstrated that boronic
acid-based saccharide sensors can be used for the
recognition of cancer-related mammalian cell surface
saccharide biomarkers.9a,b Other laboratories such as those
Herein we describe the synthesis and evaluation of one
highly fluorescent boronic acid, 6-(dimethylamino)-
naphthalene-2-boronic acid (6-DMANBA, 1, Fig. 1),
which shows ratiometric UV property changes and
significant fluorescence changes upon sugar binding. This
Keywords: Boronic acid; Fluorescent sensor; Carbohydrate; Ratiometric
UV; Boronolectin.
Corresponding author. Tel.: C1 4046510289; fax: C1 4046545827;
e-mail: wang@gsu.edu
*
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.07.035

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X. Gao et al. / Tetrahedron 61 (2005) 9111–9117
(Fig. 2A and B). For example, the ratio of the absorbance at
280 nm to that at 306 nm was 0.56:1 before addition of a
sugar and 2.7:1 after addition of 50 mM fructose. This is a
4.8-fold change. In addition, the ratio of the absorbance at
244 nm to that at 251 nm changed from 0.87:1 before
addition of a sugar to 1.1:1 after addition of 50 mM fructose.
Other carbohydrates (galactose and glucose) also exhibited
significant changes in this ratio at different concentrations
(Fig. 2B). Therefore, compound 1 can be used as a
ratiometric UV sensor for carbohydrates.
Figure 1. Structure of naphthalene-based boronic acids.
boronic acid reporter will be useful for the combinatorial
synthesis of boronolectins.
The effect of carbohydrates on the UV absorbance property
of 4-DMANBA (2) and 5-DMANBA (3) was also studied in
a fashion similar to that of 6-DMANBA (1), and no similar
changes were observed (Fig. 2C and D).
2. Results and discussions
2.1. Synthesis
We also studied the pH induced UV spectral changes of 1.
Figure 3A and B show the results in the absence of a sugar in
the pH range of 2.00–7.40 and 7.40–11.00, respectively.
When the pH was increased from 2.00 to 7.40, the
absorbance of 1 at 251 and 306 increased significantly
with an absorbance decrease at 230 nm (Fig. 3A). When the
pH was further increased from 7.40 to 11.00, the absorbance
at 306 and 251 nm decreased significantly with a significant
absorbance increase at another two wavelengths, 280 and
244 nm, respectively (Fig. 3B). It is well known that the
increase of pH from 7.40 to 11.00 can change the boronic
acid moiety from its neutral trigonal form to the anionic
tetrahedral form. These results indicate that the neutral
trigonal form of 1 has absorbance maxima of 306 and
251 nm, while the anionic tetrahedral form has absorbance
maxima of 280 and 244 nm. The absorbance changes of 1
with increasing pH from 7.40 to 11.00 are very similar to the
absorbance changes of 1 induced by addition of a sugar.
The synthesis of 6-(dimethylamino)-naphthalene-2-boronic
acid (6-DMANBA, 1) is shown in Scheme 1. The
intermediate, compound 5, was synthesized from the
commercially available 4 by substitution of the hydroxyl
group with methylamine following a literature procedure.18
Reaction of 5 with methyl iodide in the presence of NaH in
THF at 60 8C gave 6-bromo-2-(dimethylamino)-naph-
thalene (6). 6-DMANBA (1) was synthesized from 6
through lithiation and reaction with trimethylborate.
Purification of the crude product by silica gel chromato-
graphy afforded 1 as a white powder in 64% yield.
The pH induced UV absorbance property changes of
4-DMANBA (2) and 5-DMANBA (3) were also studied in
a fashion similar to that of 6-DMANBA (1). Although
significant UV absorbance changes were observed for 2 and
3 when pH increased from 3.00 to 7.40, no significant
absorbance changes were observed for them when pH
increased from 7.40 to 11.00 (Fig. 3C and D).
2.3. Fluorescent properties of 6-DMANBA (1)
Scheme 1. Synthesis of 6-DMANBA (1): (i) CH3NH2, Na2S2O5, 140 8C,
77%; (ii) CH3I, NaH, 60 8C, 84%; (iii) n-BuLi, (MeO)3B, HCl, 64%.
The fluorescence properties of 6-DMANBA (1) was studied
in neutral aqueous solution (0.1 M aqueous phosphate
buffer, pH 7.4). Under such condition, compound 1 shows
emission maximum of 432 nm with a quantum yield of 89%
(8-quinoline boronic acid was used as the reference
compound19). This is the highest quantum yield so far
among all the water-soluble fluorescent boronic reporter
compounds discovered in our laboratory.16,17
2.2. UV absorbance properties of 6-DMANBA (1)
The effect of carbohydrates on the UV absorbance property
of 6-DMANBA (1) was studied. 6-DMANBA (1) showed
two absorbance maxima at 306 and 251 nm, respectively, in
0.1 M aqueous phosphate buffer at pH 7.4. Addition of a
carbohydrate resulted in a significant absorbance change of
compound 1. For example, with the addition of 50 mM
fructose, compound 1 shifted its UV lmax from 306 and 251
to 280 and 244 nm, respectively (Fig. 2A). The two
isosbestic points for the 306/280 nm and 251/244 nm shifts
were observed at 288 and 250 nm, respectively. In addition,
there is another isosbestic point at 271 nm.
Since compound 1 shows unique UV changes upon addition
of a sugar and different excitation wavelengths would
induce different emission changes upon addition of a sugar,
we first searched for the optimal excitation wavelength for
its fluorescence studies using fructose as a model sugar.
Table 1 shows the fluorescence intensity changes of 1 at the
emission wavelength of 432 nm with different excitation
wavelengths upon addition of 50 mM fructose. The
emission intensity changes of 1 with an excitation
Significant ratiometric absorbance changes of compound 1
were observed at four wavelengths upon addition of a sugar

X. Gao et al. / Tetrahedron 61 (2005) 9111–9117
9113
Figure 2. UV absorbance changes of 1, 2, and 3 upon addition of a sugar in 0.1 M aqueous phosphate buffer at pH 7.4. (A) UV absorbance spectral changes of
6-DMANBA (1) (1.0!10K5 M) with different concentrations of D-fructose (0–50 mM). (B) Absorbance ratiometric changes (Abs (280 nm)/Abs (306 nm)) of
6-DMANBA (1) (1.0!10K5 M) as a function of sugar concentrations. (C) UV absorbance spectral changes of 4-DMANBA (2, 1.0!10K5 M) in the absence
and presence of 50 mM D-fructose. (D) UV absorbance spectral changes of 5-DMANBA (3, 1.0!10K5 M) in the absence and presence of 50 mM D-fructose.
Figure 3. UV absorbance spectral changes of 1, 2, and 3 at different pH in 0.1 M aqueous phosphate buffer. (A) 6-DMANBA (1) (1.0!10K5 M) at different pH
(from 2.00 to 7.40). (B) 6-DMANBA (1) (1.0!10K5 M) at different pH (pH from 7.40 to 10.0). (C) 4-DMANBA (2) (1.0!10K5 M) at different pH.
(D) 5-DMANBA (3) (1.0!10K5 M) at different pH.

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X. Gao et al. / Tetrahedron 61 (2005) 9111–9117
Table 1. Fluorescence intensity changes (DI/I0) of 6-DMANBA (1) at the
emission wavelength of 432 nm with different excitation wavelengths upon
addition of 50 mM D-fructose
Table 2. Apparent association constants (Ka) and fluorescence intensity
changes (DI/I0) of 6-DMANBA (1) with different sugars
Sugar
Ka (MK1
)
DI/I0 (Sugar concentration, M)
Excitation
wavelength
(lex, nm)
DI/I0
Excitation
wavelength
(lex, nm)
DI/I0
Sorbitol
Fructose
Tagatose
Galactose
Glucose
227G5
120G2
103G3
8.0G0.3
2.4G0.2
K0.825 (0.10)
K0.801 (0.05)
K0.853 (0.10)
K0.772 (1.0)
K0.610 (1.0)
250
260
270
271
280
288
K0.314
K0.588
K0.497
K0.185
K0.035
K0.366
290
300
310
320
330
340
K0.440
K0.665
K0.801
K0.743
K0.592
K0.495
wavelength at its three UV isosbestic points of 250, 271, and
288 nm were K0.314, K0.185, and K0.366, respectively
(Table 1). Understandably, excitation at the isosbestic
points did not give the largest fluorescent intensity changes
up sugar addition. Instead, the largest fluorescence changes
occurred when the excitation wavelength was at the
absorption lmax, 310 nm. Therefore, excitation at 310 nm
was used for further fluorescence studies of compound 1.
With the optimal excitation wavelength established, we
studied the concentration-dependent fluorescent changes of
1 with the addition of sugars. With the addition of fructose
and four other sugars (sorbitol, tagatose, galactose, and
glucose) at different concentrations to the solution of
6-DMANBA in 0.1 M aqueous phosphate buffer (pH 7.4),
significant decreases in fluorescent intensity were observed.
For example, with the addition of fructose, the fluorescent
intensity decreased by 80% (Fig. 4).
Figure 5. Fluorescence intensity changes (DI/I0) of 6-DMANBA (3) (1.0!
10K6 M) as a function of sugar concentrations in 0.1 M aqueous phosphate
buffer at pH 7.4; lexZ310 nm, lemZ432 nm.
Figure 4. Fluorescence spectral changes of 6-DMANBA (1) (1.0!10K6 M)
with different concentrations of D-fructose (0–50 mM) in 0.1 M aqueous
phosphate buffer at pH 7.4, lexZ310 nm.
The results of such fluorescent studies are summarily
presented in Table 2 and Figure 5. Addition of sorbitol,
fructose, and tagatose induced fluorescence intensity
changes at much lower sugar concentrations than that of
galactose and glucose due to their difference in binding
affinity. However, it is important to note that they all
eventually reach about the same level, indicating that all
these complexes have similar fluorescent intensities.
The apparent association constants (Ka) between 1 and these
five carbohydrates were determined assuming the formation
of a 1:1 complex.20 As expected, the affinity trend of various
Figure 6. Fluorescence spectral changes of 6-DMANBA (1) (1.0!10K6 M)
in the absence of sugar at different pH in 0.1 M aqueous phosphate buffer,
lexZ310 nm. (A) pH from 2.00 to 7.00; (B) pH from 7.00 to 11.00.

X. Gao et al. / Tetrahedron 61 (2005) 9111–9117
9115
sugars with 1 followed that of simple monoboronic acid in
the order of sorbitolOfructoseytagatoseOgalactoseO
glucose (Table 2). For example, the observed association
constants with different sugars show the same binding trend
as 2 and 3 as well as other monoboronic acids.6c,d,17 The
different binding properties of these sugars are attributed to
the different dihydral angles of the diol of these sugars.
The pH induced fluorescent spectral changes of 1 in the
absence and presence of fructose or glucose at a fixed
concentration (50 mM) were studied to examine the
relationship between the fluorescence intensity changes
and the different ionization states. Figure 6 shows the
fluorescence spectral changes of 1 at different pH in the
absence of a sugar. It can be seen that the fluorescence
intensity of 1 at 432 nm increased when pH increased from
2 to 7 (Fig. 6A). After that, increasing pH caused a decrease
in fluorescence intensity at 432 nm (Fig. 6B). Such results
indicate that the free form (1) is the fluorescent species (1),
and neither the protonated form (1a) nor the boron ionized
form (1b) is fluorescent (Scheme 2). With the deprotonation
of the aniline group, the proportion that exists in the neutral
form increases, which gives the increased fluorescence. At
higher pH, the boronic acid moiety becomes ionized, which
causes a drop in fluorescence.
Figure 7. pH titration of the fluorescence intensity of 6-DMANBA (1) (1!
10K6 M) at 432 nm in the absence and presence of 50 mM fructose or
glucose in 0.1 M aqueous phosphate buffer, lexZ310 nm.
requires a much higher concentration for the boronic acid to
be converted to its complexed form, which has a lower pKa
than the free boronic acid.
It should be noted that the pH induced fluorescence changes
of 1 are different from its analogs, 4-dimethylamino-
naphthaleneboronic acid (4-DMANBA, 2)17a and
5-dimethylaminonaphthaleneboronic acid (5-DMANBA,
3)17b. In 4-DMANBA, the neutral form is not fluorescent,
but the boron-ionized form is, which gives a fluorescent
intensity increase upon sugar addition. In 5-DMANBA, the
boron ionized form is also very fluorescent, but has a
different emission wavelength than the neutral form, which
gives fluorescent ratiometric sensing. It should also be noted
that 6-DMANBA only has one emission wavelength no
matter what protonation state it is in. This again is in direct
contrast to 4-DMANBA and 5-DMANBA, which also
showed an emission peak at 336 nm in the protonated form.
It is interesting to see that with the same chromophore,
naphthalene, the functional group substitution positions can
bestow very different spectroscopic properties on these
boronic acid compounds.
Scheme 2. The spectroscopic properties of 6-DMANBA (1) at its different
ionization states.
3. Conclusions
The fluorescence intensity profile of 1 versus pH in the
presence of 50 mM fructose or glucose were also studied
(Fig. 7). It can be seen from this figure that the situation with
the esters is similar to that of the free boronic acid, that is,
only the neutral form (1d, Scheme 2) is fluorescent. Neither
the protonated form (1c) nor the boron-ionized form (1e)
has appreciable fluorescence. The fluorescence intensity
changes observed with the addition of a sugar is largely due
to a decrease in the boron pKa with ester formation, which
converts the boron to the sp3 hybridized form that is non-
fluorescent. For example, the apparent pKa of the boronic
acid group of 1 in the absence of a sugar is about 9.0, while
its apparent pKa dropped to about 6.8 in the presence of
50 mM fructose. In such a case, at neutral pH the boron is
ionized in the presence of fructose and neutral in the absence
of a sugar. Since the ionized form is non-fluorescent,
addition of fructose causes a significant decrease in
fluorescent intensity. For glucose, because its binding
constant is much smaller than that of fructose (Table 1), it
One new water-soluble naphthalene-based boronic acid
fluorescent saccharide sensor, 6-DMANBA (1), was
synthesized and studied. 6-DMANBA (1) shows significant
ratiometric UV absorbance changes upon addition of a
sugar. In addition, 6-DMANBA (1) is a very fluorescent
compound with a high fluorescence quantum yield (fFZ
89%) in the absence of a sugar. Addition of a sugar
significantly decreases fluorescence intensity of 1. All these
properties make compound 1 special and useful in our effort
of synthesizing combinatorial libraries in search of new
fluorescent boronolectins.
4. Experimental
4.1. General
1H NMR spectra were recorded at 300 MHz and 13C NMR
spectra at 75 MHz with tetramethylsilane as the internal

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X. Gao et al. / Tetrahedron 61 (2005) 9111–9117
standard. Mass spectral analyses were performed by the
mass spectrometry facilities of Georgia State University.
Fluorescence spectra were recorded on a Shimadzu
RF-5301 PC spectrofluorometer. Absorption spectra were
recorded on a Shimadzu UV-1700 UV–vis spectro-
photometer. All pH values were determined by a UB-10
Ultra Basic Benchtop pH meter (Denver Instrument).
Column chromatography was performed on silica gel
(200–400 mesh) from Aldrich. Tetrahydrofuran (THF)
was distilled over Na before use.
white powder (0.055 g, 64%). 1H NMR (300 MHz, CD3OD)
d 8.11 (s, 0.5H), 7.97 (s, 0.5H), 7.55–7.70 (m, 3H), 7.16 (d,
JZ9.0 Hz, 1H), 6.91 (s, 1H), 3.00 (s, 6H); 13C NMR
(75 MHz, CD3OD) d 150.8, 137.9, 135.8, 131.7, 130.5,
127.9, 127.2, 126.3, 117.4, 107.4, 41.1; HR ESI-MS (m/z)
Calcd for C12H15BNO2 216.1196 (MCH)C, found
216.1215.
4.3. Procedures for the binding studies
Distinct solutions of the sensors (2!10K6 M for fluor-
escence and 2!10K5 M for UV absorbance) and the sugars
(various concentrations) were prepared in 0.1 M phosphate
buffer at pH 7.40. Then, 2 mL of a sensor solution was
mixed with 2 mL of a sugar solution. After stirring for
20 min, the mixture was transferred into a 1 cm quartz cell
and the fluorescence intensity or UV absorbance was
recorded immediately.
4.2. Synthesis
4.2.1. 6-(N-Methyl)-2-bromo-naphthalene (5).18 A mix-
ture of 4 (1.0 g, 4.5 mmol), Na2S2O5 (1.6 g, 8.7 mmol), and
H2O (4 mL) was placed in a pressure reactor. To the
solution was added aqueous methylamine (40%) (2.0 mL,
22 mmol). The reaction mixture was stirred at 140 8C for
4 days. After cooling, the reaction mixture was dissolved in
50 mL CH2Cl2, washed with 5% NaHCO3 (3!30 mL) and
dried over Na2SO4. After evaporation of solvent, the crude
was purified on a silica gel column, eluting with CH2Cl2/
hexanes (1:1), to give compound 5 as a white powder
Acknowledgements
1
Financial support from the National Institutes of Health
(CA88343, CA113917, and NO1-CO-27184), the Georgia
Cancer Coalition through a Distinguished Cancer Scientist
Award, and the Georgia Research Alliance through an
Eminent Scholar endowment and an Eminent Scholar
Challenge grant is gratefully acknowledged.
(0.82 g, 77%). H NMR (300 MHz, CDCl3) d 7.76 (d, JZ
1.2 Hz, 1H), 7.46–7.36 (m, 3H), 6.80–6.76 (dd, JZ8.7,
0.6 Hz, 1H), 6.54 (d, JZ2.1 Hz, 1H), 3.83 (s, 1H), 2.83 (s,
3H); 13C NMR (75 MHz, CDCl3) d 147.6, 134.1, 129.9,
129.7, 128.7, 128.2, 127.9, 119.1, 115.2, 103.7, 30.9; HR
ESI-MS (m/z) Calcd for C11H11BrN 236.0075 (MCH)C,
found 236.0079.
4.2.2. 6-(N-Dimethyl)-2-bromo-naphthalene (6). To the
solution of compound 5 (0.33 g, 1.2 mmol) and CH3I
(0.75 mL, 12 mmol) in THF (50 mL) was added NaH (60%)
(0.58 g, 14 mmol). The reaction mixture was stirred at 60 8C
overnight. The precipitate was removed by filtration. After
evaporation of solvent, the residue was dissolved in 50 mL
CH2Cl2 and washed with 5% NaHCO3 (3!30 mL) and
dried over Na2SO4. After evaporation of solvent, the crude
was purified on a silica gel column, eluting with CH2Cl2/
hexanes (1:1), to give compound 6 as a white powder
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tet.2005.07.
035. 1H and 13C NMR of 6-DMANBA (1) and two
intermediates (5 and 6) are available.
1
(0.25 g, 84%). H NMR (300 MHz, CDCl3) d 7.78 (d, JZ
1.8 Hz, 1H), 7.51 (d, JZ9.3 Hz, 1H), 7.45 (d, JZ8.6 Hz,
1H), 7.36 (dd, JZ9.0, 2.7 Hz, 1H), 7.07 (dd, JZ9.0, 2.7 Hz,
1H), 6.80 (d, JZ2.4 Hz, 1H), 2.98 (s, 6H); 13C NMR
(75 MHz, CDCl3) d 149.1, 133.8, 129.7, 129.6, 128.2,
128.1, 128.0, 117.3, 115.3, 106.3, 41.0; HR ESI-MS (m/z)
Calcd for C12H13BrN 250.0231 (MCH)C, found 250.0239.
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