New Boronic Acid Fluorescent Reporter Compounds. 2. A Naphthalene-Based On-Off Sensor Functional at Physiological pH

Article abstract of DOI:10.1021/ol035783i

(Matrix presented) A new boronlc acid fluorescent on-off reporter compound (1) was synthesized. This fluorescent sensor shows a 41-fold emission intensity increase upon addition of 50 mM fructose in 0.1 M aqueous phosphate buffer at pH 7.4.

Full text of DOI:10.1021/ol035783i

ORGANIC
LETTERS
2003
Vol. 5, No. 24
4615-4618
New Boronic Acid Fluorescent Reporter
Compounds. 2. A Naphthalene-Based
On−Off Sensor Functional at
†
Physiological pH
Xingming Gao, Yanling Zhang, and Binghe Wang*
Department of Chemistry, Georgia State UniVersity, 33 Gilmer Street S.E.,
Atlanta, Georgia 30303
wang@gsu.edu
Received September 15, 2003
ABSTRACT
A new boronic acid fluorescent on−off reporter compound (1) was synthesized. This fluorescent sensor shows a 41-fold emission intensity
increase upon addition of 50 mM fructose in 0.1 M aqueous phosphate buffer at pH 7.4.
During the past decade, a great deal of effort has been
directed toward the detection of saccharides by fluorescent
chemosensors.1 Critical to the design of sensors is the
availability of fluorescent reporter moieties that respond to
the saccharide recognition event with significant fluorescence
intensity changes. Our laboratory has been interested in the
preparation of fluorescent sensors for cell-surface poly-
saccharides for in vivo applications.2
Boronic acids have been known for decades to bind
saccharides via covalent interactions.3 During the past decade,
there has been much important progress made in the
construction of boronic acid-based sensors for carbohydrates.4
Different mechanisms have been used to induce spectroscopic
changes upon binding of the boronic acid moiety with a
saccharide.1,4i Among the most important discoveries is an
anthracene-based fluorescent reporter system developed by
Shinkai and co-workers. This system has been widely used
because of its fairly large fluorescence intensity increase upon
ester formation due to the switching of a photoinduced
electron transfer (PET) process.4b,5 Our group has also applied
the Shinkai system for the preparation of sensors for mono-
and oligosaccharides.2,6
In our continuing effort to search for more efficient and
water-soluble boronic acid fluorescent reporter compounds,7
† Part of this work was conducted at North Carolina State University.
For part 1 of this series, see ref 7.
(4) (a) Yoon, J.; Czarnik, A. W. J. Am. Chem. Soc. 1992, 114, 5874-
5875. (b) James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. Chem.
Commun. 1994, 477-478. (c) James, T. D.; Sandanayake, K. R. A. S.;
Shinkai, S. Angew. Chem., Int. Ed. Engl. 1994, 33, 2207-2209. (d) James,
T. D.; Sandanayake, K. R. A. S.; Shinkai, S. Nature (London) 1995, 374,
345-347. (e) Eggert, H.; Frederiksen, J.; Morin, C.; Norrild, J. C. J. Org.
Chem. 1999, 64, 3846-3852. (f) Adhikiri, D. P.; Heagy, M. D. Tetrahedron
Lett. 1999, 40, 7893-7896. (g) Lavigne, J. J.; Anslyn, E. V. Angew. Chem.,
Int. Ed. 1999, 38, 3666-3669. (h) Ward, C. J.; Patel, P.; Ashton, P. R.;
James, T. D. Chem. Commun. 2000, 229-230. (i) DiCesare, N.; Lakowicz,
J. R. J. Phys. Chem. A 2001, 105, 6834-6840. (j) Yang, W.; He, H.;
Drueckhammer, D. G. Angew. Chem., Int. Ed. 2001, 40, 1714-1718.
(5) (a) Sandanayake, K. R. A. S.; Shinkai, S. J. Chem. Soc., Chem.
Commun. 1994, 1083-1084. (b) James, T. D.; Sandanayake, K. R. A. S.;
Iguchi, R.; Shinkai, S. J. Am. Chem. Soc. 1995, 117, 8982-8987.
(1) (a) James, T. D.; Linnane, P.; Shinkai, S. Chem. Commun. 1996,
281-288. (b) Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley,
A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997,
97, 1515-1566. (c) Hartley, J. H.; James, T. D.; Ward, C. J. J. Chem.
Soc., Perkin Trans. 1 2000, 3155-3184. (d) Wang, W.; Gao, X.; Wang, B.
Curr. Org. Chem. 2002, 6, 1285-1317.
(2) (a) Yang, W.; Gao, S.; Gao, X.; Karnati, V. R.; Ni, W.; Wang, B.;
Hooks, W. B.; Carson, J.; Weston, B. Bioorg. Med. Chem. Lett. 2002, 12,
2175-2177. (b) Yang, W.; Hooks, W. B.; Gao, X.; Gao, S.; Karnati, V. V.
R.; Ni, W.; Carson, J.; Weston, B.; Wang, B. Chem. Bio. Submitted for
publication.
(3) (a) Sugihara, J. M.; Bowman, C. M. J. Am. Chem. Soc. 1958, 80,
2443. (b) Lorand, J. P.; Edwards, J. O. J. Org. Chem. 1959, 24, 769. (c)
Springsteen, G.; Wang, B. Tetrahedron 2002, 58, 5291-5300.
10.1021/ol035783i CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/28/2003

we are interested in taking advantage of the manipulation
of the internal charge transfer (ICT) process for the construc-
tion of boronic acid-based sensors. The ICT process is well-
known to be very sensitive to small perturbations that can
induce spectroscopic changes and has been used in the past
for the synthesis of fluorescent boronic acid reporter
compounds with a maximum intensity change of about
5-fold.8 Usually, an ICT system contains an electron donor
group and an electron acceptor group in the same chro-
mophore. Lakowicz4i,8b,c and James8a have reported a series
of boronic acid-based sensors involving the ICT mechanism.
In these sensors, the sp2-hybridized boron atom of the boronic
acid group linked directly to the fluorophore can form a
conjugated system with the aromatic moiety and act as an
electron acceptor group because of the empty p-orbital in
the boron atom. Incorporation of a donor group on the same
chromophore can result in excited-state charge transfer.
However, conversion from an sp2- to an sp3-hybridization
would leave the boron atom without the empty orbital, and
consequently switch off the ICT process. Since it is known
that conversion of a phenylboronic acid analogue to its ester
with sugars commonly results in the lowering of the pKa by
about 2-3 pH units,3b,c boronic ester formation frequently
means the conversion of the boron atom from the neutral
sp2 form to the anionic sp3 at physiological pH. Conse-
quently, addition of a sugar to a boronic acid solution could
bring about a change in the ICT states.
amino group can act as a donor and the boron atom in a sp2
state can act as an acceptor in an ICT process. We reasoned
that ester formation would change the hybridization of the
boron to sp3 and switch off the ICT process.
The effect of various carbohydrates on the fluorescent
properties of compound 1 was determined in phosphate
buffer at pH 7.4. The emission spectral change of 1 with
fructose at different concentrations in 0.1 M aqueous
phosphate buffer (pH 7.4) is shown in Figure 1. A 41-fold
Figure 1. Fluorescence spectral change of 1 (1.0 × 10-5 M) with
different concentrations of D-fructose (0-50 mM) in 0.1 M aqueous
phosphate buffer at pH 7.4, λex ) 300 nm.
In this work, we report a new water-soluble boronic acid-
based fluorescent ICT saccharide sensor (DMANBA, 1) that
shows a large fluorescence intensity increase upon binding
with a sugar in aqueous solution at physiological pH.
Compound 1 was readily synthesized from the com-
mercially available 1-bromo-4-(dimethylamino)naphthalene
through lithiation and reaction with trimethylborate (Scheme
1). Purification of the crude product by silica gel chroma-
emission intensity increase is observed in the presence of
50 mM fructose. To the best of our knowledge, such a large
emission intensity increase has never been reported for
boronic acid-based saccharide sensors involving the ICT
mechanism. The quantum yields of 1 in the absence (φF )
0.010) and in the presence of 50 mM fructose (φF ) 0.42)
in 0.1 M phosphate buffer (pH 7.4) were obtained with use
of 8-quinoline boronic acid (φF ) 0.58 in 12 M H2SO4) as
a reference compound.10 Such results indicate that indeed
addition of a carbohydrate results in a very significant change
in the spectroscopic properties of the sensor compound (1).
Scheme 1. Synthesis of DMANBA (1)
To examine the general applicability of this fluorescent
reporter compound, we have also studied the effect of four
other carbohydrates on its fluorescence intensity (Figure 2).
These carbohydrates include sorbitol, tagatose, galactose, and
glucose. From Figure 2, it is clear that all five carbohydrates
tested caused significant fluorescence intensity increases at
physiological pH with varying magnitude. Addition of
sorbitol and fructose induced the largest fluorescence inten-
sity changes, more than 40-fold, at concentrations above 50
mM. Glucose, on the other hand, induced a maximum of
tography and recrystalization from dichloromethane-hexanes
afforded 1 as a colorless crystal.9 In this compound (1), the
(6) (a) Wang, W.; Gao, S.; Wang, B. Org. Lett. 1999, 1, 1209-1212.
(b) Gao, S.; Wang, W.; Wang, B. Bioorg. Chem. 2001, 29, 308-320. (c)
Karnati, V. R.; Gao, X.; Gao, S.; Yang, W.; Ni, W.; Sankar, S.; Wang, B.
Bioorg. Med. Chem. Lett. 2002, 12, 3373-3377. (d) Yang, W.; Yan, J.;
Fang, H.; Wang, B. Chem. Commun. 2003, 792-793.
(7) Yang, W.; Yan, J.; Springsteen, G.; Wang, B. Bioorg. Med. Chem.
Lett. 2003, 13, 1019-1022.
(8) (a) Arimori, S.; Bosch, L. I.; Ward, C. J.; James, T. D. Tetrahedron
Lett. 2001, 42, 4553-4555. (b) DiCesare, N.; Lakowicz, J. R. Chem.
Commun. 2001, 2022-2023. (c) DiCesare, N.; Lakowicz, J. R. Anal.
Biochem. 2002, 301, 111-116.
(9) Selected characterization data for 4-(dimethylamino)naphthalene
boronic acid (1): colorless crystal, yield 42%; HRESI-MS calcd for C12H15-
BNO2 216.1196 (M + H)+, found 216.1187; 1H NMR (400 MHz, CD3-
OD) δ 2.87 (s, 6H), 7.10 (d, J ) 7.2 Hz, 1H), 7.45 (m, 3H), 7.76 (m, 1H),
8.21 (m, 1H); 13C NMR (400 MHz, CD3OD) δ 113.23, 124.39, 124.68,
125.71, 128.45, 128.52, 130.48. Anal. Calcd for C12H14BNO2‚3/4H2O: C,
71.51, H, 6.25, N, 6.95. Found: C, 71.80, H, 6.42, N, 6.65.
(10) Goldman, M.; Wehry, E. L. Anal. Chem. 1970, 42, 1186-1188.
4616
Org. Lett., Vol. 5, No. 24, 2003

Figure 2. Fluorescence intensity changes (∆I/I0) of 1 (1.0 × 10-5
M in 0.1 M aqueous phosphate buffer at pH 7.4) as a function of
sugar concentration at 25 °C; λex ) 300 nm, λem ) 445 nm.11
Figure 3. pH titration of the fluorescence intensity of 1 (1 × 10-5
M) in the absence and presence of sugars in 0.1 M aqueous
phosphate buffer, λex ) 300 nm, λem ) 445 nm.
17-fold fluorescence intensity increase at a much higher
concentration (1 M).
of 1 in the absence of any carbohydrate increased by 168-
fold at 445 nm upon changing the pH from 3 to 12. It is
worth mentioning that the pKa of the protonated dimethyl
amino group of 1 is about 4.6 and the emission wavelength
of 1 with the protonated dimethyl amino group at low pH is
about 338 nm. Therefore, the fluorescence intensity of 1 at
445 nm is not affected by the protonated dimethylamino
group. The fluorescence intensity changes correspond to the
boronic acid pKa (9.4) and therefore hybridization changes,
which is responsible for the fluorescence intensity changes
observed.
To examine the binding in a more quantitative fashion,
the association constants (Ka) between 1 and the five
carbohydrates were determined assuming the formation of
a 1:1 complex.4e,12 As expected, the affinity trend with 1
followed that of simple phenylboronic acid in the order of
sorbitol > fructose > tagatose > galactose > glucose (Table
1). The absolute numbers are also similar to what was
Table 1. Association Constants (Ka) and Fluorescence Intensity
Changes (∆I/I0) of 1 with Different Sugars
The pH titration curves of 1 in the presence of fructose
and glucose showed over 140-fold increases in fluorescence
intensity when pH increased from 3 to 10 (Figure 3). It is
well-known that the binding of a diol to boronic acid most
of the time lowers the pKa of the boron species.3b,c In our
case, an apparent pKa of 6.4 was observed in the presence
of 50 mM of fructose. As described earlier, the boronic acid
moiety of 1 has a pKa of about 9.4. Therefore, at physi-
ological pH, it exists in the neutral, non-ionized form.
However, upon addition of fructose, the apparent pKa of the
solution drops to about 6.4, which ensures that most of the
boron species are in the anionic tetrahedral state at physi-
ological pH. It should be noted that the pKa here is the
apparent pKa because of the tridentate binding mode of
fructose with a boronic acid.13
The apparent pKa of the mixture of 1 and glucose (50 mM)
was much higher (8.3) (Figure 3). However, it should be
noted that in this mixture only a small portion of the boronic
acid is expected to be in the ester state because of the low
association constant (4 M-1), which gives about 20%
complexation at pH 7.4. Therefore, this pKa is a reflection
of a mixture of mostly the free boronic acid and a small
portion of the ester.
∆I/I0 (sugar
concn, M)
sugar
Ka (M-1
)
sorbitol
fructose
tagatose
galactose
glucose
226 ( 5
207 ( 4
116 ( 2
12.0 ( 0.2
4.0 ( 0.1
42 (0.10)
41 (0.05)
32 (0.02)
35 (0.50)
17 (1.0)
observed with phenylboronic acid.3c For example, phenyl-
boronic acid (PBA) has a Ka of 162 M-1 for fructose and 5
M-1 for glucose.3c
To examine the relationship between the fluorescence
intensity changes and the boron ionization states, we have
also studied the pH profile of the fluorescence intensity in
the absence and presence of fructose and glucose at a fixed
concentration (50 mM) (Figure 3). The emission intensity
(11) Procedures for the Binding Studies: The sensor (1) was dissolved
in 0.1 M phosphate buffer (pH 7.40) (2 × 10-5 M) and the sugar was
dissolved in 0.1 M phosphate buffer (pH 7.40) at various concentrations.
For fluorescent binding studies, 2 mL of the sensor solution was mixed
with 2 mL of the sugar solution. After being stirred for 20 min, the mixture
was transferred into a cuvette, and the fluorescence intensity was recorded
immediately.
(12) (a) Hamai, S. Bull. Chem. Soc. Jpn. 1982, 55, 2721-2729. (b)
Sanchez, F. G.; Lopez, M. H.; Gomez, J. C. M. Analyst 1987, 112, 1037-
1040. (c) Tong, L. Cyclodextrin Chemistry-Base and Applications;
Scientific Publisher: Beijing, China, 2001; pp 145-148. (d) Fery-Forgues,
S.; Le Bris, M.-T.; Guette´, J.-P.; Valeur, B. J. Phys. Chem. 1988, 92, 6233-
7237.
To confirm the correlation of the fluorescence intensity
increase of 1 with the ionization state change of the boronic
acid, we recorded the 11B NMR spectra of 1 in the absence
and presence of fructose. The 11B chemical shift of 1 changed
from 32.5 ppm in the absence of fructose, which is
Org. Lett., Vol. 5, No. 24, 2003
4617

characteristic of the neutral trigonal form of boronic acid,13b
to 11.1 ppm in the presence of fructose (20 equiv), which is
characteristic of the anionic tetrahedron form of boronic acid
(Figure 4). Such 11B NMR spectra results are consistent with
the pH-fluorescence intensity titration results.
commercially available starting material through a one-step
conversion. We assume that an ICT process is responsible
for the low fluorescence intensity before addition of a sugar
and the removal of an ICT process upon addition of a sugar
is the reason for the increased fluorescence of compound 1.
This is essentially an on-off system. Work is underway to
use this fluorescent reporter compound for the construction
of di- and multiboronic acid sensors for the high-specificity
identification and detection of biologically important car-
bohydrates.
Acknowledgment. Financial support from National In-
stitutes of Health (CA88343 and NO1-CO-27184) and the
Georgia Cancer Coalition through a GCC Distinguished
Cancer Scientist Award is gratefully acknowledged. We
would also like to thank Dr. Sabapathy Sankar for his help
with the 11B NMR work. We also acknowledge Frontier
Scientific Corporation for providing certain boronic acid
samples in some of our related studies.
Figure 4. 11B NMR in DMSO/0.1 M aqueous phosphate buffer
(1:3, pH 7.4): (a) 1 (9 mM) alone; (b) the mixture of 1 (9 mM)
and fructose (20 equiv, 0.18 M). BF3 was used as an external
reference.
Supporting Information Available: Synthetic procedures
and 1H and 13C NMR of 1. This material is available free of
charge via the Internet at http://pubs.acs.org.
OL035783I
In conclusion, a new water-soluble fluorescent saccharide
sensor that shows large fluorescence intensity increases upon
binding with a diol was conveniently synthesized from
(13) (a) Norrild, J. C.; Eggert, H. J. Chem. Soc., Perkin Trans. 2 1996,
2583-2588. (b) Norrild, J. C. J. Chem. Soc., Perkin Trans. 2 2001, 719-
726.
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Org. Lett., Vol. 5, No. 24, 2003