The development of photometric sensors for boronic acids

Article abstract of DOI:10.1006/bioo.2001.1217

Boronic acids bind certain 1,2- and 1,3-diols with high affinity through reversible formation of boronate esters. They have been utilized as the recognition moiety for artificial receptors, particularly receptors for carbohydrates that have cis-diol moieties. Therefore, sensors for boronic acids could serve as universal reporters for monitoring boronate formation. This paper reports the design and synthesis of a series of photometric chemosensors for phenylboronic acid using diethanolamine as the recognition moiety. Diethanolamine, which binds strongly to boronic acids, has been linked to three different types of optical reporters. A photoinduced electron transfer system based on the anthracene fluorophore has been used to create sensors that show up to a fivefold increase in fluorescent intensity in the presence of millimolar concentrations of phenylboronic acid. Sensor designs based on the restriction of free rotation of extended π systems and on the perturbed electronic properties of azo dyes are also included. This work demonstrates that sensors based on several different designs can be used for the detection of boronic acids.

Full text of DOI:10.1006/bioo.2001.1217

Bioorganic Chemistry 29, 259–270 (2001)
doi:10.1006/bioo.2001.1217, available online at http://www.idealibrary.com on
The Development of Photometric Sensors for Boronic Acids1
Greg Springsteen, C. Eric Ballard, Shouhai Gao, Wei Wang, and
Binghe Wang2
Department of Chemistry, Box 8204, North Carolina State University,
Raleigh, North Carolina 27695-8204
Received May 21, 2001
Boronic acids bind certain 1,2- and 1,3-diols with high affinity through reversible formation
of boronate esters. They have been utilized as the recognition moiety for artificial receptors,
particularly receptors for carbohydrates that have cis-diol moieties. Therefore, sensors for
boronic acids could serve as universal reporters for monitoring boronate formation. This paper
reports the design and synthesis of a series of photometric chemosensors for phenylboronic
acid using diethanolamine as the recognition moiety. Diethanolamine, which binds strongly to
boronic acids, has been linked to three different types of optical reporters. A photoinduced
electron transfer system based on the anthracene fluorophore has been used to create sensors
that show up to a fivefold increase in fluorescent intensity in the presence of millimolar
concentrations of phenylboronic acid. Sensor designs based on the restriction of free rotation
of extended ␲ systems and on the perturbed electronic properties of azo dyes are also included.
This work demonstrates that sensors based on several different designs can be used for the
detection of boronic acids.
᭧ 2001 Academic Press
Key Words: boron; boronic acid; sensor; diethanolamine; PET.
INTRODUCTION
Boronic acids are capable of forming covalent linkages with diols to give boronate
esters. Although the stabilities of these esters are dependent upon many factors,
dissociation constants of boronates in aqueous solution have been reported in the
micromolar range (2). Consequently, boronate formation has been widely used to
recognize diol-containing molecules. This has led to the development of sensors
for carbohydrates and amino acids (3–7) and selective transporters of nucleosides,
saccharides, and nucleotides (8–11). Boronic acids are electron-deficient species that
have also been used as organic reagents in Suzuki cross-coupling reactions (12,13),
diol protection (14), Diels-Alder reactions (15), selective reduction of an aldehyde
in the presence of a ketone (16), and asymmetric synthesis of amino acids (17). In
1 Part of this work has been communicated (1).
2 To whom correspondence should be addressed. Fax: (919) 515-3757. E-mail: binghe wang@
ncsu.edu.
259
0045-2068/01 $35.00
Copyright ᭧ 2001 by Academic Press
All rights of reproduction in any form reserved.

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addition, compounds containing boronic acid moieties have been used as inhibitors of
proteases (18–23) and as therapeutic agents in boron neutron capture therapy (24,25).
Sensors for boronic acids could be used in a variety of applications (26). Sensors
could monitor carbohydrate/boronic acid complexation by following the disappearance
of free boronic acid, they might help elucidate the mechanisms of selective transport
of biologically important molecules, or they may prove useful in mechanistic studies
of reactions involving boronic acids or in monitoring reactions of boronic acids in
combinatorial library synthesis. Of particular interest to us is the development of
boronic acid sensors that may be used as universal reporters for monitoring boronate
formation under near physiological conditions (27). As the range and utility of boronic
acids increase in the fields of medicinal and organic chemistry, there is a growing
need for the development of sensitive chemosensors for these compounds.
A chemosensor is generally composed of a recognition moiety that interacts with
an analyte and a signaling unit that relays the recognition event. In designing our
boronic acid sensors, we utilized diethanolamine as the recognition moiety. The
diethanolamine structure displays high affinity binding with boronic acids through
the formation of a boronate ester. This boronate is stabilized by the donation of the
nitrogen lone pair electrons to the open shell of the boron atom (28,29), which allows
for the formation of two fused five-membered rings (Scheme 1, 1b). Such recognition
has been used for the stabilization, purification, and characterization of boronic
acids (30).
The second component of a chemosensor is the reporter. The reporter must provide
a facile method for determining the binding between the boronic acid and the sensor.
Photometric reporters allow sensitive analyte detection and are easy to monitor. To
attain this type of reporting system, the attachment of a chromophore that is sensitive
to the binding event is required. We have attempted to achieve this with three different
designs: (i) photoinduced electron transfer based on a 9-(aminomethyl)anthracene
system, (ii) rigidification of extended ␲ system skeletons, and (iii) electronic perturba-
tion of azo dyes. All systems show complexation with phenylboronic acid to vary-
ing degrees.
RESULTS AND DISCUSSION
Photoinduced Electron Transfer
It is well known that the nitrogen lone pair electrons of 9-(aminomethyl)anthracene
can quench the fluorescence of an anthracene moiety through photoinduced electron
transfer (PET) (3,6,31,32). Masking of the nitrogen lone-pair electrons (e.g., by
protonation) causes a suppression of this fluorescence quenching and, therefore, results
in fluorescence intensity increases (4,6,33–35). We envisioned that the diethanolamine
recognition motif could be incorporated into an anthracene molecule so that its binding
with boronic acid would lead to the formation of a boronate ester (Scheme 1, 1b–3b),
which has a boron atom ideally positioned to accept and, therefore, mask the nitrogen
lone pair electrons. This masking of the nitrogen lone-pair electrons could then lead
to an increase in fluorescence intensity of the anthracene moiety.
Three fluorescent sensors were designed, synthesized, and evaluated for their bind-
ing with phenylboronic acid (Scheme 1). In sensor 1a, (1) a diethanolamine unit is

PHOTOMETRIC SENSORS FOR BORONIC ACIDS
261
SCHEME 1. Photoinduced electron transfer sensors.
attached by a methylene linker to the 9 position of anthracene. Sensor 2a is a
constrained analog of 1a and was designed to probe the effect of entropic factors
upon the strength of binding. The bis(diethanolamine) sensor 3a was designed to
determine if two diethanolamine units would lead to more intense PET quenching in
the absence of boronic acids, and therefore display a greater on/off effect. Sensors
1a and 3a and control compound 4a were synthesized in one step from the appropriate
chloromethylanthracene and diethanolamine or diethylamine following literature pro-
cedures (34,36). Sensor 2a was synthesized in two steps by alkylation of pyrroline
with 9-(chloromethyl)anthracene and then dihydroxylation with a catalytic amount
of osmium tetroxide in the presence of N-methylmorpholine-N-oxide (NMO). The
sensors were evaluated for binding strength by titrating phenylboronic acid into a 1.0 ϫ
10Ϫ5 M solution of the sensor in methanol and measuring the increases in fluorescence
intensity at an excitation wavelength of 370 nm and emission wavelength of 419 nm.
Compound 4a was included as a control to show that the changes in fluorescence
intensity are not primarily related to changes in protonation state or due to nonspecific
binding of phenylboronic acid with the sensor amine group. Sensors 1a and 2a showed
the greatest activity compared with control 4a (Fig. 1). Compound 2a appeared to
be more sensitive to low concentrations of phenylboronic acid, but the fluorescence

262
SPRINGSTEEN ET AL.
FIG. 1. Fluorescent response of PET sensors to phenylboronic acid. I0, sensor 1a, 2a, 3a, or 4a at
1.0 ϫ 10Ϫ5 M in MeOH. I, sensor 1a, 2a, 3a, or 4a at 1.0 ϫ 10Ϫ5 M in MeOH with phenylboronic acid.
▫, Sensor, 1a; ᭡, sensor 2a; ࡗ, sensor 3a; ⅙, control 4a.
reached a maximum at a lower intensity than that of 1a. These results may be explained
by the 2a/phenylboronic acid complex having a greater binding strength, but lower
overall fluorescent intensity than the corresponding 1a complex. A stronger binding
constant for 2a may partially be explained by the constrained diol system leading to
a decrease in the negative entropy component of the complex.
An NMR titration of sensor 1a with phenylboronic acid was carried out to examine
whether the sensor binds phenylboronic acid in a 1:1 ratio as designed. Mixtures of
the sensor and phenylboronic acid with molar ratios of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1.0, 1.25, 2.0, and 10.0 in CDCl3 were equilibrated for 45 min, and
1
then H NMR spectra were recorded. The pronounced chemical shifts of the CH2O
were observed. Without the binding to PhB(OH)2, the chemical shift of the CH2OH
was about 3.5 ppm. However, with the binding, the chemical shift of the CH2OB
moved downfield to about 4.1 ppm. The ratio of [complex] to [complex ϩ sensor]
was calculated based on the ratio of integration of [complex] to that of [complex
ϩ sensor]. The molar ratio of [complex]/[complex ϩ sensor] was plotted against
[PhB(OH)2]/[sensor] (Fig. 2), and shows a distinct 1:1 binding ratio.
Sensor 3a appears to have a fluorescent profile similar to the control (Fig. 1). This
FIG. 2. Percentage of complexation as a function of the number of equivalents of phenylboronic
1
acid. Calculated based on the ratio of H NMR integration in CDCl3.

PHOTOMETRIC SENSORS FOR BORONIC ACIDS
263
SCHEME 2. Rigidification sensors.
may be caused by a lack of diester formation. A single free diethanolamine appears
to have enough quenching ability to remove any fluorescent enhancement.
Rigidification
A second approach towards sensing boronate formation is the rigidification of a
conjugated ␲ system. Restricting the free motion of an extended ␲ system can
often deter nonradiative decay pathways of the excited state, and thereby promote
fluorescence emission (37). We reasoned that properly positioned hydroxyls placed
on adjacent aryl rings might present the correct architecture for boronate formation
(Scheme 2, 5b and 6b). The covalent bonds of the ester would then hold the two
rings in a planar, restricted conformation. Sensors 5a (38) and 6a (35) (Scheme 2)
were synthesized according to literature procedures. Although the sensors have a
dipropanolamine rather than a diethanolamine recognition moiety, we rationalized
that the two six-membered rings might form a stable boronate ester (Scheme 2, 5b
and 6b). The fluorescent increases shown by compound 5a in dichloromethane (DCM)
are displayed in Fig. 3. The fluorescent intensity increased by over threefold in the
FIG. 3. Fluorescent response of 5a to phenylboronic acid. I0 sensor 5a at 1.0 ϫ 10Ϫ4 M in DCM.
I, sensor 5a at 1.0 ϫ 10Ϫ4 M in DCM titrated with indicated amount of phenylboronic acid.

264
SPRINGSTEEN ET AL.
presence of millimolar concentrations of phenylboronic acid. No intensity increases
were seen in methanol. Compound 6a showed no fluorescent intensity increases in
either solvent. The lower electron density on the phenolic hydroxyls of 6a may have
led to a decreased stability of its boronate ester.
Azo Dye Electronics Perturbation
As a third approach toward sensing phenylboronic acid, we envisioned using an
azo dye chromophore as an optical reporter. A diethanolamine moiety was attached
to the azo system with the nitrogen in the aniline position of an aryl ring (Scheme
3). It is known that perturbation of the electronic properties of the aniline nitrogen
can lead to quantifiable changes in the azo chromophore (39). We felt that boronate
formation between phenylboronic acid and a diethanolamine moiety would change
the electronic character of the aniline nitrogen (Scheme 3) and therefore shift the
␭
max of its absorbance spectrum, resulting in a colorimetric response. By varying the
substituents on the aromatic system we hypothesized that we could optimize the
system to give large changes in the ␭
.
max
To verify that N-aryldiethanolamines bind boronic acids, the affinities of three N-
aryldiethanolamine units (Fig. 4, 7–9 (40,41) for phenylboronic acid were determined
by 1H NMR studies (Table 1, 7–9). The results showed that about 20–40% complex-
ation occurred between 7 and 9 and the boronic acid. Given these promising results,
a set of potential azo sensors was prepared by coupling an N-aryldiethanolamine with
the diazonium salt of a substituted aniline (Sch. 4; Fig. 4). Commercially available
anilines were used to synthesize compounds 10–16 (Fig. 4) (42,43)). Reduction of the
respective nitro-substituted compounds gave the amine-substituted dyes 17–19 (42).
Initially 1H NMR studies were used to verify the binding between the dyes 10–19
and phenylboronic acid (Table 1). As with the N-aryldiethanolamines 7–9, the signal
for the protons on the methylene bearing the hydroxyl group typically showed the
greatest shift upon complexation (data not shown). Integrations of the signals for the
free and complexed forms of the dye were used to determine the relative ratio of free to
complexed dye. The sensors showed 20–50% complexation with excess phenylboronic
acid (Table 1, compounds 10–19).
Absorbance and fluorescence studies were also carried out on the target compounds.
SCHEME 3. N-Aryldiethanolamine azo dyes designed as sensors for boronic acids.

PHOTOMETRIC SENSORS FOR BORONIC ACIDS
265
FIG. 4. The target azo compounds.
TABLE 1
Percentage of Each N-aryldiethanolamine or Azo Dye That Complexed with Phenylboronic Acid
in CDCl3 at Room Temperature
Compound
Percentage complexationa
7
8
9
39
24
40
36
33
30
18
10
11
12
13
14
15
16
17
18
19
29
b
18
23
49
b
a Compounds 7–19 were present at ϳ5–50 mM, depending upon solubility. Excess (Ͼ1.5 equiv.)
phenylboronic acid was added.
b The complexation changed the chemical shifts of active protons, but it resulted only in broadening
of the signals for the methylene protons; therefore, the percentage of complexation could not be calculated.

266
SPRINGSTEEN ET AL.
SCHEME 4. Synthesis of azo dyes.
Dimethyl sulfoxide (DMSO) and methylene chloride were used as the solvents for
these binding studies. None of the N-aryldiethanolamines showed a change in its
UV–visible spectrum in a manner dependent upon phenylboronic acid concentration.
No changes in the ␭ of the emission spectra were recorded. Variations in fluores-
max
cence intensity were observed at high phenylboronic acid concentrations (ϳ5 ϫ 10Ϫ2
M), but these changes did not occur in a concentration-dependent manner and may
be ascribed to changes in solution properties at high solute concentrations. Although
the NMR studies showed that boronate formation occurs with the azo dye, unfortu-
nately the electronic perturbation does not seem strong enough to bring about changes
in the photometric properties of the dyes.
CONCLUSION
It is often necessary to test multiple systems to find a functional photometric sensor.
Both a strong recognition site and a linked signaling unit are required. In this paper,
three different strategies for achieving a measurable optical response to boronate
formation have been examined. Both the PET sensors 1a and 2a and the rigidification
sensor 5a showed large changes in fluorescent intensity upon binding of phenylboronic
acid. The azo dyes were able to bind phenylboronic acid, but there was no induced
change in absorbance or emission properties. Through the application of multiple
designs, this work demonstrates the successful development of various photometric
sensors for phenylboronic acid.
EXPERIMENTAL
General methods. All 1H and 13C NMR spectra were recorded at 300 and 75 MHz,
respectively, with tetramethylsilane or residual solvent as the internal standard. Column
chromatography was performed using silica gel (32–63 ␮m). Mass spectrometry
analyses were conducted by North Carolina State University Mass Spectrometry
Laboratory and University of Kansas Mass Spectrometry Laboratory. Commercially
available starting materials and reagents were purchased from Aldrich and Acros.
The methanol used for the binding studies was of ACS Certified grade from Fisher.
Fluorescent binding studies. A Shimadzu RF-5301PC fluorometer was used for the
fluorescent studies. For each measurement, 2 mL of sensor solution at the appropriate

PHOTOMETRIC SENSORS FOR BORONIC ACIDS
267
concentration was mixed with 2 mL of substrate with various concentrations for 20
min. Then 3.5 mL of the mixture was transferred into a cuvette for fluorescence
measurement. The emission spectra were recorded from 380 to 650 nm. The excitation
wavelength was set at 370 nm for the PET sensors and control (compounds 1–4) and
330 nm for sensor 5a. The experiments were carried out in triplicate.
1-Anthracen-9-ylmethyl-2,5-dihydro-1H-pyrrole (2). 9-(Chloromethyl)anthracene
(1.168 g, 5.15 mmol) and 3-pyrroline (0.50 g, 7.23 mmol) were dissolved in 30 mL
of dry acetonitrile, followed by addition of potassium carbonate (0.852 g, 6.18 mmol)
and potassium iodide (30 mg, 0.18 mmol). The reaction mixture was refluxed under
nitrogen for 22 h. After cooling to room temperature, the insoluble material was
filtered off, and the filtrate was evaporated in vacuo. The resulting residue was
dissolved in 100 mL of methylene chloride and washed with water (2 ϫ 30 mL).
The organic phase was dried over MgSO4. After solvent evaporation, the crude product
was purified on a silica gel column by eluting with ethyl acetate/hexanes (1/50–1/1)
to give a yellow solid, 1.17 g (88%). 1H NMR (CDCl3): ␦ 8.54 (2H, d, J ϭ 8.6 Hz),
8.41 (1H, s), 8.00 (2H, d, J ϭ 8.4 Hz), 7.51–7.44 (4H, m), 5.77 (2H, s), 4.77
(2H, s), 3.58 (4H, s). 13C NMR (CDCl3): ␦ 131.7, 131.0, 129.2, 128.1, 127.5, 125.8,
125.1, 125.0, 59.5, 51.1. FAB-MS: 260.2 (M ϩ H), 191.2 IR (KBr, cmϪ1): ␯ 3067,
1621, 1444, 1158, 733. Anal. Calcd for C19H17N: C, 87.99; H, 6.61; N, 5.40. Found:
C, 87.81; H, 6.65; N, 5.33.
1-Anthracen-9-ylmethyl-pyrrolidine-cis-3,4-diol (2a). To compound 2 (408 mg,
1.574 mmol) and N-methylmorpholine-N-oxide monohydrate (637 mg, 4.72 mmol)
in 5 mL of acetone and 1.5 mL of water (containing 0.1 mL of pyridine) was added
a solution of osmium tetroxide (20 mg, 0.079 mmol) in 2 mL of tert-butanol. The
reaction mixture was stirred at room temperature under nitrogen. After 36 h, 15 mL
of 10% aqueous sodium hydrosulfite was added, and the mixture was stirred for 15
min. The mixture was extracted with methylene chloride (2 ϫ 50 mL). The organic
phase was washed with water (40 mL) and saturated brine (40 mL) and dried over
MgSO4. Solvent evaporation gave a viscous yellow oil, which was purified on a silica
gel column by eluting with MeOH/CH2Cl2 (1/50) to give a yellow solid, 50 mg (20%).
1H NMR (CDCl3): ␦ 8.43–8.40 (3H, m), 8.01 (2H, d, J ϭ 8.3 Hz), 7.53–7.43
(4H, m), 4.59 (2H, s), 4.13 (2H, s), 2.88–2.83 (2H, m), 2.75–2.72 (2H, m), 2.45 (2H,
br s, exchanged with D2O). 13C NMR (CDCl3): ␦ 131.6, 131.0, 129.7, 129.3, 127.8,
126.0, 125.1, 124.7, 71.1, 60.5, 50.7. IR (KBr, cmϪ1): ␯ 3362, 2925, 1146, 884, 733.
Azo compounds. The azo compounds were synthesized according to the following
general procedure used to generate 4-(4Ј-Nitrophenylazo)-N,N-bis(2-hydroxyethyl)an-
iline (11) (42). The 4Ј-amino azo compounds were prepared from the 4Ј-nitro azo
compounds by reduction with sodium sulfide (42). A slightly modified procedure
was used to couple those primary anilines bearing a p-methyl group (43).
Representative procedure for synthesis of the azo compounds. 4-Nitroaniline (761
mg, 5.51 mmol) was partially dissolved in a mixture of 8 mL deionized water and
2 mL concentrated aqueous HCl by heating it in an oil bath. The mixture was cooled
by pouring it onto 18 mL of ice water. After additional cooling in an ice bath, a
solution of sodium nitrite (378 mg, 5.48 mmol) in 5 mL of deionized water was
added in one portion; the mixture became homogeneous. After 1 h, an ice-cold solution
of N-phenyldiethanolamine (997 mg, 5.51 mmol) in a mixture of 25 mL deionized

268
SPRINGSTEEN ET AL.
water and 2 mL concentrated aqueous HCl was added dropwise over ϳ20 min. The
dye formed from the start of this addition. After 2 h, the reaction was quenched with
1 M KOH. (Reactions involving ketones or esters were quenched by dropwise addition
of saturated K2CO3 solution.) The mixture was filtered and washed with 250 mL
deionized water and 25 mL acetone. The red powder was dried in the vacuum oven
overnight at 30ЊC. Yield of 10: 552 mg, 31%.
4-(4Ј-Nitrophenylazo)-N,N-bis(2-hydroxyethyl)aniline (10). The product was ob-
1
tained in 31% yield. mp 204–205ЊC. H NMR (CDCl3): ␦ 8.34 (2H, apparent d,
J ϭ 8.3 Hz), 7.93 (4H, apparent t, J ϭ 8.4 Hz), 6.81 (2H, apparent d, J ϭ 8.7 Hz),
3.98 (4H, t, J ϭ 4.4 Hz), 3.76 (4H, t, J ϭ 4.5 Hz), 2.17 (2H, s). UV–visible (DMSO)
␭
(␧): 509 nm (28,400).
max
3-Methyl-4-(4Ј-nitrophenylazo)-N,N-bis(2-hydroxyethyl)aniline (11). The product
was obtained in 75% yield. mp 152–154ЊC. 1H NMR (CDCl3): ␦ 8.31 (2H, apparent
d, J ϭ 9.0 Hz), 7.91 (2H, apparent d, J ϭ 8.8 Hz), 7.77 (1H, apparent d, J ϭ 4.9
Hz), 6.59 (2H, broad s), 3.97 (4H, t, J ϭ 4.5 Hz), 3.73 (4H, t, J ϭ 4.7 Hz), 2.98
(2H, s), 2.71 (3H, s). UV-visible (DMSO) ␭ (␧): 520 nm (14,300).
max
4-(4Ј-Cyanophenylazo)-N,N-bis(2-hydroxyethyl)aniline (12). The product was ob-
1
tained in 89% yield. mp 162–166ЊC. H NMR (CDCl3): ␦ 7.88 (4H, apparent d,
J ϭ 4.4 Hz), 7.76–7.74 (2H, m), 6.81–6.77 (m, 2H), 3.97 (4H, t, J ϭ 4.7 Hz), 3.75
(4H, t, J ϭ 4.7 Hz), 2.9 (2H, broad s). MS (FAB) m/z 310 (M+, 58), 311 (M ϩ H+,
80). Anal. Calcd for C17H18N4O2: C, 65.79; H, 5.85; N, 18.05. Found: C, 66.01; H,
5.77; N, 17.77. UV–visible (DMSO) ␭ (␧): 479 nm (26,700).
max
3-Methyl-4-(4Ј-carboxymethylphenylazo)-N,N-bis(2-hydroxyethyl)aniline (13). The
1
product was obtained in 72% yield. mp 163–165ЊC. H NMR (CDCl3): ␦ 8.13 (2H,
apparent d, J ϭ 8.4 Hz), 7.86 (2H, apparent d, J ϭ 8.4 Hz), 7.76 (1H, apparent d,
J ϭ 10.0 Hz), 6.59–6.58 (2H, m), 3.94 (7H, broad s), 3.71 (4H, t, J ϭ 4.78 Hz),
2.98 (2H, s), 2.71 (3H, s). MS (FAB) m/z 357 (M+, 60), 358 (M ϩ H+, 81). Anal.
Calcd for C19H23N3O4: C, 63.85; H, 6.49; N, 11.76. Found: C, 63.70; H, 6.42; N,
11.63. UV–visible (DMSO) ␭ (␧): 477 nm (35,400).
max
3-Methyl-4-(4Ј-acetylphenylazo)-N,N-bis(2-hydroxyethyl)aniline (14). The product
was obtained in 64% yield. mp 153–154ЊC. 1H NMR (CDCl3): ␦ 8.06 (2H, apparent
d, J ϭ 8.4 Hz), 7.88 (2H, apparent d, J ϭ 8.4 Hz), 7.78–7.74 (1H, m), 6.60–6.58
(2H, m), 3.96 (4H, t, J ϭ 4.7 Hz), 3.72 (4H, t, J ϭ 4.8 Hz), 2.87 (2H, s), 2.72
(3H, s), 2.65 (3H, s). MS (FAB) m/z 342 (M ϩ H+, 89). UV–visible (DMSO) ␭
max
(␧): 482 nm (53,200).
2,5-Dimethoxy-4-(4Ј-carboxymethylphenylazo)-N,N-bis(2-hydroxyethyl)aniline (15).
1
The product was obtained in 86% yield. mp 147–149ЊC. H NMR (CDCl3): ␦ 8.15
(2H, apparent d, J ϭ 8.2 Hz), 7.89 (2H, apparent d, J ϭ 8.5 Hz), 7.42 (1H, broad
s), 6.78 (1H, broad s), 4.02 (3H, s), 3.95 (3H, s), 3.90 (3H, s), 3.77 (4H, broad s),
3.51 (4H, t, J ϭ 5.0 Hz), 2.93 (2H, broad s). MS (FAB) m/z 404 (M ϩ H+, 69).
UV–visible (DMSO) ␭ (␧): 502 nm (205,000).
max
3-Methyl-4-(4Ј-toluylazo)-N,N-bis(2-hydroxyethyl)aniline (16). The product was
1
obtained in 53% yield. mp 110–115ЊC. H NMR (CD3OD): ␦ 7.70–7.63 (3H, m),
7.27 (2H, apparent d, J ϭ 8.1 Hz), 6.67–6.64 (2H, m), 3.77 (4H, t, J ϭ 5.8 Hz),
3.63 (4H, t, J ϭ 5.8 Hz), 2.65 (3H, s), 2.39 (3H, s). MS (FAB) m/z 313 (M+, 100),

PHOTOMETRIC SENSORS FOR BORONIC ACIDS
269
314 (M ϩ H+, 91). Anal. Calcd for C18H23N3O2: C, 68.98; H, 7.40; N, 13.41. Found:
C, 68.79; H, 7.48; N, 13.19. UV–visible (DMSO) ␭ (␧): 470 nm (32,700).
max
4-(4Ј-Anilino-azo)-N,N-bis(2-hydroxyethyl)aniline (17). The product was obtained
1
in 9% yield from 10. mp 160–161ЊC. H NMR (CDCl3): ␦ 7.82–7.72 (4H, m),
6.78–6.72 (4H, m), 3.94 (6H, broad s), 3.70 (4H, t, J ϭ 4.8 Hz), 2.84 (2H, s).
MS (FAB) m/z 300 (M+, 88). UV–visible (DMSO) ␭
(␧): 454 nm (58,000), 327
max
nm (10,400).
3-Methyl-4-(4Ј-anilino-azo)-N,N-bis(2-hydroxyethyl)aniline (18). The product was
1
obtained in 25% yield from 11. mp 149–152ЊC. H NMR (CDCl3): ␦ 7.74 (2H,
apparent d, J ϭ 8.5 Hz), 7.66 (1H, apparent d, J ϭ 9.7 Hz), 6.73 (2H, apparent d,
J ϭ 8.6 Hz), 6.58–6.56 (2H, m), 3.93 (6H, m), 3.68 (4H, t, J ϭ 4.5 Hz), 2.85 (2H,
broad s), 2.67 (3H, s). UV–visible (DMSO) ␭max (␧): 461 nm (40 400) 327 nm (6600).
2,5-Dimethoxy-4-(4Ј-anilino-azo)-N,N-bis(2-hydroxyethyl)aniline (19). The prod-
1
uct was obtained in 10% yield from 9 in two steps. H NMR (CD3OD): ␦ 7.66 (2H,
apparent d, J ϭ 8.6 Hz), 7.32 (1H, s), 6.83 (1H, s), 6.73 (2H, apparent d, J ϭ 8.5
Hz), 3.96 (3H, s), 3.83 (3H, s), 3.69 (4H, t, J ϭ 5.9 Hz), 3.46 (4H, t, J ϭ 6.0 Hz).
MS (FAB) m/z 361 (M ϩ H+, 45). UV–visible (DMSO) ␭ (␧): 431 nm (55,100).
max
ACKNOWLEDGMENTS
Financial support by the National Institutes of Health (DK55062 and CA88343) is gratefully acknowl-
edged. C.E.B. gratefully acknowledges a graduate fellowship from the Burroughs Wellcome Fund. W.W.
gratefully acknowledges support from the Glaxo Graduate Fellowship Fund.
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