3-[2-(Boronophenyl)benzoxazol-5-yl]alanine derivatives as fluorescent monosaccharide sensors

Article abstract of DOI:10.1016/j.tet.2012.08.085

Two 3-[2-(boronophenyl)benzoxazol-5-yl]alanine derivatives were synthesized and their potential application as fluorescent monosaccharide sensors was studied. It was found that both non-proteinogenic amino acids bound glucose and fructose at physiological pH, however, the latter much stronger. As a result they are selective sensors for fructose. Moreover, one of them (3-[2-(3-boronophenyl)benzoxazol-5-yl]alanine methyl ester) can be used to quickly distinguish, which monosaccharide is present in the solution because of the different character of fluorescence intensity changes (increase in the presence of fructose and decrease in the presence of glucose).

Full text of DOI:10.1016/j.tet.2012.08.085

Tetrahedron 68 (2012) 9240e9248
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
3-[2-(Boronophenyl)benzoxazol-5-yl]alanine derivatives as fluorescent
monosaccharide sensors
*
_ _
Katarzyna Guzow , Daria Jazdzewska, Wies1aw Wiczk
ꢀ
ꢀ
Faculty of Chemistry, University of Gdansk, Sobieskiego 18, 80-952 Gdansk, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Two 3-[2-(boronophenyl)benzoxazol-5-yl]alanine derivatives were synthesized and their potential
application as fluorescent monosaccharide sensors was studied. It was found that both non-
proteinogenic amino acids bound glucose and fructose at physiological pH, however, the latter much
Received 22 February 2012
Received in revised form 7 August 2012
Accepted 28 August 2012
stronger. As
a result they are selective sensors for fructose. Moreover, one of them (3-[2-(3-
Available online 1 September 2012
boronophenyl)benzoxazol-5-yl]alanine methyl ester) can be used to quickly distinguish, which mono-
saccharide is present in the solution because of the different character of fluorescence intensity changes
(increase in the presence of fructose and decrease in the presence of glucose).
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Benzoxazolylalanine
Phenylboronic acid
Glucose
Fructose
Fluorescence
1. Introduction
parameters are easy for the quantitative detection in comparison to
other analytical methods.
The design of receptors for neutral molecules, such as mono-
saccharides, that exhibit both high affinity and high selectivity is
recently at the center of interest of many research groups.1e12
The mechanism of monosaccharide detection by designed sen-
sors is often based on hydrogen bond formation but only in aprotic
solvents. In aqueous environment, the boronic acid is used. This
functional group interacts strongly and reversibly with 1,2- or 1,3-
diols forming five- or six-membered ring, which was first reported
in 1959.13 The result of this covalent binding with monosaccharides
is the formation of a cyclic boronic ester leading to an increase in
the Lewis acidity of the boron atom.1,2,13e17 This ester forms faster
in aqueous basic media where the boron atom is present in its
tetrahedral anionic form.13,17e20 However, the diol complexation
process is affected by common buffer components, such as phos-
phate, which may diminish the observed stability constants.17,21
Based on the above-described mechanism, many saccharide
sensing molecules have been designed and studied.1e17,21e48 These
compounds are used for saccharide detection in many analytical
methods with applications in nanostructures37,38 as well as optical
methods.1e12,17,21e37,40e50 The latter, based on fluorescence and
absorption measurements, are especially promising in the selective
detection of very low concentrations of the sensing molecule. These
In the search for new fluorescent monosaccharide sensors, we
synthesized two non-proteinogenic amino acids based on the
benzoxazol-5-ylalanine skeleton with a phenylboronic acid moiety
and studied their interactions with monosaccharides.
2. Results and discussion
2.1. Synthesis
N-Boc-3-[2-(4-boronophenyl)benzoxazol-5-yl]alanine methyl
ester (1) and N-Boc-3-[2-(3-boronophenyl)benzoxazol-5-yl]ala-
nine methyl ester (2) were obtained from N-Boc-3-aminotyrosine
methyl ester, via the intermediate Schiff base which underwent
oxidative cyclization to the heterocyclic compound in the presence
of lead tetraacetate in DMSO, according to the procedure published
previously.51,52 The removal of the Boc group was performed by
means of acidic hydrolysis giving both studied compounds (1a and
2a, Fig. 1).
2.2. Acidebase properties
2.2.1. Absorption measurements. Absorption spectra of both the
studied compounds in water lay in the near ultraviolet upto
about 340 nm and have poor vibrational structure. Their maxima
are at about 307 nm (ε¼18,000 cmꢀ1Mꢀ1
) and 302 nm
* Corresponding author. Tel.: þ48 58 5235413; fax: þ48 58 5235472; e-mail
addresses: kasiag@chem.univ.gda.pl, kguzow@wp.pl (K. Guzow).
(ε¼13,900 cmꢀ1Mꢀ1) for 1a and 2a, respectively. The position and
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.08.085

K. Guzow et al. / Tetrahedron 68 (2012) 9240e9248
9241
Fig. 1. Structures of synthesized compounds.
shape of these spectra do not change significantly with an increase
in pH (Figs. 2 and 3). Only a slight increase of the absorbance is
observed in the studied pH range (from 3.7 to 10.6). From these
results, pKa values in the ground state were determined using
global analysis, which takes into account absorbance changes at
different wavelengths. For compound 1a, pKa is equal to 8.05ꢁ0.9
whereas for 2a e 8.09ꢁ0.16. These values are comparable to these
determined for other monoboronic acid derivatives.5,30,45,49
In order to check the stability of these compounds at different
pH, the measurements were repeated for all solutions after 24 and
48 h (Figs. 1Se6S). It was observed that the absorbance increases
more with time, especially in the strongly alkaline environment (for
pH >9.5, Figs.1S and 4S). Moreover, it results in slight decline of pKa
value (7.86ꢁ0.20 and 8.05ꢁ0.22 for 1a and 2a, respectively, after
24 h and 7.68ꢁ0.21 and 7.89ꢁ0.22 for 1a and 2a, respectively, after
48 h, Table 1S). The observed changes are smaller in the case of
compound 2a than 1a indicating on its higher stability.
The influence of pH on the position and shape of the absorption
spectra of both compounds in the presence of 50 mM of fructose or
glucose is similar as in the aforementioned case. However, pKa
values of compounds studied in the presence of fructose decrease
significantly to the value of 5.89ꢁ0.11 for 1a and 5.54ꢁ0.12 for 2a.
The influence of glucose is smaller as pKa values are equal to
7.86ꢁ0.14 and 7.29ꢁ0.16 for 1a and 2a, respectively. Such changes
are in agreement with those observed for other monoboronic acid
esters with monosaccharides.5,13,30,45,49
The stability experiments reveal that for both compounds
studied for pH >9 after 24 h or more, a new absorption band ap-
pears with a maximum at 340 nm, at the expense of parent com-
pound absorption, regardless of the monosaccharide used. It is
worth mentioning that for the same pH and monosaccharide
concentration, the absorption spectra of 2a changes to a lesser
extent than those of 1a (Figs. 2S, 3S, 5S and 6S). The observed new
band may be a result of instability of the benzoxazole moiety in an
alkaline environment leading to oxazole ring opening with the
restoration of the Schiff base,53 or the dissociation of boronic acid.
However, the data presented in the literature concerning the sta-
bility of boronic acid derivatives in alkaline solution do not confirm
this supposition.
Fig. 2. Absorption spectra of 1a in water solutions of different pH in the absence (at
the top) and in the presence of 50 mM of fructose (in the middle) or 50 mM of glucose
(at the bottom).
fluorescence intensity with a small hypsochromic shift of the
spectrum as a result of the change in the electronic properties and
the geometry at the boron atom (Figs. 4 and 5).1,2,13,17 Moreover, for
pH >9 a diffuse vibrational structure appears. The apparent pKa
values in the excited state, calculated from the titration curves, are
equal to 7.32ꢁ0.03 for 1a and 7.26ꢁ0.03 for 2a (Table 1, Fig. 6). Thus,
in the excited state both compounds are stronger acids than in the
2.2.2. Fluorescence measurements. Both studied compounds are
fluorescent with emission maxima in water at about 360 nm and
fluorescence quantum yields equal to 0.13 and 0.09 for 1a and 2a,
respectively. In both cases the increase in pH causes a decrease in

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K. Guzow et al. / Tetrahedron 68 (2012) 9240e9248
Fig. 4. Fluorescence spectra of 1a in water solution of different pH in the absence (at
the top) and in the presence of 50 mM of fructose (in the middle) or 50 mM of glucose
(at the bottom).
Fig. 3. Absorption spectra of 2a in water solutions of different pH in the absence (at
the top) and in the presence of 50 mM of fructose (in the middle) or 50 mM of glucose
(at the bottom).
1a, a simultaneous blue shift is observed. For solutions with pH >7,
the diffuse vibrational structure of the emission band appears. It is
more pronounced in the presence of fructose suggesting a stiffen-
ing of the compound structure probably as a result of interactions
between the boronic group and the monosaccharide. The observed
quenching effect of the fluorescence intensity in the presence of
a monosaccharide is much smaller in comparison with pure
ground state. Moreover, regardless of the position of boronic group
in the phenyl ring, both derivatives show very similar acidebase
properties.
The pH titration of both compounds in the presence of 50 mM of
monosaccharide (fructose or glucose) reveals that increase of pH
causes a decrease in fluorescence intensity. Moreover, in the case of

K. Guzow et al. / Tetrahedron 68 (2012) 9240e9248
9243
Table 1
The pKa values of compounds studied in the ground and excited state (pKa*) in the
absence and presence of 50 mM of fructose or glucose determined using global
analysis (pKa) or single data file analysis (pKa*)
pKa
R2
pKa*
r2
1a
8.05ꢁ0.09
5.89ꢁ0.11
7.86ꢁ0.14
8.07ꢁ0.16
5.54ꢁ0.12
7.29ꢁ0.16
0.9997
0.9680
0.9217
0.9988
0.9462
0.9514
7.32ꢁ0.03
6.51ꢁ0.11
6.74ꢁ0.09
7.26ꢁ0.03
6.01ꢁ0.10
6.88ꢁ0.06
0.9982
0.9806
0.9865
0.9976
0.9838
0.9943
1aþ50 mM of fructose
1aþ50 mM of glucose
2a
2aþ50 mM of fructose
2aþ50 mM of glucose
Fig. 6. The dependence of fluorescence intensity on pH for 1a (at the top) and 2a (at
the bottom) in the absence (black points) and in the presence of 50 mM of fructose
(blue points) or glucose (green points). The red points were disregarded in the pKa
calculations.
visible in the spectra recorded after 24 h, Fig. 7S). It is probably
a result of the benzoxazole ring opening and formation of a weak
fluorescent Schiff base whose emission band maximum is about
400 nm. In the case of 2a, in the presence of glucose the fluores-
cence intensity is totally quenched, whereas in the presence of
fructose it is equal to about 90% of initial intensity (Fig. 8S).
The calculated pKa values in the presence of monosaccharides
are smaller than those for the pure compounds (Table 1, Fig. 6)
indicating that both compounds studied are stronger acids in their
presence, especially fructose. It is probably a result of interaction
between the compound and monosaccharide resulting in forma-
tion of an electron-rich boronic anion.13,45e48
Fig. 5. Fluorescence spectra of 2a in water solutions of different pH in the absence (at
the top) and in the presence of 50 mM of fructose (in the middle) or 50 mM of glucose
(at the bottom).
compounds (for 1a: about 14% in the presence of fructose and 54%
in the presence of glucose; for 2a: about 60% in the presence of
fructose and 20% in the presence of glucose). However, for solutions
with pH >8, a violent reduction in fluorescence intensity is ob-
served. For compound 1a the reduction is about 40% of initial in-
tensity in the presence of fructose and 25% in the presence of
glucose, together with the red shift of the emission band (clearly

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K. Guzow et al. / Tetrahedron 68 (2012) 9240e9248
Table 2
2.3. Interaction with monosaccharides
The binding constants values of compounds studied with fructose and glucose in
phosphate buffer (pH¼7.5)
2.3.1. Absorption measurements. The presence of a monosaccharide
in solution (pH¼7.5, phosphate buffer) does not influence the shape
and position of the absorption band of both compounds studied,
however, an increase in monosaccharide concentration results in
an increase in absorbance (Fig. 7, Figs. 9Se11S). The titration curves
reveal a linear increase in absorbance for high monosaccharide
concentration, which is connected with the light scattering by
precipitated monosaccharide microcrystals (Fig. 7, Figs. 9Se11S).
This effect was confirmed by an independent experimentdspec-
trophotometric titration of the buffer with monosaccharide (not
shown). Thus, binding constant calculations were performed as-
suming the presence of two equilibrium constants, one denoting
the actual binding constant with monosaccharide, while the other
was added to include the impact of scattered light and has no
physical meaning. However, such an approach (two equilibrium
constants) significantly improves the quality of the fit.
1a
2a
Fructose
Glucose
Fructose
Glucose
K [Mꢀ1] (absorption)
895ꢁ71
0.9942
24.9ꢁ0.8
0.9988
847ꢁ34
0.9993
14.2ꢁ0.1
0.9964
R2
K [Mꢀ1] (fluorescence)
1419ꢁ19
27.7ꢁ0.8
1135ꢁ26
17.0ꢁ0.3
R2
0.9999
0.9995
0.9995
0.9999
Wang et al. for dibenzofuran-4-boronic acid (514 Mꢀ1 for fructose
and 0.6 Mꢀ1 for glucose),30 6-(N,N-dimethylamino)-naphthalene-2-
boronic acid (120 Mꢀ1 and 2.4 Mꢀ1 for fructose and glucose, re-
spectively),35 or 5-(N,N-dimethylamino)-naphthalene-1-boronic
acid (311 Mꢀ1 and 3.6 Mꢀ1 for fructose and glucose, respectively),37
but comparable with those obtained for some of stilbene de-
rivatives of boronic acid, published by Lakowicz et al.45
2.3.2. Fluorescence measurements. For both compounds, an in-
crease in the monosaccharide concentration results in a small blue
shift of the emission band, as well as appearance of its diffuse vi-
brational structure (Figs. 8e10). Moreover, the significant changes
of fluorescence intensity are observed but their character depends
on the monosaccharide used.
Fig. 7. Absorption spectra of 2a in phosphate buffer (pH¼7.5) in the presence of in-
creasing concentrations of fructose (at the top) and the obtained binding curve (at the
bottom).
The obtained binding constants (Table 2) indicate that fructose is
more strongly bound by both compounds than glucose. Moreover,
compound 1a interacts sligthly stronger with both monosaccharides.
Such effects of monosaccharides binding is characteristic for mono-
boronic acid derivatives,1,2,13,30,35,37,47 however, the obtained binding
constants for these compounds are higher than those reported by
Fig. 8. Fluorescence spectra of 1a in phosphate buffer (pH¼7.5) in the presence of
increasing concentrations of fructose (at the top) or glucose (at the bottom).

K. Guzow et al. / Tetrahedron 68 (2012) 9240e9248
9245
Fig. 10. Fluorescence spectra of 2a in phosphate buffer (pH¼7.5) in the presence of
increasing concentrations of glucose (at the top) and obtained binding curve (at the
bottom).
Fig. 9. Fluorescence spectra of 2a in phosphate buffer (pH¼7.5) in the presence of
increasing concentrations of fructose (at the top) and obtained binding curve (at the
bottom).
The presence of fructose in both cases results in an increase in
the fluorescence intensity (Figs. 8 and 9, and Fig. 12S) but the bigger
changes are observed for compound 1a (about 2.4- and 1.5-fold
fluorescence enhancement for 1a and 2a, respectively, Fig. 11).
Also, the calculated binding constant value is higher for 1a than 2a
indicating a stronger interaction of this compound with fructose.
Moreover, for both compounds, the obtained binding constants in
the excited state are significantly higher than those in the ground
state obtained from the absorption measurements (Table 2).
The presence of glucose in the solution causes an increase in the
fluorescence intensity of 1a (about 1.9-fold, Figs. 8 and 11) whereas
the fluorescence intensity of 2a is quenched (by about 50%, Figs. 8
and 10). Such behavior of isomers may be a result of the different
structure of the anionic ester of boronic acid with glucose. For
compound 2a, the structure of the complex formed probably allows
glucose hydroxyl groups to interact with the heteroatom(s) of the
oxazole moiety through the hydrogen bonds resulting in fluores-
cence quenching. The calculated binding constants of glucose in the
excited state are small and comparable to these obtained from the
absorption titration (Table 2).
Fig. 11. Relative fluorescence intensity (I/I0) versus saccharide (fructose or glucose)
concentration profile of 1a or 2a in phosphate buffer (pH¼7.5).
The observed stronger interaction of both compounds with
fructose is consistent with the literature: for all simple monoboronic
mentioning that both compounds enabled the determination of
small fructose concentrations as the maximal changes in their
fluorescence intensity are observed up to about 10 mM of fructose
(Fig. 11). Compound 1a saturates with glucose at concentrations
higher than 100 mM (Fig. 11).
acids the binding constants increase as follows:
D-fructose>D-gal-
actose>
D
-glucose.1,5,13,15e17,26,54 Also, greater fluorescence en-
hancement on the addition of fructose than glucose (Fig. 11) has
been observed for other saccharide sensors.1,22,23,25,49,50 It is worth

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K. Guzow et al. / Tetrahedron 68 (2012) 9240e9248
The interaction of benzoxazolylalanine derivatives with mono-
obtained compounds was checked by means of analytical RP-HPLC
(Kromasil column, C-8, 5
tection at 223 nm. Melting points (mp) were determined in capil-
saccharides was also studied by means of time-resolved fluores-
cence spectroscopy. It was found that the fluorescence intensity
decays of both pure compounds in phosphate buffer at pH¼7.5 are
heterogeneous (Table 3). This heterogeneity of the fluorescence
intensity decay decreases in the presence of fructose, especially in
the case of 1a (the fluorescence intensity decay becomes mono-
exponential), in contrast to the presence of glucose. These data
also indicate that the binding of monosaccharide causes the
shortening of the decay times, however, these are minor changes
only. Thus, these compounds cannot be used as lifetime sensitive
monosaccharide sensors.
m
m, 250 mm long, ID¼4.6 mm) with de-
lary tubes using Gallenkamp Griffin MPA-350.MB2.5 apparatus and
are uncorrected. Specific rotation ([a]
20) was determined on a Per-
D
kin Elmer 343 polarimeter. 1H and 13C NMR spectra were recorded
on a Varian, Mercury-400 BB spectrometer (400 and 100 MHz,
respectively) in DMSO-d6. Infrared spectra were recorded on
a Bruker IFS-66 instrument. Mass spectra were recorded on
a Bruker Biflex III instrument (MALDI-TOF). Elemental analysis
were taken on a Carlo Erba CHNSeO EA1108 instrument.
4.1.2. N-Boc-3-[2-(4-boronophenyl)benzoxazol-5-yl]alanine methyl
ester (1) and N-Boc-3-[2-(3-boronophenyl)benzoxazol-5-yl]alanine
methyl ester (2). Both compounds were obtained according to the
procedure published previously.51,52
Table 3
The fluorescence lifetimes (s), pre-exponential factors (a
) and values of c2R parameter
for 1a and 2a in the phosphate buffer (pH¼7.5) in the absence and presence of
fructose or glucose
A mixture of N-Boc-3-nitro-L-tyrosine methyl ester (0.69 g,
Compound
si [ns]
ai
cR2
7.02
2.04 mmol) and 5% palladium on active carbon in MeOH (20 mL)
was stirred under a hydrogen atmosphere at rt for about 90 min.
The catalyst was filtered off and the solvent evaporated in vacuo to
give a brownish oily product, which was dissolved in absolute EtOH
(3 mL) and mixed with a solution of appropriate aldehyde
(4-formyphenylboronic acid or 3-formylphenylboronic acid, 0.31 g,
2.06 mmol) in absolute EtOH (7 mL). The mixture was stirred at rt
overnight (TLC monitoring). After this time the solvent was re-
moved by evaporation and the obtained Schiff base (brown oil,
Rf¼0.57 (AcOEt) for (1), Rf¼0.76 (AcOEt/petroleum ether 2:1) for
(2)) was dissolved in DMSO (10 mL) and lead tetraacetate (1.36 g,
3.07 mmol) was added. The mixture was stirred at rt for an hour
(TLC monitoring) and then dissolved in ethyl acetate (AcOEt, 30 mL)
and washed in turn with a saturated aqueous solution of NaCl (3ꢂ).
The combined organic layers were dried over anhydrous MgSO4
and evaporated in vacuo giving the product, which was isolated by
means of column chromatography ((1)dAcOEt/petroleum ether
2:1; (2)dAcOEt). The crude product was recrystallized from AcOEt/
petroleum ether giving compound (1) as a yellowish solid (0.52 g,
1.18 mmol, 58%) or compound (2) as a brown solid (0.26 g,
0.59 mmol, 29%). The purity of the obtained compound was
checked by means of TLC ((1): Rf¼0.61 (AcOEt/petroleum ether
2:1); (2): Rf¼0.64 (AcOEt)) and analytical RP-HPLC (tR¼52.5 min (1),
tR¼52.6 min (2), the mobile phase was a gradient running from 0%
to 100% of B over 60 min (A¼0.01% water solution of trifluoroacetic
acid (TFA), B¼80% of acetonitrile in A)).
1a (c¼15
m
M)
1.41
1.57
0.28
1.16
1.14
1.36
0.58
1.54
1.69
0.51
1.31
1.39
0.51
1.06
1.56
0.42
1.00
0.30
0.70
1.00
1.00
0.38
0.62
1.00
0.54
0.46
1.00
0.67
0.33
1.00
0.14
0.86
1.02
1a (c¼15
1a (c¼15
m
m
M)þfructose (c¼50 mM)
M)þglucose (c¼50 mM)
1.15
4.21
1.09
2a (c¼15
m
M)
3.55
0.94
2a (c¼9
2a (c¼5
m
M)þfructose (c¼37 mM)
M)þglucose (c¼442 mM)
1.76
0.99
m
45.71
1.12
3. Conclusion
The studies of interactions of two 3-[2-(borophenyl)benzoxazol-
5-yl]alanine derivatives with monosaccharides revealed that these
non-proteinogenic amino acids may be used as efficient fluorescent
fructose sensors in water. Despite some limitations, such as low
stability in alkaline environment as well as absorption in the UV
range, both of them show, at physiological pH, significant fluores-
cence intensity changes after binding fructose, which is very strong
(high binding constant value). Moreover, small value of glucose
binding constant makes them selective sensors for fructose in the
presence of glucose. Additionally, because of the different character
of fluorescence intensity changes, compound 2a can be used to
quickly distinguish, which monosaccharide (glucose or fructose) is
present in the solution. Due to the presence of an amino acid
moiety these compounds may be used in biological applications as
monosaccharide sensor residues in peptides.
Compound (1): [found: C, 59.62; H, 6.06; N, 6.03. C22H25BN2O7
requires C, 60.02; H, 5.72; N, 6.36%]; mp 276e278 ꢃC (de-
20
composition); [
a
]
þ24.6 (c 0.013, MeOH); nmax (KBr) 3612.1,
D
3425.4, 3342.2, 3071.4, 2981.5, 2927.5, 1738.6, 1686.4, 1571.4,
1548.5, 1503.6e1407.0, 1372.7e1163.4, 1057.9, 926.0e709.8 cmꢀ1
;
dH (400 MHz, DMSO-d6) 1.30 (9H, s, (CH3)3), 2.96e3.18 (2H, m,
CbH2), 3.63 (3H, s, OCH3), 4.22e4.28 (1H, m, CaH), 7.30e7.33 (2H, m,
C6H, NH), 7.70 (2H, d, J 8.01 Hz, C4H, C7H), 8.00 (2H, dd, J 2.00,
0
0
0
0
4. Experimental
4.1. Synthesis
6.61 Hz, C2 H, C6 H), 8.15 (2H, dd, J 2.00, 6.41 Hz, C3 H, C5 H), 8.21
(2H, s, B(OH)2); dC (100 MHz, DMSO-d6) 28.02 (CH3)3, 36.30 Cb,
51.75 OCH3, 55.44 Ca, 78.25 0Ct-Bu, 110.36 C7, 120.27 C4, 126.04 C6,
0
0
0
0
0
126.74 C2 , C6 , 127.57 C3 , C5 , 134.50 C1 , C4 , 134.75 C5, 141.25 C9,
149.06 C8, 155.37 NHCO, 162.57 C2, 172.44 CO; m/z (MALDI): MHþ,
found 441. C22H25BN2O7 requires 440.
4.1.1. General. N-Boc-3-nitro-
L-tyrosine methyl ester was prepared
according to literature procedure.55,56 4-Formylphenylboronic acid
and 3-formylphenylboronic acid were purchased from Lancaster
whereas lead tetraacetate from SigmaeAldrich and used as re-
ceived. TLC was carried out on Merck silica gel plates (Kieselgel 60
F254). The spots were revealed using a UV lamp (254 nm, 366 nm).
All solvent ratios are in volume parts. The purification was carried
out by means of column chromatography (Merck, Silica gel 60,
0.040e0.063 mm) and semi-preparative RP-HPLC (Kromasil col-
Compound (2): [found: C, 59.95; H, 5.85; N, 6.02. C22H25BN2O7
requires C, 60.02; H, 5.72; N, 6.36%]; mp 178e180 ꢃC (de-
20
composition); [
a
]
D
þ22.8 (c 0.016, MeOH); nmax (KBr) 3462.9,
3348.1, 2971.1, 1737.3, 1683.2, 1548.8, 1519.9, 1475.3, 1438.4,
1366.6e1166.9, 1066.6, 1018.4, 971.4e699.1 cmꢀ1
; dH (400 MHz,
DMSO-d6) 1.30 (9H, s, (CH3)3), 2.97e3.18 (2H, m, CbH2), 3.64 (3H, s,
OCH3), 4.22e4.28 (1H, m, CaH), 7.31 (1H, dd, J 2.000, 8.21 Hz, C6H),
umn, C-8, 5
column, C-18, 5
m
m, 250 mm long, ID¼20 mm or SupelcosilÔ SPLC-ABZ
7.34 (1H, s, NH), 7.58 (1H, t, J 8.01, 7.61 Hz, C5 H), 7.69 (1H, d, J
0
m
m, 250 mm long, ID¼10 mm). The purity of the
2.00 Hz, C7H), 7.71 (1H, s, C4H), 8.02 (1H, dt, J 1.20, 7.61 Hz, C4 H),

K. Guzow et al. / Tetrahedron 68 (2012) 9240e9248
9247
0
8.22 (1H, dt, J 1.60, 8.01 Hz, C6 H), 8.29 (2H, s, B(OH)2), 8.65 (1H, s,
and carbonate buffer for pH 9.46e10.60. The pH of the solution was
measured using Hanna Instruments HI 9321 pH-meter with
a combined glass electrode.
0
C2 H); dC (100 MHz, DMSO-d6) 28.02 (CH3)3, 36.29 Cb, 51.74 OCH3,
0
55.44 Ca,0 78.23 Ct-Bu, 110.29 C7, 120.15 C4, 125.60 C6, 126.53 C1 ,
0
0
0
0
128.30 C2 , C5 , 128.71 C6 , 132.94 C4 , 134.41 C3 , 137.44 C5, 141.56 C9,
149.06 C8, 155.34 NHCO, 162.81 C2, 172.44 CO; m/z (MALDI): MHþ,
found 441. C22H25BN2O7 requires 440.
All calculations were performed using Origin v. 6.1 software.
4.2.2. pH titrations. Three series of thirteen samples of the same
concentration of each compound were prepared in appropriate
buffer solution (pH from 3.50 to 10.60). Two of them contained
additionally 50 mM of monosaccharide (glucose or fructose). For
absorption measurements the concentration of compound was
4.1.3. 3-[2-(4-Boronophenyl)benzoxazol-5-yl]alanine methyl ester
(1a) and 3-[2-(3-boronophenyl)benzoxazol-5-yl]alanine methyl ester
(2a). To magnetically stirred solution of (1) (50 mg, 0.11 mmol) or
(2) (100 mg, 0.23 mmol) in CH2Cl2 (2 mL) trifluoroacetic acid (2 mL)
was added. After 1 h solvent was evaporated in vacuo. The oily
residue was twice dissolved in toluene and evaporated in vacuo to
remove traces of TFA. The crude products were purified by means of
semi-preparative RP-HPLC ((1a)dKromasil column, mobile pha-
sedgradient running from 0% to 60% of B over 120 min, detection at
223 nm; (2a)d1. Kromasil column, mobile phasedgradient run-
ning from 0% to 70% of B over 120 min, detection at 223 nm, 2.
Supelcosil column, mobile phasedgradient running from 20% to
60% of B over 120 min, detection at 304 nm, A¼0.01% water solution
of TFA, B¼80% of acetonitrile in A). Compound (1a) was obtained as
grey solid (40 mg, 0.09 mmol, 86%) whereas compound (2a) as
a beige solid (22.5 mg, 0.05 mmol, 22%).
about 44
measurements the concentration of both compounds was equal to
15 M. The spectra of each series were measured just after prepa-
mM for 1a and 56 mM for 2a whereas for fluorescence
m
ration and additionally after about 24 and 48 h.
Titration curves were fitted and pKa values (pKa¼ꢀlogKa) were
obtained using the equation:
ꢀ
ꢁ
Iacid þ Ibase ꢂ 10ðpHꢀpK Þ
a
I ¼
10ðpHꢀpK Þ þ 1
a
where: Iacid and Ibase are the absorbance or fluorescence intensity
limits in the acid and basic region, respectively. The single data file
and global analysis (simultaneous analysis of all data files with pKa
as common parameter) were used to determine pKa values.
In the case of fluorometric titrations the measured fluorescence
intensity of each sample was corrected for differences in absor-
bance according to the equation:
Compound (1a): tR¼27.8 (gradient running from 0% to 100%B
over 60 min); mp 290e293 ꢃC (decomposition); m/z (MALDI): MHþ,
found 341. C17H17BN2O5 requires 340.
Compound (2a): tR¼31.0 (gradient running from 0% to 100%B
over 60 min); mp >360 ꢃC (decomposition); m/z (MALDI): MHþ,
found 341. C17H17BN2O5 requires 340.
ꢂ
ꢃ.ꢂ
ꢃ
Ip ¼ I ꢂ 1 ꢀ 10ꢀA
1 ꢀ 10ꢀA
wz
pr
where: I is measured fluorescence intensity of the sample, Ipd
fluorescence intensity corrected for the sample absorbance, Awz and
Apr are absorbances of the standard and the sample at the excitation
wavelength, respectively.
4.2. UVevis and fluorescence measurements
4.2.1. General. Monosaccharide
-(ꢀ)-fructose, were purchased from SigmaeAldrich.
Absorption spectra were measured on a PerkineElmer Lambda
used,
D
-(þ)-glucose
and
D
4.2.3. Monosaccharide binding experiments. In the monosaccharide
titration experiments a solution of compound in phosphate buffer
pH¼7.5 was treated with increasing amounts of a solution of
monosaccharide (glucose or fructose) containing a fluorophore at
the same concentration as in the cuvette. After each addition, using
a Hamilton microsyringe with micrometer screw, the solution was
stirred for a few minutes to reach equilibrium and the UVevis or
fluorescence spectrum was subsequently recorded. All titrations
were performed twice.
40P spectrophotometer at 25 ꢃC, stabilized by a P-1 Peltier system
with magnetic stirrer. Fluorescence spectra were measured on
a PerkineElmer LS-50B spectrofluorometer with magnetic stirrer in
the cuvette holder (Electronic Stripper Model 300). The tempera-
ture of the solution was maintained using Julabo F26-MP re-
frigerated circulator. Quantum yields were calculated using 2-
aminopyridine in 0.05 M H2SO4 (QY¼0.605) as a reference and
were corrected for different refractive indices of solvents.41 In all
fluorometric measurements, the optical density of the solution
does not exceed 0.1.
Titration curves against monosaccharide were fitted, and bind-
ing constants (KD) were obtained from global analysis of data ob-
tained from two independent titrations using the equation:
The fluorescence lifetimes were measured with
a time-
correlated single-photon counting apparatus Edinburgh CD-900.
The excitation source was a NanoLed N15 (lex¼277 nm) from IBH.
The half-width of the response function of the apparatus, measured
using a Ludox solution as a scatter, was about 1.0 ns. The emission
wavelengths were isolated using a monochromator (about 12 nm
spectral band-width). Fluorescence decay data were fitted by the
iterative convolution tothe sum of exponents according to equation:
ðI0 þ I1KD½CꢄÞ ðI0 þ I2KS½CꢄÞ
I ¼
þ
1 þ KD½Cꢄ
1 þ KS½Cꢄ
where: I denotes absorbance or integral fluorescence intensity and
I0, I1 and I2 are the initial (no monosaccharide), intermediate and
final (plateau) intensity on the titration curve. The second part of the
equation is added to take into account the effect of light scattering in
absorption measurements and KS has no physical meaning. This part
of the equation was omitted in fluorescence data analysis.
X
IðtÞ ¼
aiexpðꢀt=si
Þ
i
Acknowledgements
where ai is the pre-exponential factor obtained from the fluores-
cence intensity decay analysis and si the decay time of the i-th
component, using software supported by the manufacturer. The
adequacy of the exponential decay fitting was judged by visual
inspection of the plots of weighted residuals as well as by the
statistical parameter c2R and shape of the autocorrelation function
of the weighted residuals and serial variance ratio (SVR).57
This work was financially supported by the Polish Ministry of
Science and Higher Education under grant DS 8441-4-0132-12.
Supplementary data
Titration curves against pH were measured in buffer solutions:
acetate buffer for pH 3.50e5.78, phosphate buffer for pH 5.90e8.56
Spectrophotometric and spectrofluorometric pH titrations of
compounds studied measured after 24 and 48 h as well as

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