Synthesis, evaluation, and computational studies of naphthalimide-based long-wavelength fluorescent boronic acid reporters

Article abstract of DOI:10.1002/chem.200701785

Boronic acids that change fluorescence properties upon sugar binding are very useful for the synthesis of carbohydrate sensors. Along this line, boronic acids that fluoresce beyond 500 nm are especially useful. A series of boronic acid fluorescent reporte

Full text of DOI:10.1002/chem.200701785

FULL PAPER
DOI: 10.1002/chem.200701785
Synthesis, Evaluation, and Computational Studies of Naphthalimide-Based
Long-Wavelength Fluorescent Boronic Acid Reporters
Shan Jin, Junfeng Wang, Minyong Li, and Binghe Wang*[a]
Abstract: Boronic acids that change
fluorescence properties upon sugar
binding are very useful for the synthe-
sis of carbohydrate sensors. Along this
line, boronic acids that fluoresce
beyond 500 nm are especially useful. A
series of boronic acid fluorescent re-
porter compounds based on the 4-
have been synthesized (1a–d) and eval-
uated under near physiological condi-
tions. These compounds showed good
water solubility and significant changes
in fluorescence properties after binding
with sugars, with the emission wave-
length being at around 570 nm. Ana-
logues in this series with different sub-
stitutions showed similar properties.
We have also examined the mechanism
of the observed fluorescence changes
for these compounds.
Keywords: boronic acids · carbohy-
drates · fluorescence · sensors
amino-1,8-naphthalimide
structure
Introduction
and the addition of a 4-amino groupto this compound has
been shown to significantly change its fluorescence proper-
ties.[12,37–39] In our recent work, 4-amino-1,8-naphthalimide
was used as a template for a series of long-wavelength fluo-
rescent boronic acid reporters that showed fluorescence in-
tensity increases upon sugar binding.[13] Somewhat related
work was also reported by Trupp and co-workers. However,
in their work, an added aliphatic linker with an appended
amino group was used to modulate the fluorescence proper-
ties of a naphthalimide fluorophore.[12] In our work, the phe-
nylboronic acids are directly attached to the naphthalimide
fluorophore to modulate its fluorescence. In this series, an
amino groupwas positioned in a 1,5 relationshipto the bor-
onic acid, which helps to modulate its pKa and electronic
states.[6,40–43] The change in the ionization state of the boron
atom upon sugar binding from the neutral sp2 form to the
anionic sp3 state was thought to be the reason for the ob-
served fluorescence changes. In our previous work, we ex-
amined the effect of N substitution on the fluorescence
properties of this series of compounds. Herein, we would
like to report our work on the design, synthesis, and evalua-
tion of 1b–d, compounds that have electron-donating or
electron-withdrawing groups at the para position on the
phenyl boronic acid moiety (Scheme 1). These groups in-
clude a methoxy group( 1b), a methoxycarbonyl group( 1c),
and a fluoro group( 1d). We were interested in studying
whether changes in the phenylboronic acid substitution
would have a significant effect on the fluorescence and bind-
ing properties of these compounds. Moreover, we also used
computational chemistry to study the theoretical basis for
Boronic acids are known to bind 1,2- and 1,3-diol-containing
structures through reversible covalent interactions.[1–4] Such
interactions have been used for the preparation of fluores-
cent sensors[5–23] and transporters[24,25] for carbohydrates, as
well as lectin mimics (termed boronolectins) for cell-surface
carbohydrate recognition.[26–30] Critical to such sugar-sensing
efforts is the availability of boronic acid reporter compounds
that change fluorescence properties upon sugar binding. Par-
ticularly desirable are boronic acids that emit fluorescence
at long wavelengths beyond the UV region (above 500 nm),
which would have less background interference and higher
penetrating power for cellular applications.
Some long-wavelength fluorescent boronic acid reporters
have been reported.[7,9,15,31–35] Among those compounds, a
boron–dipyrromethane (BODIPY) dye functionalized with
a phenylboronic acid group (510 nm) was the only water-
soluble fluorescent probe that showed fluorescence intensity
increases upon sugar binding.[36] Long-wavelength emission
has been observed with the 1,8-naphthalimide fluorophore,
[a] S. Jin, Dr. J. Wang, Dr. M. Li, Prof. B. Wang
Department of Chemistry and
Center for Biotechnology and Drug Design
Gerogia State University
Atlanta, GA 30302-4089 (USA)
Fax : (+1)404-413-5543
E-mail: wang@gsu.edu
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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sulted in chelation of the palladium catalyst in the boryla-
tion stepand subsequently diminished borylation efficien-
cy.[13] Therefore, in the first stepof protection, 4-amino-1,8-
naphthalimide (2) was treated with sodium methoxide/meth-
anol in DMF at room temperature and was then treated
with benzyl bromide to afford 4-amino-N-benzyl-naphthali-
mide 3 (Scheme 2). Among the alkylating agents 5a–e, three
were not commercially available and were prepared through
benzylic bromination of the corresponding bromotoluene
analogues 4c–e by treatment with AIBN and NBS in CCl4
at 708C (reflux conditions). Subsequent alkylation of 3 with
benzylbromide analogues 5 gave the desired arylbromide
compounds 6 except with 5e, which did not undergo alkyla-
Scheme 1. Structures of long-wavelength boronic acid reporters. Bn:
benzyl.
the observed spectroscopic property changes. The com-
pounds synthesized will be very useful for the preparation
of fluorescent carbohydrate sensors and lectin mimics (boro-
nolectins),[29] and the understanding of the substituent ef-
fects gained will also be very useful for the future design of
other fluorescent boronic acid compounds.[29]
tion. Borylation of 6a was carried out with PdCl2(dppf) as
A
the catalyst to generate 1a.[44–46] Boronic acids 1b–d were
prepared from 6b–d by following similar procedures. It
should be noted that deprotection of the boronic acids oc-
curred spontaneously during the borylation reaction and the
subsequent workup. Therefore, no separate deprotection
stepwas needed. Purification of the final products by using
C-18 reversed-phase (RP) HPLC afforded free boronic
acids 1a (30%), 1b (24%), 1c (14%), and 1d (21%).
Results and Discussion
Synthesis: For the synthesis of the analogues designed, our
general approach was to couple the 4-amino-1,8-naphthali-
mide core (2) with an appropriately substituted 2-bromo-
benzylbromide (5a–e, Scheme 2). For this, we needed to
protect the imide nitrogen atom of 2 to minimize side reac-
tions. A benzyl protecting group was used. In our previous
work, we also studied other protecting groups and found
that use of the methoxymethyl (MOM) protecting group re-
Fluorescence binding studies: Since the compounds were de-
signed to change their fluorescence properties upon sugar
binding, we studied their fluorescence properties under vari-
ous conditions. However, before such studies, we wanted to
make sure that these boronic acids have sufficient water sol-
ubility for the study. Therefore, we took seven UV spectra
of each boronic acid in a concentration range of 1”10ꢀ4
(1% methanol in phosphate buffer, pH 7.4) to 1”10ꢀ6 m.
Good linear relationships between concentrations and UV
absorbance were obtained for all four boronic acids, a result
indicating that these compounds were completely soluble
and aggregation was not an issue under the conditions stud-
ied. As a result, we selected a concentration range of 1”
10ꢀ5 to 5”10ꢀ6 m for the binding tests at pH 7.4 (1% MeOH
in phosphate buffer).
The boronic acids themselves showed an excitation wave-
length (lex) of 493 nm and an emission wavelength (lem) of
567 nm. Upon binding with a model sugar, fructose, the
emission blue shifted to 550 nm and the fluorescence inten-
sity increased by about 2-fold for all these boronic acids
(Figure 1). Subsequent extensive binding studies were then
conducted at a sensor concentration of 1”10ꢀ5 m for boronic
acids 1a, 1b, and 1d and 5”10ꢀ6 m for boronic acid 1c.
The emission shift and the fluorescence intensity changes
of the boronic acids upon binding with sorbitol followed the
same trend as those with fructose, and the fluorescence in-
tensity increased to an even larger degree (about fourfold;
Figure 2). Such results are consistent with our earlier obser-
vations that that carbohydrates that bind more tightly tend
to bring on greater maximal fluorescence intensity
changes.[47–52] Similar studies were conducted with glucose.
The apparent association constants (Ka) between the bor-
onic acids and the three sugars were determined. The affini-
ty trend with the boronic acids followed the order d-sorbi-
Scheme 2. a) NaOCH3/CH3OH, BnBr, DMF, room temperature; b) NBS,
AIBN, CCl4, reflux, hn; c) NaH, DMF, 5a–d, room temperature; 4) neo-
pentylglycol diboron, [PdCl2(dppf)], KOAc, DMSO, 908C. DMF: N,N-di-
A
methylformamide; NBS: N-bromosuccinimide; AIBN: azobisisobutyroni-
trile; dppf: 1,1’-bis(diphenylphosphanyl)ferrocene; DMSO: dimethylsulf-
oxide.
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Fluorescent Boronic Acid Reporters
FULL PAPER
ous carbohydrates. It is interest-
ing to note that the four ana-
logues with varying substituents
on the phenylboronic acid
moiety showed very similar
binding constants (Table 1).
Such results further confirm our
earlier findings that the sub-
stituent effect on the binding
affinity of a phenylboronic acid
moiety is hard to predict,[4] es-
pecially when there is an amino
[19]
groupin a 1,5 relationship.
The newly synthesized com-
pounds (1b–d) showed very
similar properties to those of
the original compound 1a. It
should be noted that, in our
previous work, methanol con-
centrations affected the binding
constants of 1a. For example,
the binding constant of 1a with
fructose was 57mꢀ1 in 0.1%
methanol/buffer solution and
changed to 25mꢀ1 in 1% metha-
nol/buffer.[13] However, the
binding constants with other
sugars showed little change
with this minor change in meth-
anol concentration.
Figure 1. Fluorescence spectra of boronic acids upon addition of d-fructose (0–500 mm) in 0.1m phosphate
buffer at pH 7.4 with 1% MeOH; lex =493 nm. A) 1a (1”10ꢀ5 m); B) 1b (1”10ꢀ5 m); C) 1c (5”10ꢀ6 m); D) 1d
(1”10ꢀ5 m).
pH titration and apparent pKa
determination: With the aim of
understanding the basic mecha-
nism through which fluores-
cence intensity changes occur,
we studied the pH profiles of
the fluorescence intensity in the
absence and presence of sugars
(500 mm). When tested in the
absence of any sugar, the emis-
sion intensity of 1a increased
nine-fold at 550 nm upon
changing the pH value from 2
to 12 (Figure 3), with an appar-
ent pKa value of about 6.0,
which was assigned to the bor-
onic acid moiety (Table 2).
Figure 2. Fluorescence spectra of boronic acids upon addition of d-sorbitol (0–500 mm) in 0.1m phosphate
buffer at pH 7.4 with 1% MeOH; lex =493 nm. A) 1a (1”10ꢀ5 m); B) 1b (1”10ꢀ5 m); C) 1c (5”10ꢀ6 m); D) 1d
(1”10ꢀ5 m).
When the pH value was
changed from 2 to 12, the fluo-
rescence intensity of 1a at
550 nm increased 33-, 11-, and
tol>d-fructose>d-glucose, which is consistent with previ-
ous literature reports for other monoboronic acids
(Table 1).[1,4] Such intrinsic preference for different diols by
monoboronic acids dictates the need for more than a single
boronic acid in the construction of selective sensors for vari-
40-fold with the addition of fructose, glucose, and sorbitol,
respectively. The apparent pKa values observed were 3.9,
5.5, and 4.1 for the esters of fructose, glucose, and sorbitol,
respectively. The apparent pKa values of 1a in 1% metha-
nol/buffer were slightly different from those of previous
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B. Wang et al.
Table 1. Apparent association constants (Ka) of the boronic acids with
Table 2. Apparent pKa values of the boronic acids in the absence and
different sugars.[a]
presence of sugars.[a]
Fructose
Ka [mꢀ1 DIf[b]
(fold)
Glucose
]
Sorbitol
Ka [mꢀ1 DIf[b]
(fold)
pKa
Boronic acid
Fructose
Glucose
Sorbitol
]
Ka [mꢀ1
DIf[b]
(fold)
]
1a[b]
1b[b]
1c[c]
1d[b]
6.0Æ0.2
6.3Æ0.2
5.6Æ0.1
5.8Æ0.1
3.9Æ0.4
4.2Æ0.2
3.7Æ0.2
3.6Æ0.1
5.5Æ0.3
4.9Æ0.2
4.7Æ0.1
5.5Æ0.3
4.1Æ0.3
3.9Æ0.1
3.6Æ0.2
3.5Æ0.3
G
G
ACHTREUNG
1a[c]
1b[c]
1c[d]
1d[c]
25Æ2
28Æ1
27Æ1
24Æ2
2.5
2.4
1.7
1.8
1.4Æ0.3
1.1Æ0.2
1.4Æ0.2
0.9Æ0.3
1.1
1.1
1.2
1.1
110Æ1
117Æ3
103Æ2
98Æ6
2.6
2.5
2.2
2.4
[a] All experiments were performed in duplicate; sugar concentration:
500 mm in 0.1m buffer solution at pH 7.4 with 1% MeOH. [b] [boronic
acid]=1”10ꢀ5 m. [c] [boronic acid]=5”10ꢀ6 m.
[a] Experiments in duplicate were conducted in 0.1m buffer solution at
pH 7.4 with 1% MeOH. [b] DIf[a] (fold): represents maximum intensity
change over initial intensity. [c] [boronic acid]=1”10ꢀ5 m. [d] [boronic
acid]=5”10ꢀ6 m.
In order to achieve a good understanding of the origin of
the fluorescence intensity changes, we took 1a as an exam-
ple to study the pH profile of UV absorbance in the absence
and presence of fructose (500 mm). For boronic acid 1a
alone, the lmax value changed from 440 to 492 nm when the
solution pH value increased from 4.5 to 7.5; the UV intensi-
ty decreased by half at 440 nm and increased 3-fold at
493 nm. For the boronic ester of 1a with fructose, the UV
wavelength changed from 440 nm to 479 nm when the solu-
tion pH value increased from 2.5 to 5.0; the absorbance in-
tensity showed a slight decrease in the wavelength range
400–425 nm and the intensity increased 2-fold at 479 nm
(Figures 4 and 5). Such results suggest that the observed
fluorescence intensity changes upon variation in the pH
value could, at least partially, be attributed to the increased
absorption at the excitation wavelength. The apparent pKa
values of 1a and the 1a ester form were 6.0 and 3.9 from
the UV studies; these values
studies in 0.1% methanol/buffer.[13] The trend of the appar-
ent pKa values with the other three boronic acids (1b–d) fol-
lowed the order boronic acid alone>d-glucose>d-fruc-
tose>d-sorbitol (Table 2), as expected.[1,4,13] These apparent
pKa values showed a general Hammett correlation, as ex-
pected from our earlier publication (data not shown).[4]
The shift to the left in the pH-titration traces (Figure 3)
for the sorbitol and fructose esters compared with those for
the glucose ester and boronic acids alone is consistent with
previous findings that sorbitol and fructose esters have
lower apparent pKa values than the free boronic acid and
the glucose ester.[1,4,13] The overall lower apparent pKa
values for those compounds with an electron-withdrawing
group( 1c and 1d) than for those with no such substituents
or with an electron-donating substituent (1a and b) is to be
expected.[4]
are consistent with the results
of the fluorescence studies
(Figure 3 and Table 2). As
shown in Figure 4 and 5B, the
UV lmax value of 1a changed
from 492 to 479 nm after fruc-
tose addition at a near physio-
logical pH value.
Fluorescence quantum yield
studies: The fluorescence quan-
tum yields for boronic acids
1a–d and their sugar complexes
were also determined with fluo-
rescein as
a reference com-
pound.[53,54] The quantum yields
of the boronic acids were deter-
mined according to Equa-
tion (1), where Q is quantum
yield, A is UV absorbance, OD
is optical density (fluorescence),
and subscript R indicates the
reference compound.[55,56]
Figure 3. pH profile of the fluorescence intensity of boronic acids in the absence and presence of sugars in
0.1m aqueous phosphate buffer with 1% MeOH, [sugar]=500 mm, lex =493 nm, lem =550 nm. Boronic acid
Q ¼ QRðA=ARÞðODR=ODÞ
ð1Þ
~
&
^
alone ( ), in the presence of d-fructose ( ), in the presence of d-glucose ( ), and in the presence of d-sorbitol
(”). A) 1a (1”10ꢀ5 m); B) 1b (1”10ꢀ5 m); C) 1c (5”10ꢀ6 m); D) 1d (1”10ꢀ5 m).
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Fluorescent Boronic Acid Reporters
FULL PAPER
Figure 4. UV spectra of pH studies of boronic acid 1a (1”10ꢀ5 m) in the
absence and presence of fructose in 0.1m aqueous phosphate buffer with
1% MeOH and pH value changes from 2.0 to 12.0. A) Boronic acid 1a
alone; B) 1a in the presence of d-fructose (500 mm). All experiments
were performed in duplicate.
The results showed that the quantum yields of the boronic
acids in the presence of fructose and sorbitol were higher
than those of the boronic acids alone and in the presence of
glucose, a fact that is consistent with the trend of fluores-
cence intensity changes (Table 3). However, these quantum
yields are not directly correlated with the apparent pKa
value of each compound. This is understandable since many
other factors, such as flexibility, solvation, and excited-state
electron-density distribution, are expected to affect the
quantum yields of these compounds as well.
Figure 5. pH profile of the UV absorbance lmax and intensity of boronic
acid 1a (1”10ꢀ5 m) in the absence and presence of fructose in 0.1m aque-
ous phosphate buffer with 1% MeOH and [fructose]=500 mm. A) Ab-
sorbance intensity changes with varying pH. Boronic acid alone at
&
^
440 nm ( ), boronic acid alone at 493 nm ( ), in the presence of d-fruc-
tose (500 mm) at 415 nm (+), and in the presence of d-fructose (500 mm)
at 479 nm (”); B) absorbance lmax changes with different pH values. Bor-
~
*
onic acid alone ( ) and in the presence of d-fructose (500 mm; ). All
experiments were performed in duplicate.
emission wavelength for both compounds was observed
(Tables 4 and 5). Furthermore, both the UV and fluores-
cence intensities increased with decreases in the solvent po-
larity. Such results are consistent with an excited-state inter-
nal charge-transfer (ICT) mechanism,[15,57–65] in which polar
solvents helpto stabilize the excited-state charge separation
and therefore lower the energy gapbetween the ground and
excited states.[65,66] Other possibilities may also include a
change in the specific species formed when going from an
aqueous environment to an organic solution.[10,40,41,43,67–70]
Solvent studies and proposed mechanism for the fluores-
cence property changes: In order to understand the reason
for the observed spectroscopic property changes upon sugar
addition and variation of the pH value, we also studied sol-
vent effects on boronic acid 1b and a reference compound
3. When the solvent polarity decreased from water to ethyl
acetate, a significant blue shift in the lmax value and the
Table 3. Fluorescence quantum yields of the boronic acids alone and in
the presence of various sugars.[a]
Table 4. Solvent effect on the spectroscopic properties of compound 3.[a]
Boronic acid
Fructose
Glucose
Sorbitol
H2O
MeOH
Ethyl
acetate
1a[b]
1b[b]
1c[c]
1d[b]
0.026Æ0.003
0.037Æ0.002
0.048Æ0.002
0.038Æ0.003
0.081Æ0.003
0.095Æ0.002
0.090Æ0.004
0.093Æ0.002
0.035Æ0.004
0.052Æ0.001
0.059Æ0.001
0.053Æ0.002
0.084Æ0.005
0.101Æ0.001
0.103Æ0.007
0.093Æ0.002
lmax [nm]
434Æ1 433Æ1
1.0
542Æ1 527Æ1
1.0
417Æ2
UV intensity[b]
2.6Æ0.3 2.9Æ0.2
emission wavelength [nm]
emission intensity relative to that in water
494Æ1
6.9Æ0.2 10.6Æ0.4
[a] Sugar concentration: 500 mm in 0.1m buffer solution at pH 7.4 with
1% MeOH; all experiments were performed in duplicate. [b] [boronic
acid]=1”10ꢀ5 m. [c] [boronic acid]=5”10ꢀ6 m.
[a] [compound 3]=1”10ꢀ5 m. All experiments were performed in dupli-
cate. [b] Relative to that in water.
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B. Wang et al.
Table 5. Solvent effect on the spectroscopic properties of 1b.[a]
charged functional groupnext to the fluorohpore should
helpto stabilize the excited-state charge seapration and
should result in a bathochromic shift of the UV spectra with
increased intensity. To a certain extent, the effect of the neg-
atively charged boron atom is similar to increased solvent
polarity on the stabilization of the excited-state charge sepa-
ration.
Through a combination of all of the results stated above,
one can summarize the structural changes of 1a–d and their
relationship to the fluorescence properties by using
Scheme 3. At low pH values, boronic acids 1a–d exist in the
H2O
MeOH
Ethyl
acetate
color
red
orange
yellow
466Æ3
lmax [nm]
492Æ1 486Æ2
1.0
567Æ1 559Æ1
1.0
UV intensity[b]
1.3Æ0.1 1.4Æ0.1
emission wavelength [nm]
emission intensity relative to that in water
[a] [boronic acid 1b]=5”10ꢀ6 m. All experiments were performed in du-
537Æ1
5.3Æ0.2 14.4Æ1.1
plicate. [b] Relative to that in water.
ꢀ
The similar solvent effects on compound 3, which does
not have a boronic acid moiety, and boronic acid 1b indicate
that the phenylboronic acid moiety itself does not directly
participate in the generation of fluorescence, although the
formation of the anionic boronate species (Scheme 3) may
influence the fluorescence properties of 1b (and 1a, 1c, and
1d), which will be discussed in the next section. The similar
behaviors among 1a–d in terms of lmax, emission wave-
length, pH profiles, and sugar-induced fluorescence changes
also suggest that the phenylboronic acid moiety plays only
an auxiliary role in fluorescence modulation and is not part
of the fluorophore that is responsible for fluorescence gener-
ation.
neutral trigonal form, 1, with a possible B N bond through
donation of the nitrogen lone-pair electrons to the boron
open shell.[40,41,43,71] It is understood that the aromatic amine
groupinvolved is not a strong Lewis base because of the
substitution of two electron-withdrawing imide functional
groups on the naphthalene ring. Therefore, the exact
ꢀ
strength (or existence) of the B N bond is not clear. How-
ever, in either case, it does not affect the subsequent analy-
sis of the observed fluorescence changes. As the solution pH
value increases toward the respective pKa values (5.6–6.0;
Table 2), the boron atom is converted into its anionic tetra-
hedral form, 7. As discussed earlier, this generation of a net
negative charge helps to stabilize the excited-state charge
transfer and therefore causes a bathochromic shift of the
UV lmax value with increased intensity (Figures 4 and 5).
The increased absorbance can be at least a partial contribu-
ting factor for the observed fluorescence intensity increase.
With the addition of a carbohydrate, the pKa value of the
boronic ester 8 becomes even lower than that of the boronic
acid species (Table 2).[1,2,4] Similarly, an increased pH value
would result in the generation of the anionic boronate ester
9, which should also helpto stabilize the excited-state
charge transfer in much the same way as the boronate spe-
cies 7 and would explain the bathochromic shift in the UV
lmax value. As presented in Figures 1–3 and Tables 1 and 3,
the boronate esters 9 are much more fluorescent than the
corresponding boronates 7. Complexation with a sugar will
most likely add steric hindrance and decrease the freedom
ꢀ
of rotation around the C N bond of the aniline nitrogen
atom, which can increase the fluorescence quantum yield
and consequently the fluorescence intensity. Sugar addition
to the boronic acid solution at the physiological pH value
also causes a hypsochromic shift in both the UV lmax value
(Figures 4 and 5B) and fluorescence emission wavelength
(Figures 1 and 2). Since at the physiological pH value, both
the boronic acid 1 and the boronic ester 8 should be in their
respective ionized states, 7 and 9, there is no difference in
their net charge. Therefore, the observed hypsochromic shift
is most likely due to the perturbation of the position and/or
orientation of the anionic boronate esters 9 relative to that
of the boronate species 7. It is reasonable to expect that ad-
dition of a sugar moiety would increase steric hindrance,
which in turn could increase the distance between the anion-
ic boronate functional groupand the fluorophore. The dis-
tance between the boron and aniline nitrogen atoms is espe-
cially important because the lone-pair electrons of the nitro-
Scheme 3. Equilibrium between different forms of boronic acids 1a–d in
the absence and presence of sugars at different pH values.
The proposed internal charge-transfer mechanism is also
consistent with the pH profiles presented in Figure 4. As the
pH value increases, the boronic acid moiety becomes ion-
ized, which gives a net ꢀ1 charge. The positioning of a
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Chem. Eur. J. 2008, 14, 2795 – 2804

Fluorescent Boronic Acid Reporters
FULL PAPER
gen atom are most likely involved in the excited-state
charge transfer.[72–77] If this is indeed the case, the increased
ꢀ
B–N distance (this is simply the B–N distance, not a B N
ꢀ
bond length as there is no B N bond here) could lessen the
stabilizing effect of the negatively charge boronate in 9 on
the excited-state charge-transfer species, which in turn
should lead to a hypsochromic shift.
To examine structural aspects of the sugar–boronic acid
complex, we performed a series of density function theory
(DFT)[78,79] calculations in a polarizable continuum model
(PCM)[80] to investigate the structures of these boronic acids
and the corresponding esters with fructose. Electronic struc-
ture calculations were performed by using the Gaussian 03
series of programs[81] on a Linux-based 40-node cluster. The
DFT method B3LYP and the 6ꢀ31+G
C
zations and frequency calculations (PCM(opt)). All calcula-
G
tions with the PCM solvent model employed the united-
atom topological model for Hartree–Fock (UAHF) atomic
radii during construction of the solvent cavity, as recom-
mended in the Gaussian 03 userꢁs reference manual. All ge-
ometries were fully optimized, and the characters of the sta-
tionary points found were confirmed by a harmonic frequen-
cy calculation at the same level of theory to ensure a mini-
mum was located. The results are shown in Table 6. Figure 6
shows the minimized structures of 1a in different forms and
the B–N distances.
Figure 6. Modeled structures of boronic acid 1a in the neutral form (A),
the anionic form (B), and the boronic ester form (C).
open shell to accommodate the lone-pair electrons of the ni-
ꢀ
trogen atom and therefore there cannot be B N bond for-
mation. The calculated B–N distances in boronate fructose
complexes 9 with 1a–d were 0.13–0.29 Š longer than those
in the corresponding boronates 7. The lengthened B–N dis-
tance should result in a lessened stabilization of the excited-
state charge separation and then a hypsochromic shift.
Therefore, the computational results are consistent with the
observed hypsochromic shifts of the UV lmax value and fluo-
rescence emission wavelength as discussed above.
Table 6. Calculated B–N distances of boronic acids 1a–d in the neutral
form (1), anionic form (7), and complexed boronate ester form (9).
B–N distance [Š]
neutral
anionic
complex
1a
1b
1c
1d
1.85
1.85
1.91
1.84
3.19
3.02
3.05
3.01
3.33
3.31
3.31
3.17
ꢀ
The B–N distance for a strong B N bond is usually
Conclusion
around 2 Š.[82–84] In the neutral trigonal form (1, Scheme 3),
ꢀ
the calculated B N bond lengths of 1a–d were 1.84–1.91 Š;
We have synthesized and evaluated a series of long-wave-
length (around 570 nm) boronic acid fluorescent reporters,
which showed good water solubility and significant fluores-
cence changes upon sugar addition. Variation of the para-
position substituents on the phenylboronic acid moiety had
very little effect on either the binding affinity or the spectro-
scopic properties. Mechanistic studies suggest that these flu-
orophores involve excited-state charge transfer, which can
be stabilized by the appended boronic acid group once it is
ionized. Such stabilization is consistent with the observed
fluorescence and UV pH profiles. The increased fluores-
cence intensities of these boronic acids upon sugar addition
can be explained by the increased B–N distance in the boro-
nate complex state, 9; this conclusion is supported by exten-
sive computational results. The boronic acids synthesized
these values suggest the possibility of B–N interactions to
some degree. Furthermore, the solvent-bridging effect ob-
served in the crystal state for some boronic acids is another
ꢀ
factor that could influence the interpretation of the B N
bond strength and/or existence.[43] It should be noted that
ꢀ
N
our grouprecently reported the reexamination of the B
ꢀ
bond issue and concluded that B N bond hydrolysis after
complexation with a diol was the dominate pathway in most
cases in an aqueous environment.[40,41] Such results were fur-
ther supported by studies from the Anslyn group[43] and are
consistent with the present study. Upon conversion of the
boron atom into its anionic form 7, the B–N distances in-
creased to about 3 Š. This is understandable since the tetra-
hedral form of the boronic acid functional grouphas no
Chem. Eur. J. 2008, 14, 2795 – 2804
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2801

B. Wang et al.
13C NMR (CDCl3): d=176.6, 175.3, 148.7, 141.3, 139.3, 137.8, 134.5,
133.9, 131.4, 128.8, 128.4, 127.2, 125.9, 125.1, 123.3, 120.8, 115.6, 114.5,
111.3, 105.2, 55.5 (OCH3), 43.4 (CH2), 29.7 ppm (CH2); MS (ESI): m/z
(%): 500.9 [MꢀH]+, 501.8 [M+], 502.8 [M+H]+, 503.8 [M+2H]+.
3-[(2-Benzyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-ylamino)-
methyl]-4-bromobenzoic acid methyl ester (6c): Column chromatography
(hexanes/ethyl acetate 8:1) gave 6c (25%) as a yellow powder. TLC
(hexanes/ethyl acetate 1:2): Rf =0.45; 1H NMR (CDCl3): d=8.65 (d, J=
.0 Hz, 1H), 8.48 (d, J=8.4 Hz, 1H), 8.18 (d, J=7.6 Hz, 1H), 8.07 (s, 1H),
7.89 (m, J=8.4 Hz, 1H), 7.76 (d, J=8.4 Hz, 1H), 7.69 (t, J=7.6 Hz, 1H),
7.55 (d, J=6.8 Hz, 2H), 7.23 (m, 3H), 6.71 (d, J=8.4 Hz, 1H), 5.39 (s,
2H; CH2), 4.77 (s, 2H; CH2), 3.88 ppm (s, 3H; CH3); MS (ESI): m/z
(%): 529.0 [M+], 531.0 [M+2H]+.
(1a–d) will be very useful for the preparation of long-wave-
length sensors and boronolectins[29] for carbohydrates, glyco-
lipids, and glycoproteins. Work on incorporating these bor-
onic acids into DNA for fluorescent aptamer development
is in progress.[85]
Experimental Section
General: 1H and 13C NMR spectra were recorded on a Bruker 400 MHz
spectrophotometer in deuterated choloroform (CDCl3) or deuterated
[D6]DMSO ((CD3)2SO) with either tetramethylsilane (TMS; d=
0.00 ppm) or the NMR solvent as the internal reference unless otherwise
specified. HPLC purification was carried out with a Shimadzu LC-10AT
VP system and a Zobax C18 reversed-phase column (4.6 mm”25 cm).
Fluorescence spectra were recorded on a Shimadzu RF-5301 PC spectro-
fluorometer. Absorption spectra were recorded on a Shimadzu UV-1700
UV/Vis spectrophotometer. Quartz cuvettes were used in all fluorescence
and UV studies. All pH values were determined by a UB-10 Ultra basic
benchtoppH meter (Denver Instruments). Analytical thin-layer chroma-
tography (TLC) was performed on Merck silica gel 60 plates (0.25 mm
thickness with F-254 indicator). Sugars, buffers, and diols were bought
from Aldrich or Acros and were used as received. Water used for the
binding studies was doubly distilled and further purified with a Milli-Q
filtration system. Solvents for extraction and chromatography were used
as received. Drying solvents (DMF, DMSO) were purchased from Acros.
1H, 13C NMR and MS spectra of selected compounds are given in the
Supporting Information.
2-Benzyl-6-(2-bromo-5-fluorobenzylamino)-benzo[de]isoquinoline-1,3-
dione (6d): Column chromatography (hexanes/ethyl acetate 12:1) gave
6d (27%) as a yellow powder. TLC (hexanes/ethyl acetate 1:2): Rf =0.50;
1H NMR ([D6]acetone, 400 MHz): d=8.72 (d, J=8.4 Hz, 1H), 8.57 (d,
J=7.2 Hz, 1H), 8.37 (d, J=8.4 Hz, 1H), 7.79–7.72 (m, 3H), 7.46 (d, J=
7.2 Hz, 2H), 7.31–7.20 (m, 4H), 7.11–7.06 (m, 1H), 6.71 (d, J=8.4 Hz,
1H), 5.33 (s, 2H; CH2), 4.81 ppm (s, 2H; CH2); 13C NMR ([D6]acetone):
d=178.2, 176.4, 163.3, 149.7, 138.4, 134.5, 134.1, 130.9, 128.2, 127.6, 126.9,
124.9, 120.8, 116.0, 115.7, 110.4, 104.8, 47.2 (CH2), 42.7 ppm (CH2); MS
(ESI): m/z (%): 489.0 [M+], 490.1 [M+H]+.
General procedure for synthesis of compounds 1: According to a typical
borylation procedure,
borane (64.2 mg, 0.284 mmol, 1.2 equiv), [PdCl2
6
(0.237 mmol, 1.0 equiv), bis(neopentylglycol)
(dppf)] (23.5 mg,
AHCTREUNG
0.029 mmol, 0.12 equiv), and potassium acetate (70.5 mg, 0.718 mmol,
3 equiv) were mixed at room temperature under an N2 atmosphere. This
was followed by addition of anhydrous DMSO (2 mL) with a syringe.
The solution was heated at 908C for 10 h and then cooled to room tem-
perature. Ethyl acetate (20 mL) and water (15 mL) were added to the re-
action mixture. The separated organic phase was washed with water (2”
10 mL). After drying over sodium sulfate and solvent evaporation, the
residue was purified by column chromatography (hexanes/ethyl acetate
3:2, to ethyl acetate/methanol 10:1) and further purified by HPLC (C18
RP column). Elution conditions: CH3CN/MeOH (1 mLminꢀ1); 0–10 min
(CH3CN 100%), 10–20 min (CH3CN 100!0%), 20–29 min (CH3CN
0%), 29–30 min (CH3CN 0!100%).
2-Benzyl-6-(2-boronic acid-5-methoxy-benzylamino)-benzo[de]isoquino-
line-1,3-dione (1b): 1b (24%) was obtained as a red powder. HPLC: tR =
20 min; 1H NMR (CD3OD, 400 MHz): d=8.22 (d, J=7.2 Hz, 1H), 8.05
(d, J=8.4 Hz, 1H), 7.59 (d, J=6.8 Hz, 1H), 7.35 (d, J=8.4 Hz, 2H),
7.17–7.27 (m, 4H), 7.02 (d, J=7.6 Hz, 2H), 6.74 (t, J=10.8 Hz, 1H), 6.17
(d, J=8.4 Hz, 1H), 5.33 (s, 2H; CH2), 4.52 (s, 2H; CH2), 3.73 ppm (s,
3H; CH3); 13C NMR (CD3OD): d=166.3, 164.6, 163.2, 159.8, 141.8,
138.6, 137.3, 132.0, 129.3, 128.7, 127.8, 127.6, 127.2, 126.3, 124.8, 119.6,
117.9, 112.5, 111.8, 102.3, 100.3, 54.2 (OCH3), 46.3 (CH2), 42.5 ppm
(CH2); MS (ESI): m/z (%): 466.2 [M+], 467.2 [M+H]+, 468.2 [M+2H]+.
General procedure for synthesis of compounds 5: Dry CCl4 (100 mL) was
added to
a mixture of 4 (10 mmol, 1.0 equiv), AIBN (0.5 mmol,
0.05 equiv), and NBS (10 mmol, 1.0 equiv). The mixture was heated at
708C (reflux) under white light (IR: 115–125 V, 175 W) with stirring for
2.5 h. Then solvent was evaporated under vacuum.
4-Bromo-3-bromomethylbenzoic acid methyl ester (5c): Column chroma-
tography (hexanes/ethyl acetate 8:1) gave 5c (38%) as white crystals.
TLC (hexanes/ethyl acetate 10:1): Rf =0.50; 1H NMR (CDCl3): d=8.08
(s, 1H), 7.77 (t, J=8.4 Hz, 1H), 7.62 (d, J=8.4 Hz, 1H), 4.58 (s, 2H;
CH2), 3.89 ppm (s, 3H; CH3); 13C NMR (CDCl3): d=165.8, 137.5, 133.6,
132.2, 130.8, 130.1, 129.8 (COOCH3), 52.5 (OCH3), 32.6 ppm (CH2); MS
(ESI): m/z (%): 310 [M+].
1-Bromo-2-bromomethyl-4-fluorobenzene (5d): Column chromatography
(hexanes) gave 5d (39%) as white crystals. TLC (hexanes/ethyl acetate
10:1): Rf =0.50; 1H NMR (CDCl3): d=7.48–7.52 (m, 1H), 7.16–7.19 (m,
1H), 6.86–6.91 (m, 1H), 4.52 ppm (s, 2H; CH2); 13C NMR (CDCl3): d=
163.1, 160.6, 138.9, 134.5, 118.1, 117.3, 32.5 ppm (CH2); MS (ESI): m/z
(%): 268 [M+], 270 [M+2H]+.
1-Bromo-2-bromomethyl-4-nitrobenzene (5e): Column chromatography
(hexanes/ethyl acetate 20:1) gave 5e (21%) as white crystals. TLC (hex-
anes/ethyl acetate 20:1): Rf =0.50; 1H NMR (CDCl3): d=8.31 (s, 1H),
8.00 (m, J=11.6 Hz, 1H), 7.76 (d, J=8.4 Hz, 1H), 4.61 ppm (s, 2H;
CH2); 13C NMR (CDCl3): d=139.0, 134.5 (2C), 131.6, 125.9, 124.4,
31.5 ppm (CH2); MS (ESI): m/z (%): 295 [M+], 297 [M+2H]+.
3-[(2-Benzyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-ylamino)-
methyl]-4-boronic acid-benzoic acid methyl ester (1c): 1c (14%) was ob-
tained as a red powder. HPLC: tR =22 min; 1H NMR (CD3OD): d=8.25
(d, J=6.8 Hz, 1H), 8.15 (s, 1H), 8.08 (d, J=8.4 Hz, 1H), 7.88 (d, J=
7.2 Hz, 1H), 7.72 (d, J=8.0 Hz, 1H), 7.62 (d, J=6.8 Hz, 1H), 7.36–7.43
(m, 3H), 7.26–7.29 (m, 2H), 7.20 (d, J=8.8 Hz, 1H), 6.18 (d, J=8.8 Hz,
1H), 5.35 (s, 2H; CH2), 4.62 (s, 2H; CH2), 3.88 ppm (s, 3H; CH3); MS
(ESI): m/z (%): 493.1 [MꢀH]+, 495.1 [M+H]+, 522.1 [Mꢀ2H+2CH3]+.
General procedure for synthesis of compounds 6: Dry DMF (25 mL) was
added to a mixture of 3 (1.00 mmol, 1.0 equiv) and sodium hydride
(60%, 1.03 mmol, 1.03 equiv). The mixture was stirred for 10 min, then a
solution of 5 (1.00 mmol, 1.0 equiv) in anhydrous DMF (15 mL) was
added dropwise under N2 at room temperature with stirring. Afterwards,
the stirring was continued at room temperature for 12 h. The solvent was
then evaporated under vacuum.
2-Benzyl-6-(2-boronic acid-5-fluorobenzylamino)-benzo[de]isoquinoline-
1,3-dione (1d): 1d (21%) was obtained as a red powder. HPLC: tR
=
21 min; 1H NMR (CD3OD, 400 MHz): d=8.55 (d, J=7.2 Hz, 1H), 8.47
(d, J=8.4 Hz, 1H), 7.66 (d, J=7.6 Hz, 1H), 7.42–7.48 (m, 3H), 7.21–7.37
(m, 6H), 7.07–7.14 (m, 2H), 5.35 (s, 2H; CH2), 4.55 ppm (s, 2H; CH2);
13C NMR (CD3OD): d=174.0, 164.7, 164.2, 140.5, 137.67, 133.8, 130.12,
128.4, 128.0, 127.7, 126.8, 124.0, 123.0, 122.3, 114.4, 110.5, 107.4, 104.9,
48.3 (CH2), 42.8 ppm (CH2); MS (ESI): m/z (%): 453.1 [MꢀH]+, 454.1
[M+].
2-Benzyl-6-(2-bromo-5-methoxybenzylamino)-benzo[de]isoquinoline-1,3-
dione (6b): Column chromatography (hexanes/ethyl acetate 12:1) gave
6b (31%) as a yellow powder. TLC (hexanes/ethyl acetate 1:2): Rf =0.50;
1H NMR (CDCl3): d=8.64 (d, J=6.4 Hz, 1H), 8.49 (d, J=8.4 Hz, 1H),
8.10 (d, J=8.4 Hz, 1H), 7.61–7.64 (m, 1H), 7.49–7.52 (m, 3H), 7.26–7.28
(m, 2H), 7.21 (d, J=7.2 Hz, 1H), 6.91 (d, J=4.2 Hz, 1H), 6.68–6.75 (m,
2H), 5.35 (s, 2H; CH2), 4.63 (s, 2H; CH2), 3.70 ppm (s, 3H; CH3);
Procedures for the binding studies (with 1a as an example): Solutions of
1a (1”10ꢀ5 m) and 1a (1”10ꢀ5 m) with sugar (0.5m) were prepared in
0.1m phosphate buffer at pH 7.40. These two solutions were then mixed
2802
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 2795 – 2804

Fluorescent Boronic Acid Reporters
FULL PAPER
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Acknowledgements
Financial support from the National Institutes of Health (grant nos.:
CA113917, CA88343, and NO1-CO-27184), the Georgia Cancer Coali-
tion through a Distinguished Cancer Scientist Award, and the Georgia
Research Alliance through an Eminent Scholar endowment and an Emi-
nent Scholar Challenge grant is gratefully acknowledged.
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Published online: January 28, 2008
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