Synthesis and evaluation of aryl boronic acids as fluorescent artificial receptors for biological carbohydrates

Article abstract of DOI:10.1016/j.bioorg.2011.11.003

Carbohydrates in various forms play a vital role in numerous critical biological processes. The detection of such saccharides can give insight into the progression of such diseases such as cancer. Boronic acids react with 1,2 and 1,3 diols of saccharides

Full text of DOI:10.1016/j.bioorg.2011.11.003

Bioorganic Chemistry 40 (2012) 137–142
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and evaluation of aryl boronic acids as fluorescent artificial receptors
for biological carbohydrates
Sandra Craig
Department of Chemistry and Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30302-4098, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 September 2011
Available online 25 November 2011
Carbohydrates in various forms play a vital role in numerous critical biological processes. The detection of
such saccharides can give insight into the progression of such diseases such as cancer. Boronic acids react
with 1,2 and 1,3 diols of saccharides in non-aqueous or basic aqueous media. Herein, we describe the
design, synthesis and evaluation of three bisboronic acid fluorescent probes, each having about ten linear
steps in its synthesis. Among these compounds that were evaluated, 9b was shown to selectively label
Keywords:
Boronic acid
Carbohydrate
Saccharides
Antibody mimics
Fluorescence
Receptor
HepG2, liver carcinoma cell line within a concentration range of 0.5–10
normal fibroblast cell line.
lM in comparison to COS-7, a
Ó 2011 Elsevier Inc. All rights reserved.
Smart probe
Sensor
1. Introduction
are engineered genetically near 100% human phenotype, there is al-
ways a possibility of eliciting an adverse immunogenic response.
There is an evident need to continue the efforts in designing target-
specific fluorophores to aid in the detection of tumorigenesis, pres-
ence metastasis, and in addition, provide visual guide in the removal
of tumor masses.
Carbohydrates are essential for cell–cell recognition and various
biological responses such as inflammation, lymphocyte homing,
regulation of metabolic pathways, among others. In addition, the
cross-talk between cell surface carbohydrates and cellular recep-
tors has also been associated with the metastatic behavior of var-
ious cancer types. Thus far, the detection of the over-expression of
such glycoproteins or lipids on membrane surfaces have been
accomplished through biological receptors such as antibodies
[1,2], aptamers [3,4], peptides or proteins [5], or metabolites
[6,7]) linked to a fluorochrome (Fig. 1) allowing the biomarker to
be detected by a light source.
Namely, dueto their high affinity for theirtarget and/or biomarker
of interest, monoclonal antibodies are widely used as biological
probes in fluorescence imaging. There are several monoclonal
antibodies available for in vivo fluorescence imaging applications,
anti-PSMA (prostate specific membrane antigen) [8] labels prostate
tumor cells, anti-VEGF (vascular endothelial growth factor) [9] labels
tumor cells associated with angiogenesis, and anti-HER-2 (human
epidermal growth factor recptor-2) [10] labels breast, ovary, and
other carcinomas. However, the disadvantages in using monoclonal
antibody conjugates as biological imaging probes are contributed
to their size. Large biomolecules tend to exhibit lower penetration
in tissue of host animal and longer clearance time, allowing back-
ground fluorescence interference. Although monoclonal antibodies
We are interested in the design and synthesis of small organic
molecules with the ability to recognize specific oligosaccharide pat-
terns. Boronic acid moieties, since the 1940s, have been known to
form rapid reversible complexes with 1,2 and 1,3 cis diols [11–13].
Much development has been geared toward sensory design
[14,15,21–24,26,27,29–33] and cell labeling [34–36] for biological
carbohydrates with the use of boronic acid serving as the artificial
receptor, which make boronic acid an ideal biological probe for the
detection of a cell surface carbohydrates over-expressed on various
cancer types. Aryl boronic acid scaffolds targeting cell surface carbo-
hydrates can be considered antibody mimics as they have high affin-
ity and selectivity as antibodies. The advantages of having smaller
molecules present creates faster clearance time, higher tissue
penetration, and the structural framework can be altered to enhance
the pharmacodynamics and pharmacokinetics, leading to a more
lucrative imaging probe candidate for in vivo applications.
An ideal in vivo biosensor for carbohydrates consists of: (1) a
recognition moietywithhigh affinityandspecificityand(2)aspectro-
scopic reporter, which gives off a measurable signal upon binding.
Numerous of peer-reviewed articles have provided insight in the de-
sign of biomolecular sensors that contain an ‘on’ and ‘off’ state
through a fluorescence quenching mechanism [37–39]. This attribute
E-mail address: scraig30@gmail.com
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doi:10.1016/j.bioorg.2011.11.003

138
S. Craig / Bioorganic Chemistry 40 (2012) 137–142
Fig. 1. The design of a site-specific fluorescence probe.
of ‘turning on’ only at the target site has been termed activatable or
‘smart’. Several mentionable molecular quenching mechanisms that
have been used in literature are; photoinduced electron transfer
(PET), internal charge transfer (ICT), metal–ligand charge transfer
(MLCT), and most commonly used in the development of smart
probes, fluorescence resonance electron transfer (FRET). The advan-
tages of using molecular fluorescence can be summarized, high
sensitivity of detection, and a signal generated only after being
bound to the specific biomarker resulting in low to no background
noise.
bond is labile; as a result it is hydrolyzed. The amine is then pro-
tonated, stopping the quenching process.
When designing a boronic acid scaffold as an artificial probe for
a carbohydrate of interest, the appropriate spatial arrangement is
imperative for optimal binding [41,45]. In continuing the efforts
of developing fluorescent artificial receptors, we have synthesized
a series of rigid dianthracene acid compounds. Our goal is to obtain
the framework of previously synthesized diboronic acid [46], het-
eroatom(s) were added within the di-carboxylic acid linker in com-
pound 4 in hopes of increasing the hydrophilicity. The tertiary
amine attached to the carbonyl group was changed to a secondary
amine to evaluate how a slight change in electronic properties and
reduction of a possible steric effect may alter and/or enhance selec-
tivity. With that in place, three di-anthracene boronic acids were
synthesized. Since 4 labeled HepG2, hepatocellular liver carcinoma
In our initial design an anthracene–boronic acid system (Fig. 2)
was chosen as the fluorescent probe. This system was first intro-
duced in 1992 by the Czarnik group (3, Fig. 3) [40], and was later
used by Shinkai. In Shinkai system a 1, 5 relationship between an
amine and boron was incorporated to create more electron density
around the boron center. In doing so, they developed monoboronic
acid 1, which is intrinsically selective for fructose and a diboronic
derivative also selective for glucose [41]. In this system the amine
regulates the fluorescence intensity. The anthracene moiety is
quenched by an excited state photoinduced electron transfer
(PET), which is considered to be the ‘off’ state of the sensor. Upon
addition of a diol, the fluorescence intensity increases, which rep-
resent the ‘on’ state of the sensor 2; therefore, creating a smart or
‘activatable’ probe for the detection of cell surface carbohydrates
with low background fluorescence. There are two proposed mech-
anisms in literature that have been introduced as the mechanisms
which stop the quenching process of the anthracene motif [41,42].
Shinkai and co-workers proposed that there is a B–N bond forma-
tion which stops the quenching process. Upon addition of a diol,
leading to the formation of a boronic ester, the pKa of the boron
species is decreased. This causes the amine to react with the boron,
forming a B–N bond, stopping the quenching process. Later, the
Wang group published a paper with detailed experiments provid-
ing additional insight as to the mechanism in which the quenching
process is eliminated in aqueous medium. They proposed the
mechanism is stopped through a hydrolysis mechanism. The B–N
cells, at 1 lM, concentrations between 0.5 and 10 lM were used in
fluorescent cell labeling studies. To evaluate the ability of the new-
ly synthesized bisboronic acid derivatives selectively labeling can-
cer cell only, COS-7, a normal fibroblast mammalian cell line was
used in parallel.
2. Materials and methods
2.1. Biology
2.1.1. Cell culture
Cell lines were purchased from ATCC. HEPG2 and COS7 cells
were maintained in RPMI with 10% FBS (fetal bovine serum), 1%
L-glutamine, and 0.5% gentamicin sulfate (50 mg/ml) (MediaTech).
All cells were maintained at 37 °C in a 5% CO2 incubator. Remaining
materials were purchased from Media-Tech unless otherwise
noted.
2.1.2. Fluorescent labeling studies
HEPG2 and COS7 cells were harvest in six well plates in growth
medium until ca. 50% confluency. Cells were then washed with PBS
following fixation with 4% paraformaldehyde at 4 °C for 25 min or
in 1:1 solution of MeOH/PBS for 25 min. After fixation the cells
were washed with PBS twice. Next, 1 ml of 1:1 MeOH/PBS was
placed in each well, followed by the desired concentration of
anthracene boronic acid derivative (0.5–10 lM). The six well plates
were placed at 4 °C for 45 min. The cells were viewed with a blue
emission filter.
R2
R1
OH
B
O
O
(HO)2B
R1
R2
N
. .
N
HO
OH
H
hv'
hv
hv'
hv
1
2
2.1.3. Imaging
Boronic acid
Weakly fluorescent
Boronic ester
Strongly fluorescent
Phase contrast and fluorescence overlay images were taken
with Carl Zeiss Axiovert 200 M by the process imaging software
Axiovision with the use of a blue long pass filter (emission
wavelength: 397 nm).
Fig. 2. Signaling unit for anthracene based photoinduced electron transfer (PET)
system.

S. Craig / Bioorganic Chemistry 40 (2012) 137–142
139
(HO)2B
N
B(OH)2
N
(HO)2B
N
B(OH)2
N
N
C
O
C
O
1
3
4
Fig. 3. Anthracene-based fluorescent chemosensors for saccharides.
2.2. Chemistry
2.2.1. General
(sigmoidal–dose response) curve an overlaid with one-site binding
curve (fructose) and two-site binding curve (sorbitol) by the soft-
ware GraphPrism 4.0. Triplicate measurements were taken and cor-
relation coefficients were P0.95 for each fit.
All 1H and 13C NMR were recorded at 400 MHz and 100 MHz,
respectively, with tetramethylsilane as the internal reference. Ele-
mental and mass spectral analyses were performed at Georgia State
University Analytical Facilities. All commercial reagents were used
without further purification unless otherwise noted. Acetonitrile
(CH3CN) and dichloromethane (CH2Cl2) were distilled from CaH2.
Tetrahyrofuran (THF) was distilled from Na and benzophenone.
2.3. Synthesis and structural analysis
2.3.1. Preparation of (10-azidomethyl-anthracen-9-ylmethyl)-methyl
carbamic acid tert-butyl ester (6)
Triphenylphosphine (717 mg, 2.74 mmol), carbon tetrachloride
(1 ml), and 2 ml of dry DMF were added to a round bottom flask
followed by alcohol derivative 5 (300 mg, 0.856 mmol), in 3 ml of
dry DMF. After disappearance of 5 as monitored by TLC, sodium
azide (208 mg, 3.16 mmol) was added. The reaction was allowed
to stir at room temperature until completion as indicated by TLC
and GC–MS analysis. Ice water (10 ml) was added to reaction and
the reaction mixture was stirred for 5 min. Then the reaction solu-
tion was diluted with ether (50 ml). The organic layer was washed
(2 Â 10 ml) with water and brine, dried over anhydrous magne-
2.2.2. Fluorescent binding studies
A constant concentration of (sensor) bisboronic acid (2 Â 10À6
M
in MeOH) was mixed with various sugars solution in 0.1 M phos-
phate buffer (pH 7.4) with increasing concentrations at equal vol-
umes. The mixture was allowed to mix for 20 min and
fluorescence intensity was recorded with a Shimadzu RF-5301PC
fluorometer. The fluorescent intensity readings were normalized
with the sensor solution only and fitted to non-linear regression
NBoc
NBoc
NBoc
b
a
OH
N3
NH2
5
7
6
NBoc
NBoc
d
c
N
H
N
R
H
O
O
8a-c
Br
(HO)2B
10 =
B(OH)2
B
O
O
N
N
N
H
R
N
H
O
O
N
R=
N
N
9a-c
c
a
b
Scheme 1. Synthesis of bis-anthracene boronic acid derivatives. (a) DMF, PPh3, CCl4, NaN3, RT; 90%; (b) (aq.) THF, PPh3, RT; 85%; (c) CH2Cl2, DMF, EDCI, HOBt, TEA,
HOOCRCOOH, 0 °C ? RT; 50–80%; (d) i. TFA, CH2Cl2, ii. K2CO3, cat. KI, CH3CN, 10, iii. 10%NaHCO3, CH2Cl2, H2O, RT; 15–30%.

140
S. Craig / Bioorganic Chemistry 40 (2012) 137–142
sium sulfate, and concentrated. The residue was purified by flash
chromatography with ethyl acetate/hexanes (15:85) to produce a
yellow oil, (277 mg, 90% yield).
1H NMR (CDCl3, 400 MHz) d: 8.52–8.36 (m, 4H), 7.64–7.59 (m,
4H), 5.57 (s, 2H), 5.37 (s, 2H), 2.51 (s, 3H), 1.59 (s, 9H); 13C NMR
(CDCl3, 100 MHz): 155.8, 131.0, 130.4, 127.1, 126.5, 126.1, 126.0,
125.1, 124.3, 79.9, 46.5, 42.7, 31.8, 28.5; ESI MS: [M+(Na)] calcu-
lated 400.2, found 400.1.
2.3.2. Preparation of (10-Aminomethyl-anthracen-9-ylmethyl)-
methyl-carbamic acid tert-butyl ester (7)
Compound 6 (154 mg, 0.410 mmol) and triphenylphosphine
(268 mg, 1.02 mmol) in aqueous THF (1:100) was stirred at RT
for 16 h. The solution was then concentrated and purified by
means of flash chromatography with CH2Cl2/MeOH (90:10) to give
122 mg of a yellow solid, 85% yield. 1H NMR (CDCl3, 400 MHz) d:
8.45–8.38 (m, 4H), 7.57–7.52 (m, 4H), 5.50 (s, 2H), 4.83 (s, 2H),
2.47 (s, 3H), 1.55 (s, 9H); 13C NMR (CDCl3): 155.8, 135.8, 131.2,
129.0, 128.5 125.8, 125.7, 125.1, 124.5, 79.7, 42.5, 38.4, 31.7,
28.5; MS(EI) calculated 350, found 350.
2.3.3. General procedure for preparation of Boc-protected diamides (8)
The di-acid (0.543 mmol, 1 equivalent), N-hydroxybenzotria-
zole (HOBt, 1.9 mmol, 1.47 mg), 1-(2-dimethylaminopropyl)-
3-ethylcarbodiimide (EDCI, 1.07 mmol, 213 mg), and then compound
7 (1.14 mmol, 400 mg) was added to round bottom flask, followed by
the addition of 30 ml of dry CH2Cl2. The solution was allowed to
mix for 30 min at 0 °C, then triethylamine (TEA) was added to ob-
tain a slight basic solution. Then the reaction temperature was
slowly raised to room temperature and allowed to stir for 18 h.
The reaction mixture was washed with 5% sodium bicarbonate
(10 ml), 5% citric acid (10 ml), and brine (10 ml). The organic layer
was dried over anhydrous magnesium sulfate, gravity filtered, and
concentrated. The crude product was purified by flash chromatog-
raphy with CH2Cl2/MeOH or precipitation from CH2Cl2/hexanes.
[10-({[4-({10-[(tert-Butoxycarbonyl-methyl-amino)-methyl]-ant
hracen-9-ylmethyl}-methyl-carbamoyl)-benzoyl]-aminomethyl)-an
thracen-9-ylmethyl]-methyl-carbamic acid tert-butyl ester (8a). 80%
yield. 1H NMR (CDCl3, 400 MHz) d: 8.49–8.39 (m, 8H), 7.70–7.59 (m,
8H), 7.70–7.69 (s, 12H), 5.64 (s, 4H), 5.54 (s, 4H), 2.49 (s, 6H), 1.57
(s, 18H).
[10-({[4-({10-[(tert-Butoxycarbonyl-methyl-amino)-methyl]-anth
racen-9-ylmethyl}-methyl-carbamoyl)-pyridine-2-carbonyl]-
anthracen-9-ylmethyl]-methyl-carbamic acid tert-butyl ester (8b).
60% yield. 1H NMR (CDCl3, 400 MHz) d: 8.68 (s, 1H), 8.42–8.40 (m,
2H), 8.35–8.31 (m,6H), 8.12 (s, 1H), 8.05 (s, 1H), 7.53–7.44 (m, 8H),
5.58 (d, J = 4.4 Hz, 2H), 5.45 (s, 4H), 5.31 (s, 2H), 2.42 (s, 3H), 2.36 (s,
6H), 1.51 (s, 18H).
[10-({[4-({10-[(tert-Butoxycarbonyl-methyl-amino)-methyl]-ant
hracen-9-ylmethyl}-methyl-carbamoyl)-pyrazine-2-carbonyl]-ami-
nomethyl)-anthracen-9-ylmethyl]-methyl-carbamic acid tert-butyl
ester (8c). 50% yield 1H NMR (CDCl3, 400 MHz) d: 9.22–9.20 (m,
1H), 8.51–8.40 (m, 8H), 8.05–8.04 (m, 1H), 7.2–7.56 (m, 8H), 5.67 (s,
4H), 5.54 (s, 4H), 2.52 (s, 6H), 1.66 (s, 18H).
Fig. 4. (a) Fluorescence intensity changes (I/I0) of 9c as
a function of sugar
concentration at room temperature: 1.0 Â 10À6 M in 50% MeOH/0.1 M aqueous
phosphate buffer at pH 7.4: kex = 370 nm, kem = 423 nm.
was evaporated in vacuo. The resulting residue was dissolved in
CH2Cl2, 20 ml of 10% sodium bicarbonate, and 8 ml of water for
the removal of protecting group of the boronate motif. The mixture
was stirred for 4 h. The organic phase was washed with brine and
dried over anhydrous magnesium sulfate. The solvent was re-
moved under reduced pressure. The crude material was precipi-
tated from THF/hexanes.
Diboronic acid (9a). 28% yield. 1H NMR (CD3OD + CDCl3,
400 MHz) d: 8.56–8.54 (m, 4H), 8.37 (m, 4H), 7.62–7.56 (m, 12H),
7.38–7.31 (m, 8H), 5.38 (s, 4H), 5.03 (s, 4H), 4.37 (s, 4H), 2.42 (s,
6H); HRMS(+H/D)[ÀH2O] calculated 882.4124, found 882.4105.
Diboronic acid (9b). 25% yield. 1H NMR (CD3OD, 400 MHz) d:
8.71–8.70 (m, 1H), 8.51–8.49 (m, 4H), 8.46–8.44 (m, 4H), 8.24–
8.19 (m, 1H), 7.69–7.66 (m, 2H), 7.57–7.51 (m, 9H), 7.37–7.32
(m, 4H), 7.29–7.26 (m, 2H), 5.59 (s, 1H), 5.54 (s, 1H), 5.49 (s, 2H),
5.00 (s, 4H), 4.31 (s, 4H), 2.38 (s, 6H). HRMS(+H)[ÀH2O] calculated
882.3997, found 882.4001.
2.3.4. General procedure for preparation of diboronic acids (9)
Deprotection of the amine moiety of diamide 8 was accom-
plished by dissolving it in dry CH2Cl2 (15 ml) followed by trifluoro-
acetic acid addition and stirring at room temperature 15 min. After
removal of Boc-protected group, the residue was concentrated and
dried in vacuo for 3 h. The reaction mixture was then subsequently
placed in a round bottom flask. Then dry acetonitrile (35 ml),
potassium carbonate (2.2 mmol, 305 mg), catalytic amount of
potassium iodide, and compound 10 (0.88 mmol, 251 mg) were
added to the same flask. The reaction mixture was allowed to stir
for 18 h. The insoluble materials were filtered, and the filtrate
Table 1
Binding constants for the complex of sensor and saccharide.
Sensors
Ka (MÀ1) fructose
Ka (MÀ1) glucose
Ka (MÀ1) sorbitol
9a
9b
9c
212
266
504
28
1
2
__a
__a
1051
Ka values were obtained using a non-linear regression curve fitting with the soft-
ware GraphPad Prism 4.0.
a
Binding constant not determined.

S. Craig / Bioorganic Chemistry 40 (2012) 137–142
141
Fig. 5. Fluorescent labeling studies of a liver carcinoma cell line HepG2 and a normal fibroblast mammalian cell line COS-7 with compounds 9a–c. The negative control
contains buffer only.
Diboronic acid (9c). 20% yield. 1H NMR (CD3OD, 400 MHz) d: 9.09
(s, 2H), 8.53–8.51 (m, 4H), 8.25–8.23 (m, 4H), 7.71–7.69 (m, 2H),
7.60–7.52 (m, 8H), 7.37–7.33 (m, 4H), 7.30–7.28 (m, 2H), 5.60 (s,
4H), 5.49 (s, 4H), 4.29 (s, 4H), 2.37 (s, 6H). HRMS(+H)[ÀH2O] calcu-
lated 883.3951, found 883.3978.
for the binding of monoboronic acids [10], one could possibly state
that 9c displays a two-binding site model between the interactions
of hydroxyl groups of the saccharide (substrate) and the bisboronic
acid units (receptor).
3.3. Evaluation of fluorescent labeling studies
3. Results and discussion
To explore the capability of the bisboronic acids labelling carci-
noma cell lines, we studied their ability to stain HepG2, liver carci-
noma cells as oppose to a normal fibroblast cell line, COS-7 [44].
Briefly, cells were cultured in six-well plates with 1 Â 106 M per well
and incubated at 37 °C in 5% CO2 for 48 h. The media was then re-
moved and cells were washed with PBS. The cells were fixed with
methanol/PBS. After fixation, cells were washed twice with PBS.
3.1. Synthesis of artificial receptors
The preparation of a dianthracene boronic acid for the develop-
ment of a fluorescent probe for biological saccharides begin with 5,
which was prepared from a previous published paper [44]. The hy-
droxyl moiety of 5 was converted to an azide to give 6 in 90% yield
using a mild Mitsunobu type reaction [47]. The reduction of the
azide was achieved by the addition of triphenylphosphine in aque-
ous THF to generate amine 7 in 81% yield. The amidation reaction
of 7 with various di-acids was performed by treatment with 1-(2-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI)
along with N-hydroxybenzotriazole (HOBt) to afford compounds
8a–c in 50–60% yield. After deprotection of derivatives 7 with tri-
fluoroacetic acid (TFA), the unprotected free amines were then re-
acted with aryl boronic ester 10 [41] in the presence of potassium
carbonate with a catalytic amount of potassium iodide. Then
deprotection of the boronate produced compounds (9a–c,
Scheme 1) in yields of 15–30%.
The bis-anthracene boronic acids at 0.5–10 lM were added to each
well that contained 1 ml of 1:1 MeOH/PBS, and incubated for
45 min at 4 °C. The staining of the fluorescent probes was observed
using a fluorescent microscope with a blue optical filter. Images
were shown as overlay images of phase contrast and fluorescent
microscopy. In this way, the non-labeled cells appear as a gray scale
image and labeled cells are blue1 in color. The staining results are
shown in Fig. 5.
Bisboronic acids 9a and 9b stained the HEPG2 cell line at similar
concentration, 1 lM. However, 9a as well stained COS-7 cell line,
diminishing the selectivity of the lead compound 4 toward hepato-
cellular carcinoma line versus normal fibroblast cells. The pyrazine
compound 9c at concentrations between 0.5 and 10 lM showed
weak or no binding affinity for either cell line.
3.2. Determination of binding constants
4. Conclusion
To validate the complexation of the synthesized boronic acids
and diol, various biological sugars were used to obtain the apparent
bindingconstant. Briefly, 2 mlof sensorsolutioninMeOH wasmixed
with 2 ml of aqueous phosphate buffer solution containing saccha-
ride of interest with increasing concentration. Next, the fluorescent
intensity was obtained, and normalized by sensor solution only. As
shown in Fig. 4, there is a fluorescent intensity increase with increas-
ing concentration of carbohydrate. The apparent binding constants
are displayed in Table 1, as shown there is a low binding affinity
for glucose with each di-boronic acid. However there is a higher
binding affinity for sugars that bind to boronic acids in a trivalent
fashion, such as fructose and sorbitol [48]. The binding constants
for fructose are 9c (504 MÀ1) > 9b (266 MÀ1) > 9a (212 MÀ1) respec-
tively. Compound 9c showed the highest binding affinity for fruc-
tose; therefore sorbitol was tested with this compound only,
showing a binding affinity of 1051 MÀ1. Such results show a pattern
Three dianthracene diboronic acids were synthesized and eval-
uated for the labeling of liver carcinoma cell line, HepG2 as oppose
to a normal mammalian fibroblast cell line, COS-7. Compound 9b
showed similar staining concentration compared to model com-
pound 4. However 9a, the removal of the methyl group from the
amino group attached to the linker, seemed to diminish the selec-
tivity. Compound 9c showed weak or no selectivity for either cell
line. One could speculate that there are physiochemical parameters
governing non-labeling for either cell line of compound 9c. How-
ever it is beyond the scope of this article, additional studies are
underway.
1
For interpretation of color in Fig. 5, the reader is referred to the web version of
this article.

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S. Craig / Bioorganic Chemistry 40 (2012) 137–142
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Acknowledgments
We gratefully acknowledge the financial support in our lab by
the National Institutes of Health (NO1-CO-27184, CA88343, and
CA113917), the Georgia Cancer Coalition through a Distinguished
Cancer Scientist Award, and the Georgia Research Alliance through
an Eminent Scholar endowment and a Challenge grant. We also we
would like to give special thanks to Shafiq Khan and lab members
at Clark Atlanta University for the use of his cell culture lab. Dr.
Binghe Wang at Georgia State University, Dr. Irene Lee at Case
Western Reserve University for the helpful suggestions and critiqu-
ing of this manuscript, and GAANN fellowship through Georgia
State University.
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