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Addressing the Biochemical Foundations of a Glucose-Based “Trojan
Horse”-Strategy to Boron Neutron Capture Therapy: From Chemical
Synthesis to In Vitro Assessment
Jelena Matović
,
ϕ
Juulia Järvinen,
ϕ
Helena C. Bland, Iris K. Sokka, Surachet Imlimthan,
Ruth Mateu Ferrando, Kristiina M. Huttunen, Juri Timonen, Sirpa Peräniemi, Olli Aitio,
Anu J. Airaksinen, Mirkka Sarparanta, Mikael P. Johansson, Jarkko Rautio, and Filip S. Ekholm*
Cite This: Mol. Pharmaceutics 2020, 17, 3885−3899
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sıSupporting Information
ABSTRACT: Boron neutron capture therapy (BNCT) for cancer is on the rise
worldwide due to recent developments of in-hospital neutron accelerators which are
expected to revolutionize patient treatments. There is an urgent need for improved
boron delivery agents, and herein we have focused on studying the biochemical
foundations upon which a successful GLUT1-targeting strategy to BNCT could be
based. By combining synthesis and molecular modeling with affinity and cytotoxicity
studies, we unravel the mechanisms behind the considerable potential of appropriately
designed glucoconjugates as boron delivery agents for BNCT. In addition to addressing
the biochemical premises of the approach in detail, we report on a hit glucoconjugate
which displays good cytocompatibility, aqueous solubility, high transporter affinity, and,
crucially, an exceptional boron delivery capacity in the in vitro assessment thereby
pointing toward the significant potential embedded in this approach.
KEYWORDS: boron neutron capture therapy, cancer therapeutics, carbohydrates, glucose transporters, medicinal chemistry, drug delivery
1. INTRODUCTION
As one of the leading causes of morbidity and mortality on a
global scale, cancer is a significant societal economic burden
with annual global costs of 723−930 billion euros. Head and
neck cancers account for up to 10% of all cancers with 630 000
new cases annually diagnosed worldwide.
1,2
Despite traditional
treatments featuring surgery, radiation, and chemotherapy, all
of which are arduous for the patients, many of these cancers
recur. In head and neck cancers, the inoperable recurrent ones
are accompanied by a poor survival rate with a mean survival
time of only a few months.
3
A number of novel treatment
strategies have recently gained ground. These include anti-
body−drug conjugates,
4
proton therapy,
5,6
and, especially,
boron neutron capture therapy (BNCT).
7
BNCT represents
one of the most promising noninvasive binary treatment
modalities for head and neck cancers since it can eradicate
cancer cells while simultaneously sparing healthy cells (the
basis of our approach is displayed in Figure 1).
8,9
The
selectivity in BNCT arises from a 2-fold effect. First, only cells
with a sufficient concentration of 10B atoms are destroyed and,
second, the external neutron beam can be applied to a narrow
and highly specific area where malignant cells are present.
Previously, the applications of, and interest in, clinical BNCT
have been hampered by the need for nuclear reactors, as a
neutron source, and the poor properties of clinically used
boron delivery agents. In recent years, new in-hospital neutron
accelerators
10
have emerged thus revolutionizing the clinical
aspects of patient treatments; a renewed interest in the BNCT
Received: June 13, 2020
Revised: August 5, 2020
Accepted: August 11, 2020
Published: August 11, 2020
Figure 1. Principles of our approach to BNCT. Blue dots represent
boron atoms while gray dots represent carbon atoms in the ortho-
carboranylmethyl moiety.
Article
pubs.acs.org/molecularpharmaceutics
© 2020 American Chemical Society 3885
https://dx.doi.org/10.1021/acs.molpharmaceut.0c00630
Mol. Pharmaceutics 2020, 17, 3885−3899
This is an open access article published under a Creative Commons Attribution (CC-BY)
License, which permits unrestricted use, distribution and reproduction in any medium,
provided the author and source are cited.
field has been invokednow, the final challenge to solve is
that of developing improved boron delivery agents.
An optimally functioning delivery agent for BNCT should
display a minimal systemic toxicity, a cellular uptake of 20−35
μg/g of tumor (i.e., ppm range), and tumor/normal tissue (T/
N) and tumor/blood (T/B) ratios above 3:1, with higher
ratios naturally desirable. Combining these different aspects
into one single delivery agent has proved challenging. Despite
the large number of delivery agents (amino acids, carbohy-
drates, porphyrins, antibody−boron conjugates, polymers,
peptides, liposomes, and nanoparticles) evaluated in the
literature,
11−13
only three are in clinical use. These are sodium
borocaptate (BSH), boronophenylalanine (BPA and its
fructose-complex) and decahydrodecaborate (GB-10). None
of them exhibit optimal properties. BPA has poor water
solubility, contains only one boron atom/delivered molecule,
and gives poor T/B- and T/N-ratios.
14,15
BSH
16
and GB-10
17
lack active targeting and uptake mechanisms and have an ionic
nature which may cause undesired interactions with other
biomolecules in a biological context.
The intrinsic properties of carbohydrates, i.e., high aqueous
solubility, low systemic toxicity, and high biocompatibility
make them seemingly ideal candidates for clinical BNCT.
Polysaccharide and oligosaccharide carriers are, however,
suboptimal from a BNCT-perspective: polysaccharides are
constrained to the extracellular matrix while oligosaccharides
display low lectin-binding affinities. Thus, we have chosen to
focus on monosaccharides in combination with carbohydrate
transporters, glucose transporters (GLUTs and SGLTs) in
particular.
Glucose is an essential nutrient for mammalian cells. An
increased expression of GLUTs and SGLTs, especially
GLUT1, has been observed in head and neck cancers.
18
The
basis for this increase is the switch in glucose metabolism
which in cancer cells proceeds by an inefficient aerobic
glycolysis route in contrast to the oxidative metabolism in
healthy noncancerous cells.
19
This inefficient metabolic
pathway leads to a substantial increase in glucose uptake
which allows the cancer cells to grow rapidly and proliferate.
20
Exploiting this “Warburg effect”, named after Nobel laureate
Otto Heinrich Warburg, provides the foundation for the
development of novel glucose-based “Trojan horses”for
clinical BNCT. Before reaching the end stages of the
development process (in vivo-studies with/without neutron
sources), the biochemical foundations of the approach need to
be addressed in detail. In this regard, it is important to note
that concerns have been raised regarding the effects of
glucoconjugates on glucose metabolism in healthy cells, the
possible incorporation of metabolic products into other
biomolecules, and the competition for the transporters with
the high glucose levels found in blood.
21
Therefore, for a
GLUT1 targeting approach to be successful, it is crucial to
address these issues already at the design stage.
To this end, we have designed and synthesized three
glucoconjugates bearing an ortho-carboranylmethyl substituent.
The carboranyl provides ten boron nuclei per delivery
molecule in a charge-neutral, chemically stable form, and is
thus highly suitable for the purposes of BNCT. In addition, a
neutral and hydrophobic boron cluster should be advantageous
when aiming for transport through a transmembrane protein
since possible unfavorable interactions between charged boron
clusters and amino acids can be avoided.
22
Figure 2 shows the
three 6-O-carboranylmethyl glucoconjugates targeted: the
hemiacetal and both methyl glycosides. The attachment of
boron clusters at the sixth position in glucose is rare in the
scientific literature, and conjugates with charge-neutral boron
clusters have not been prepared earlier.
23,24
A modification at
this site will, however, remove the concerns regarding
interference with glucose metabolism and incorporation into
other biomolecules through the glycolysis route; the 6-O-
carboranylmethyl glucoconjugates are no longer substrates for
the glycolysis route in which the first transformation is a
phosphorylation at the sixth position.
25
In addition to synthesizing the glucoconjugates and
conducting the most detailed structural characterization of
such conjugates to date, we have addressed the biochemical
foundations of the GLUT1-targeting approach through a
preliminary, yet, comprehensive in vitro evaluation study
featuring cytotoxicity, computational/experimental receptor
affinity, and cellular uptake experiments in the relevant
human head and neck cancer cell line CAL 27 (oral
adenosquamous carcinoma cell line). To our satisfaction, the
new glucoconjugates display a significantly stronger binding
affinity to GLUT1 than glucose. This shows that the previous
fear regarding their competition with the high levels of free
glucose in blood has been unfounded. Moreover, the
glucoconjugates display a boron delivery capacity 40 times
higher than the best agents currently in clinical useshowing
that there is considerable potential embedded in this
alternative approach.
2. EXPERIMENTAL SECTION
2.1. Synthesis and Structural Characterization. Re-
action solvents were purified by the VAC vacuum solvent
purification system prior to use when dry solvents were
needed. All reactions containing moisture- or air-sensitive
reagents were carried out under an argon atmosphere. All
reagents were purchased from commercial sources. The NMR
spectra were recorded with a Bruker Avance III NMR
spectrometer operating at 500.13 MHz (1H: 500.13 MHz,
Figure 2. Two of the delivery agents in clinical use (left box) and the 6-O-ortho-carboranylmethyl glucoconjugates prepared in the current study
(right box). Blue dots represent boron atoms while gray dots represent carbon atoms in the carboranyl moiety.
Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics Article
https://dx.doi.org/10.1021/acs.molpharmaceut.0c00630
Mol. Pharmaceutics 2020, 17, 3885−3899
3886
13C: 125.76 MHz, 11B: 160.46 MHz). The probe temperature
during the experiments was kept at 23 °C. All products were
characterized by utilization of the following 1D-techniques:
1H, 13C, 11B, and 1D-TOCSY and the following 2D-
techniques: Ed-HSQC, HMBC, and COSY by using pulse
sequences provided by the instrument manufacturer. Chemical
shifts are expressed on the δscale (in ppm) using TMS
(tetramethylsilane), residual chloroform, methanol, or 15%
BF3in CDCl3(11B NMR) as internal standards. Coupling
constants have been obtained through spectral simulations
with the Perch Peak Research software, are given in Hz, and
are provided only once, when first encountered. Coupling
patterns are given as s (singlet), d (doublet), t (triplet), etc.
HRMS were recorded using Bruker Micro Q-TOF with ESI
(electrospray ionization) operated in positive mode. The
purity of the compounds was determined to be >95% in all
cases. TLC was performed on aluminum sheets precoated with
silica gel 60 F254 (Merck). Flash chromatography was carried
out on silica gel 40. Spots were visualized by UV, followed by
spraying the TLC plates with a solution of H2SO4:MeOH
(1:4) and heating.
General Experimental Procedures. General Procedure for
Selective Silylation of the 6-OH Group in Glucopyranose.
tert-Butyldimethylsilyl chloride (1.35 equiv) was added
portion-wise to a solution of D-glucose (1 equiv) in pyridine
(10 mL/g of starting material) at 0 °C. The mixture was
brought to rt and stirred for 21 h. The solvent was removed in
vacuo, and the crude product was purified by column
chromatography (DCM:MeOH 7:1), the solvents were
removed, and the corresponding silylated glycopyranose was
dried on the vacuum line.
General Procedure for Selective Silylation of the 6-OH
Group in Glucopyranosides. The corresponding methyl D-
glucopyranoside (1 equiv) was dissolved in dry DMF (10 mL/
g of starting material) at 0 °C under an atmosphere of argon.
Imidazole (1.5 equiv) and the tert-butyldimethylsilyl chloride
(1.35 equiv) were added, and the reaction mixture was brought
to rt. The mixture was stirred o/n. The reaction was quenched
by the addition of MeOH (0.25 mL/g of starting material) and
concentrated in vacuo. The crude product was purified by
column chromatography (DCM:MeOH 7:1), the solvents
were removed, and the corresponding silylated glycoside was
dried on the vacuum line.
General Procedure for Alkylation of Free Hydroxyl
Groups. The partially protected glucoside/glucopyranose (1
equiv) was dissolved in dry DMF (2 mL/100 mg of starting
material) under an atmosphere of argon. The solution was
cooled on an ice bath, and NaH (1.9 equiv) was added. The
reaction mixture was stirred for 15 min and then brought to rt
and stirred for a further 10 min. The corresponding bromide
(1.5 equiv/free OH-group) was added, and the resulting
mixture was stirred for 1−4 h, quenched with MeOH (0.4 mL/
mmol of starting material), diluted with DCM (4 mL/100
mg), and washed with a satd. NaHCO3-solution. The organic
phase was separated, and the aqueous phase was extracted with
DCM (3 ×3 mL/100 mg). The organic phases were
combined and washed with brine (3 mL/100 mg), dried
over Na2SO4,filtered, and concentrated. The crude product
was purified by column chromatography (EtOAc:hexane 1:8),
the solvents were removed, and the corresponding alkylated
glycoside was dried on the vacuum line.
General Procedure for Deprotection of Silyl Protective
Groups. To a solution containing the protected glycoside (1
equiv) in dry THF (3 mL/200 mg of starting material) at 0 °C,
HF-pyridine (18 μL/0.03 mmol of starting material) was
added. The resulting mixture was brought to rt and stirred for
20 h. The reaction mixture was diluted with DCM (30 mL/0.5
g of starting material) and quenched by the addition of a satd.
NaHCO3-solution (20 mL/200 mg of starting material). The
aqueous phase was extracted with DCM (3 ×20 mL), and the
organic phases were combined and washed with brine (20 mL/
500 mg of starting material). The combined organic phase was
dried over Na2SO4,filtered, and concentrated. The crude
product was purified by column chromatography (hexane:E-
tOAc 2:1), the solvents were removed, and the corresponding
deprotected glycoside was dried on the vacuum line.
General Procedure for Coupling Reaction with Decabor-
ane. B10H14 (1.8 equiv) in dry ACN (5 mL/150 mg) under
argon was heated to 60 °C and stirred for 1 h. Meanwhile, the
propargylated glycoside (1 equiv) was dissolved in dry toluene
(5 mL/150 mg) and added after the first hour. The reaction
mixture was stirred for 15−18 h at 80 °C. The mixture was
quenched by the addition of dry methanol (1.8 mL/200 mg
starting material) and allowed to stir for 30 min at 80 °C. The
solvent was removed, and the crude product was purified by
column chromatography (EtOAc:hexane 1:3), the solvents
were removed, and the corresponding carboranyl glycoside was
dried on the vacuum line.
General Procedure for Deprotection of Benzyl Groups.
The corresponding protected glucoside was dissolved in
EtOAc:MeOH 7:1 (1 mL/10 mg of starting material). Pd/C
(10% Pd, 1 weight equiv) was added, and the reaction mixture
was stirred in an autoclave under H2(3−5 bar) for 4−6 h. The
resulting mixture was filtered through Celite, washed with
EtOAc:MeOH 7:1 (3 ×10 mL), and concentrated under
vacuum. The crude product was purified by column
chromatography (DCM:MeOH 5:1), the solvents were
removed, and the product was dried on the vacuum line to
give the corresponding deprotected glucoside/glucopyranose.
Substrate Specific Analytical Data. 6-O-(tert-Butyldime-
thylsilyl)-D-glucopyranose. Synthesized from D-glucose (9.99
g/55.5 mmol), according to the general procedure for selective
silylation of the 6-OH group in glucopyranoses. This reaction
yielded an off-white powder (10.64 g, 69%; α:β58:42). Rf=
0.61 (DCM:MeOH 5:1).
1H NMR of the α-anomer (500.13 MHz; CD3OD): δ5.08
(d, 1H, J1,2 = 3.7 Hz, H-1), 3.85 (dd, 1H, J6a,5 = 2.1, J6a,6b =
−11.2 Hz, H-6a), 3.84 (dd, 1H, J6b,5 = 4.6 Hz, H-6b), 3.75
(ddd, 1H, J5,4 = 9.8 Hz, H-5), 3.67 (dd, 1H, J3,4 = 9.1, J3,2 = 9.6
Hz, H-3), 3.35 (dd, 1H, H-4), 3.33 (dd, 1H, H-2), 0.90 (s, 9H,
6-OSi(CH3)2C(CH3)3) and 0.08 and 0.07 (each s, each 3H, 6-
OSi(CH3)2C(CH3)3) ppm.
13C NMR of the α-anomer (125.76 MHz; CD3OD): δ93.9
(C-1), 74.9 (C-3), 73.8 (C-2), 73.2 (C-5), 71.6 (C-4), 64.1
(C-6), 26.4 (6-OSi(CH3)2C(CH3)3), 19.3 (6-OSi-
(CH3)2C(CH3)3) and −5.0 and −5.1 (6-OSi(CH3)2C(CH3)3)
ppm.
1H NMR of the β-anomer (500.13 MHz; CD3OD): δ4.44
(d, 1H, J1,2 = 7.8 Hz, H-1), 3.94 (dd, 1H, J6a,5 = 2.0, J6a,6b =
−11.2 Hz, H-6a), 3.78 (dd, 1H, J6b,5 = 5.4 Hz, H-6b), 3.33 (dd,
1H, J3,4 = 8.9, J3,2 = 9.8 Hz, H-3), 3.30 (dd, 1H, J4,5 = 9.4 Hz,
H-4), 3.26 (ddd, 1H, H-5), 3.11 (dd, 1H, H-2), 0.90 (s, 9H, 6-
OSi(CH3)2C(CH3)3) and 0.08 and 0.07 (each s, each 3H, 6-
OSi(CH3)2C(CH3)3) ppm.
13C NMR of the β-anomer (125.76 MHz; CD3OD): δ98.1
(C-1), 78.2 (C-3, C-5), 76.2 (C-2), 71.5 (C-4), 64.3 (C-6),
Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics Article
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3887
26.4 (6-OSi(CH3)2C(CH3)3), 19.3 (6-OSi(CH3)2C(CH3)3)
and −5.1 (6-OSi(CH3)2C(CH3)3) ppm.
HRMS: m/zcalcd. for C12H26O6SiNa [M + Na]+317.1397;
found 317.1385.
1,2,3,4-Tetra-O-benzyl-6-O-(tert-butyldimethylsilyl)-D-glu-
copyranose (4). Synthesized from 6-O-(tert-butyldimethylsil-
yl)-D-glucopyranose (0.95 g, 3.2 mmol), NaH (0.90 g, 37.4
mmol), and BnBr (4.90 g, 28.6 mmol) according to the general
procedure for alkylation of free hydroxyl groups to give a white
solid (1.85 g, 88%; α:β31:69). TLC: Rf: 0.39 (EtOAc:Hex
1:8).
1H NMR of the α-anomer (500.13 MHz; CDCl3): δ7.42−
7.23 (m, 20H, arom. H), 5.00 and 4.84 (each d, each 1H, J=
−10.6 Hz, 3-OCH2Ph), 4.89 and 4.66 (each d, each 1H, J=
−11.0 Hz, 4-OCH2Ph), 4.83 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.69
and 4.56 (each d, each 1H, J=−11.9 Hz, 1-OCH2Ph), 4.66
and 4.58 (each d, each 1H, J=−11.7 Hz, 2-OCH2Ph), 4.06
(dd, 1H, J3,4 = 9.1, J3,2 = 9.8 Hz, H-3), 3.78 (dd, 1H, J6a,5 = 4.6,
J6a,6b =−11.4 Hz, H-6a), 3.74 (dd, 1H, J6b,5 = 1.7 Hz, H-6b),
3.71 (ddd, 1H, J5,4 = 10.0 Hz, H-5), 3.56 (dd, 1H, H-4), 3.52
(dd, 1H, H-2), 0.90 (s, 9H, 6-OSi(CH3)2C(CH3)3) and 0.06
and 0.05 (each s, each 3H, 6-OSi(CH3)2C(CH3)3) ppm.
13C NMR of the α-anomer (125.76 MHz; CDCl3): δ139.0−
127.7 (arom. C), 95.2 (C-1), 82.3 (C-3), 80.5 (C-2), 78.0 (C-
4), 76.0 (3-OCH2Ph), 75.2 (4-OCH2Ph), 73.1 (2-OCH2Ph),
71.9 (C-5), 68.8 (1-OCH2Ph), 62.3 (C-6), 26.1 (6-OSi-
(CH3)2C(CH3)3), 18.5 (6-OSi(CH3)2C(CH3)3) and −5.0 and
−5.2 (6-OSi(CH3)2C(CH3)3) ppm.
1H NMR of the β-anomer (500.13 MHz; CDCl3): δ7.42−
7.23 (m, 20H, arom. H), 4.97 and 4.73 (each d, each 1H, J=
−10.8 Hz, 2-OCH2Ph), 4.94 and 4.67 (each d, each 1H, J=
−11.6 Hz, 1-OCH2Ph), 4.92 and 4.81 (each d, each 1H, J=
−10.8 Hz, 3-OCH2Ph), 4.86 and 4.69 (each d, each 1H, J=
−10.8 Hz, 4-OCH2Ph), 4.51 (d, 1H, J1,2 = 7.8 Hz, H-1), 3.90
(dd, 1H, J6a,5 = 1.7, J6a,6b =−11.4 Hz, H-6a), 3.86 (dd, 1H, J6b,5
= 4.4 Hz, H-6b), 3.65 (dd, 1H, J3,2 = 8.9, J3,4 = 9.0 Hz, H-3),
3.63 (dd, 1H, J4,5 = 9.5 Hz, H-4), 3.49 (dd, 1H, H-2), 3.30
(ddd, 1H, H-5), 0.93 (s, 9H, 6-OSi(CH3)2C(CH3)3) and 0.12
and 0.10 (each s, each 3H, 6-OSi(CH3)2C(CH3)3) ppm.
13C NMR of the β-anomer (125.76 MHz; CDCl3): δ139.0−
127.7 (arom. C), 102.4 (C-1), 84.9 (C-3), 82.7 (C-2), 77.8
(C-4), 76.0 (C-5, 3-OCH2Ph), 75.2 (4-OCH2Ph), 75.1 (2-
OCH2Ph), 71.0 (1-OCH2Ph), 62.4 (C-6), 26.1 (6-OSi-
(CH3)2C(CH3)3), 18.5 (6-OSi(CH3)2C(CH3)3) and −4.8
and −5.2 (6-OSi(CH3)2C(CH3)3) ppm.
HRMS: m/zcalcd. for C40H50O6SiNa [M + Na]+677.3275;
found 677.3300.
1,2,3,4-Tetra-O-benzyl-D-glucopyranose. Synthesized from
1,2,3,4-tetra-O-benzyl-6-O-(tert-butyldimethylsilyl)-D-gluco-
pyranose (1.73 g, 2.6 mmol) and HF-pyridine (1.6 mL, 17.8
mmol) according to the general procedure for deprotection of
silyl protective groups to give a white solid (1.42 g, 99%; α:β
33:67). TLC: Rf: 0.8 (EtOAc:Hex 1:1).
1H NMR of the α-anomer (500.13 MHz; CDCl3): δ7.41−
7.25 (m, 20H, arom. H), 5.01 and 4.84 (each d, each 1H, J=
−10.8 Hz, 3-OCH2Ph), 4.89 and 4.64 (each d, each 1H, J=
−11.0 Hz, 4-OCH2Ph), 4.80 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.68
and 4.56 (each d, each 1H, J=−11.9 Hz, 2-OCH2Ph), 4.68
and 4.55 (each d, each 1H, J=−12.4 Hz, 1-OCH2Ph), 4.06
(dd, 1H, J3,4 = 9.2, J3,2 = 9.5 Hz, H-3), 3.71 (ddd, 1H, J5,6a =
2.7, J5,6b = 2.8, J5,4 = 9.5 Hz, H-5), 3.70 (ddd, 1H, J6a,6‑OH = 5.7,
J6a,6b =−11.1 Hz, H-6a), 3.68 (ddd, 1H, J6a,6‑OH = 7.0 Hz, H-
6b), 3.54 (dd, 1H, H-4), 3.50 (dd, 1H, H-2) and 1.57 (dd, 1H,
6-OH) ppm.
13C NMR of the α-anomer (125.76 MHz; CDCl3): δ139.0−
127.7 (arom. C), 95.7 (C-1), 82.1 (C-3), 80.2 (C-2), 77.6 (C-
4), 75.9 (3-OCH2Ph), 75.2 (4-OCH2Ph), 73.2 (2-OCH2Ph),
71.1 (C-5), 69.4 (1-OCH2Ph) and 62.0 (C-6) ppm.
1H NMR of the β-anomer (500.13 MHz; CDCl3): δ7.41−
7.25 (m, 20H, arom. H), 4.95 and 4.73 (each d, each 1H, J=
−10.9 Hz, 2-OCH2Ph), 4.93 and 4.81 (each d, each 1H, J=
−10.9 Hz, 3-OCH2Ph), 4.92 and 4.69 (each d, each 1H, J=
−11.9 Hz, 1-OCH2Ph), 4.86 and 4.64 (each d, each 1H, J=
−11.0 Hz, 4-OCH2Ph), 4.57 (d, 1H, J1,2 = 7.9 Hz, H-1), 3.87
(ddd, 1H, J6a,5 = 2.9, J6a,6‑OH = 5.8, J6a,6b =−11.5 Hz, H-6a),
3.70 (ddd, 1H, J6b,5 = 4.9, J6b,6‑OH = 7.7 Hz, H-6b), 3.67 (dd,
1H, J3,2 = 9.2, J3,4 = 9.2 Hz, H-3), 3.57 (dd, 1H, J4,5 = 9.7 Hz,
H-4), 3.49 (dd, 1H, H-2), 3.36 (ddd, 1H, H-5) and 1.84 (dd, 1
H, 6-OH) ppm.
13C NMR of the β-anomer (125.76 MHz; CDCl3): δ139.0−
127.7 (arom. C), 103.0 (C-1), 84.7 (C-3), 82.5 (C-2), 77.7
(C-4), 75.9 (3-OCH2Ph), 75.2 (C-5, 4-OCH2Ph), 75.1 (2-
OCH2Ph), 71.8 (1-OCH2Ph) and 62.2 (C-6) ppm.
HRMS: m/zcalcd. for C34H36O6Na [M + Na]+563.2410;
found 563.2395.
1,2,3,4-Tetra-O-benzyl-6-O-propargyl-D-glucopyranose
(5). Synthesized from 1,2,3,4-tetra-O-benzyl-D-glucopyranose
(1.42 g, 2.6 mmol, 1.0 equiv), NaH (0.15 g, 6.0 mmol), and
propargyl-bromide (0.56 g, 4.7 mmol) according to the general
procedure for alkylation of free hydroxyl groups to give a white
solid (1.26 g, 83%; α:β31:69).
1H NMR of the α-anomer (500.13 MHz; CDCl3): δ7.42−
7.23 (m, 20H, arom. H), 5.00 and 4.84 (each d, each 1H, J=
−10.9 Hz, 3-OCH2Ph), 4.87 and 4.68 (each d, each 1H, J=
−11.2 Hz, 4-OCH2Ph), 4.83 (d, 1H, J1,2 = 3.5 Hz, H-1), 4.68
and 4.55 (each d, each 1H, J=−12.2 Hz, 1-OCH2Ph), 4.67
and 4.56 (each d, each 1H, J=−12.1 Hz, 2-OCH2Ph), 4.20
(dd, 1H, JCH2a,CH =−2.4, JCH2a,CH2b =−16.0 Hz, 6-OCH2aC
CH), 4.12 (dd, 1H, JCH2b,CH =−2.4 Hz, 6-OCH2bCCH),
4.04 (dd, 1H, J3,4 = 9.0, J3,2 = 9.7 Hz, H-3), 3.83 (dd, 1H, J6a,5 =
3.4, J6a,6b =−10.0 Hz, H-6a), 3.81 (ddd, 1H, J5,6b = 1.6, J5,4 =
10.3 Hz, H-5), 3.63 (dd, 1H, H-4), 3.58 (dd, 1H, H-6b), 3.54
(dd, 1H, H-2) and 2.37 (dd, 1H, 6-OCH2CCH) ppm.
13C NMR of the α-anomer (125.76 MHz; CDCl3): δ139.1−
127.7 (arom. C), 95.9 (C-1), 82.2 (C-3), 79.9 (C-2), 79.6 (6-
OCH2CCH), 77.6 (C-4), 75.8 (3-OCH2Ph), 75.2 (6-
OCH2CCH), 75.1 (4-OCH2Ph), 73.1 (2-OCH2Ph), 70.3
(C-5), 69.4 (1-OCH2Ph), 68.1 (C-6) and 58.7 (6-OCH2C
CH) ppm.
1H NMR of the β-anomer (500.13 MHz; CDCl3): δ7.41−
7.25 (m, 20H, arom. H), 4.97 and 4.66 (each d, each 1H, J=
−11.9 Hz, 1-OCH2Ph), 4.95 and 4.72 (each d, each 1H, J=
−10.9 Hz, 2-OCH2Ph), 4.92 and 4.79 (each d, each 1H, J=
−11.0 Hz, 3-OCH2Ph), 4.86 and 4.68 (each d, each 1H, J=
−10.8 Hz, 4-OCH2Ph), 4.51 (d, 1H, J1,2 = 7.8 Hz, H-1), 4.26
(dd, 1H, JCH2a,CH =−2.4, JCH2a,CH2b =−15.9 Hz, 6-OCH2aC
CH), 4.20 (dd, 1H, JCH2b,CH =−2.4 Hz, 6-OCH2bCCH),
3.83 (dd, 1H, J6a,5 = 4.5, J6a,6b =−10.7 Hz, H-6a), 3.79 (dd,
1H, J6b,5 = 2.1 Hz, H-6b), 3.64 (dd, 1H, J3,4 = 9.0, J3,2 = 9.2 Hz,
H-3), 3.62 (dd, 1H, J4,5 = 9.6 Hz, H-4), 3.51 (dd, 1H, H-2),
3.47 (ddd, 1H, H-5) and 2.39 (dd, 1H, 6-OCH2CCH) ppm.
13C NMR of the β-anomer (125.76 MHz; CDCl3): δ139.0−
127.7 (arom. C), 102.8 (C-1), 84.8 (C-3), 82.4 (C-2), 79.8 (6-
OCH2CCH), 77.7 (C-4), 75.8 (3-OCH2Ph), 75.1−74.9 (C-
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2, 4-OCH2Ph, 2-OCH2Ph, 6-OCH2CCH), 74.8 (C-5), 71.3
(1-OCH2Ph), 68.5 (C-6) and 58.8 (6-OCH2CCH) ppm.
HRMS: m/zcalcd. for C37H38O6Na [M + Na]+601.2566;
found 601.2628.
1,2,3,4-Tetra-O-benzyl-6-O-(o-carboranylmethyl)-D-glu-
copyranose (6). Synthesized from 1,2,3,4-tetra-O-benzyl-6-O-
propargyl-D-glucopyranose (0.91 g, 1.6 mmol) and B10H14
(0.33 g, 2.7 mmol) according to the general procedure for
coupling reaction of decaborane with propargylated glucosides
to give a colorless oil (0.61 g, 55.1%; α:β25:75). TLC: Rf: 0.55
(EtOAc:Hex 1:3).
1H NMR of the α-anomer (500.13 MHz; CDCl3): δ7.39−
7.23 (m, 20H, arom. H), 5.01 and 4.81 (each d, each 1H, J=
−10.6 Hz, 3-OCH2Ph), 4.90 and 4.55 (each d, each 1H, J=
−11.1 Hz, 4-OCH2Ph), 4.79 (d, 1H, J1,2 = 3.7 Hz, H-1), 4.68
and 4.56 (each d, each 1H, J=−11.8 Hz, 2-OCH2Ph), 4.66
and 4.53 (each d, each 1H, J=−12.2 Hz, 1-OCH2Ph), 4.04
(dd, 1H, J3,4 = 9.1, J3,2 = 9.4 Hz, H-3), 3.87 and 3.79 (each d,
each 1H, J=−10.7 Hz, 6-OCH2-carborane), 3.81 (br s, 1H,
carborane-CH), 3.74 (ddd, 1H, J5,6b = 1.7, J5,6a = 4.6, J5,4 = 10.1
Hz, H-5), 3.63 (dd, 1H, J6a,6b =−11.5 Hz, H-6a), 3.49 (dd, 1H,
H-2), 3.46 (dd, 1H, H-6b), 3.40 (dd, 1H, H-4) and 2.99−1.50
(br m, 10H, carborane-BH) ppm.
13C NMR of the α-anomer (125.76 MHz; CDCl3): δ138.7−
127.9 (arom. C), 95.6 (C-1), 82.1 (C-3), 80.2 (C-2), 77.5 (C-
4), 76.0 (3-OCH2Ph), 75.1 (4-OCH2Ph), 73.2 (2-OCH2Ph),
72.9 (6-OCH2-carborane), 71.0 (C-6, carborane-C), 70.6 (C-
5), 69.4 (1-OCH2Ph) and 57.6 (carborane-CH) ppm.
1H NMR of the β-anomer (500.13 MHz; CDCl3): δ7.39−
7.23 (m, 20H, arom. H), 4.96 and 4.73 (each d, each 1H, J=
−10.8 Hz, 2-OCH2Ph), 4.93 and 4.78 (each d, each 1H, J=
−10.8 Hz, 3-OCH2Ph), 4.88 and 4.66 (each d, each 1H, J=
−11.9 Hz, 1-OCH2Ph), 4.87 and 4.55 (each d, each 1H, J=
−11.2 Hz, 4-OCH2Ph), 4.49 (d, 1H, J1,2 = 7.9 Hz, H-1), 3.93
and 3.87 (each d, each 1H, J=−10.7 Hz, 6-OCH2-carborane),
3.91 (br s, 1H, carborane-CH), 3.67 (dd, 1H, J6a,5 = 4.8, J6a,6b =
−11.4 Hz, H-6a), 3.64 (dd, 1H, J6b,5 = 1.9 Hz, H-6b), 3.63 (dd,
1H, J3,4 = 9.0, J3,2 = 9.2 Hz, H-3), 3.47 (dd, 1H, H-2), 3.44 (dd,
1H, J4,5 = 9.9 Hz, H-4), 3.36 (ddd, 1H, H-5) and 2.99−1.50
(br m, 10H, carborane-BH) ppm.
13C NMR of the β-anomer (125.76 MHz; CDCl3): δ138.7−
127.9 (arom. C), 102.8 (C-1), 84.7 (C-3), 82.4 (C-2), 77.4
(C-4), 76.0 (3-OCH2Ph), 75.1 (2-OCH2Ph, 4-OCH2Ph), 74.8
(C-5), 72.9 (6-OCH2-carborane), 71.5 (1-OCH2Ph), 71.0 (C-
6, carborane-C) and 57.7 (carborane-CH) ppm.
11B NMR (160.46 MHz; CDCl3): δ−2.56, −4.38, −8.64, −
11.24 and −12.91 ppm.
HRMS: m/zcalcd. for C37H48B10O6Na [M + Na]+
721.4258; found 721.4327.
6-O-(o-Carboranylmethyl)-D-glucopyranose (1). Synthe-
sized from 1,2,3,4-tetra-O-benzyl-6-O-(o-carboranylmethyl)-D-
glucopyranose (0.11 g, 0.02 mmol) and Pd/C (0.17 g, 0.02
mmol) according to the general procedure for hydrogenolysis
to give a colorless oil (0.04 g, 79%; α:β60:40). TLC: Rf: 0.56
(EtOAc:MeOH 5:1).
1H NMR of the α-anomer (500.13 MHz; CD3OD): δ5.10
(d, 1H, J1,2 = 3.7 Hz, H-1), 4.59 (br s, 1H, carborane-CH),
4.04 and 4.02 (each d, each 1H, J=−10.8 Hz, 6-OCH2-
carborane), 3.86 (ddd, 1H, J5,6b = 1.8, J5,6a = 4.9, J5,4 = 10.1 Hz,
H-5), 3.75 (dd, 1H, J6a,6b =−11.3 Hz, H-6a), 3.73 (dd, 1H, H-
6b), 3.66 (dd, 1H, J3,4 = 9.0, J3,2 = 9.6 Hz, H-3), 3.35(dd, 1H,
H-2), 3.29 (dd, 1H, H-4) and 3.00−1.50 (br m, 10H,
carborane-BH) ppm.
13C NMR of the α-anomer (125.76 MHz; CD3OD): δ93.9
(C-1), 75.2 (carborane-C), 74.8 (C-3), 73.9 (6-OCH2-
carborane), 73.7 (C-2), 72.5 (C-6), 72.2 (C-5), 71.6 (C-4)
and 60.5 (carborane-CH) ppm.
1H NMR of the β-anomer (500.13 MHz; CD3OD): δ4.63
(br s, 1H, carborane-CH), 4.46 (d, 1H, J1,2 = 7.8 Hz, H-1),
4.05 and 4.03 (each d, each 1H, J=−10.8 Hz, 6-OCH2-
carborane), 3.79 (dd, 1H, J6a,5 = 2.0, J6a,6b =−11.3 Hz, H-6a),
3.72 (dd, 1H, J6b,5 = 5.2 Hz, H-6b), 3.37 (ddd, 1H, J5,4 = 10.3
Hz, H-5), 3.34 (dd, 1H, J3,2 = 9.0, J3,4 = 9.3 Hz, H-3), 3.30 (dd,
1H, H-4), 3.12 (dd, 1H, H-2) and 3.00−1.50 (br m, 10H,
carborane-BH) ppm.
13C NMR of the β-anomer (125.76 MHz; CD3OD): δ98.2
(C-1), 78.0 (C-3), 76.9 (C-5), 76.2 (C-2), 75.2 (carborane-C),
73.9 (6-OCH2-carborane), 72.2 (C-6), 71.3 (C-4) and 60.6
(carborane-CH) ppm.
11B NMR (160.46 MHz; CD3OD): δ−2.09, −4.01, −8.31,
−10.48 and −12.17 ppm.
HRMS: m/zcalcd. for C9H24B10O6Na [M + Na]+361.2401;
found 361.2382.
Methyl 6-O-tert-Butyldimethylsilyl-α-D-glucopyranoside.
Synthesized from methyl-α-D-glucopyranoside (9.91 g, 51
mmol), according to the general procedure for selective
silylation of the 6-OH group in glucopyranosides. This
reaction yielded a white solid (12.61 g, 80%). Rf= 0.52
(DCM:MeOH 7:1).
1H NMR (500.13 MHz; CDCl3): δ4.74 (d, 1H, J1,2 = 3.7
Hz, H-1), 3.88 (dd, 1H, J6a,5 = 5.0, J6a,6b =−10.8 Hz, H-6a),
3.83 (dd, 1H, J6b,5 = 5.0 Hz, H-6b), 3.74 (dd, 1H, J3,2 =J3−4=
8.9, 9.6 Hz, H-3), 3.60 (ddd, 1H, J5,4 = 9.8 Hz, H-5), 3.52 (dd,
1H, H-2), 3.52 (dd, 1H, H-4), 3.42 (s, 3H, 1-OCH3), 0.91 (s,
9H, 6-OSi(CH3)2C(CH3)3) and 0.10 (s, 6H, 6-OSi(CH3)2C-
(CH3)3) ppm.
13C NMR (125.76 MHz; CDCl3): δ99.3 (C-1), 74.8 (C-3),
72.4−72.3 (C-2, C-4), 70.6 (C-5), 64.3 (C-6), 55.4 (1-OCH3),
26.0 (6-OSi(CH3)2C(CH3)3), 18.4 (6-OSi(CH3)2C(CH3)3)
and −5.3 (6-OSi(CH3)2C(CH3)3) ppm.
HRMS: m/zcalcd. for C13H28O6SiNa [M + Na]+331.1553;
found 331.1553.
Methyl 2,3,4-Tri-O-benzyl-6-O-tertbutyldimethylsilyl-α-D-
glucopyranoside (7). Synthesized from methyl 6-O-tertbutyl-
dimethylsilyl-α-D-glucopyranoside (0.60 g, 1.93 mmol),
according to the general procedure for the alkylation of free
hydroxyl groups. This reaction yielded a white solid (0.70 g,
78%). Rf= 0.32 (EtOAc:hexane 1:8).
1H NMR (500.13 MHz; CDCl3): δ7.37−7.26 (m, 15H,
arom. H), 4.97 and 4.82 (each d, each 1H, J=−10.7 Hz, 3-
OCH2Ph), 4.88 and 4.64 (each d, each 1H, J=−10.9 Hz, 4-
OCH2Ph), 4.79 and 4.68 (each d, each 1H, J=−12.1 Hz, 2-
OCH2Ph), 4.61 (d, 1H, J1,2 = 3.6 Hz, H-1), 3.99 (dd, 1H, J3,4 =
9.0, J3,2 = 9.6 Hz, H-3), 3.78 (m, 2H, H-6a and H-6b), 3.61
(ddd, 1H, J5,6a = 3.1, J5,6b = 3.1, J5,4 = 10.0 Hz, H-5), 3.53 (dd,
1H, H-4), 3.50 (dd, 1H, H-2) and 3.36 (s, 3H, 1-OCH3), 0.88
(s, 9H, 6-OSi(CH3)2C(CH3)3) and 0.04 and 0.03 (each s,
each 3H, 6-OSi(CH3)2C(CH3)3) ppm.
13C NMR (125.76 MHz; CDCl3): δ139.3−127.6 (arom.
C), 98.0 (C-1), 82.3 (C-3), 80.4 (C-2), 77.9 (C-4), 76.0 (3-
OCH2Ph), 75.1 (4-OCH2Ph), 73.5 (2-OCH2Ph), 71.6 (C-5),
62.4 (C-6), 55.0 (1-O-CH3), 26.1 (6-OSi(CH3)2C(CH3)3),
18.4 (6-OSi(CH3)2C(CH3)3)and−5.1 (6-OSi(CH3)2C-
(CH3)3) ppm.
HRMS: m/zcalcd. for C34H46O6SiNa [M + Na]+601.2962;
found 601.2983.
Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics Article
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Mol. Pharmaceutics 2020, 17, 3885−3899
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Methyl 2,3,4-Tri-O-benzyl-α-D-glucopyranoside. Synthe-
sized from methyl 2,3,4-tri-O-benzyl-6-O-tert-butyldimethylsil-
yl-α-D-glucopyranoside (2.6 g, 4.49 mmol), according to the
general procedure for deprotection of silyl protective groups.
This reaction yielded a white solid (1.70 g, 82%). Rf= 0.60
(EtOAc:hexane 1:1).
1H NMR (500.13 MHz; CDCl3): δ7.37−7.28 (m, 15H,
arom. H), 4.99 and 4.84 (each d, each 1H, J=−10.9 Hz, 3-
OCH2Ph), 4.88 and 4.64 (each d, each 1H, J=−11.0 Hz, 4-
OCH2Ph), 4.80 and 4.67 (each d, each 1H, J=−12.1 Hz, 2-
OCH2Ph), 4.56 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.00 (dd, 1H, J3,4 =
8.9, J3,2 = 9.6 Hz, H-3), 3.76 (ddd, 1H, J6a,5 = 2.7, J6a,6‑OH = 5.3,
J6a,6b =−11.9 Hz, H-6a), 3.69 (ddd, 1H, J6b,5 = 4.1, J6b,6‑OH =
7.6 Hz, H-6b), 3.65 (ddd, 1H, J5,4 = 9.9 Hz, H-5), 3.52 (dd,
1H, H-4), 3.50 (dd, 1H, H-2), 3.37 (s, 3H, 1-OCH3) and 1.62
(dd, 1H, 6-OH) ppm.
13C NMR (125.76 MHz; CDCl3): δ139.7−127.7 (arom.
C), 98.3 (C-1), 82.1 (C-3), 80.1 (C-2), 77.5 (C-4), 75.9 (3-
OCH2Ph), 75.2 (4-OCH2Ph), 73.6 (2-OCH2Ph), 70.8 (C-5),
62.0 (C-6) and 55.3 (1-OCH3) ppm.
HRMS: m/zcalcd. for C28H32O6Na [M + Na]+487.2097;
found 487.2084.
Methyl 2,3,4-Tri-O-benzyl-6-O-propargyl-α-D-glucopyra-
noside (8). Synthesized from methyl 2,3,4-tri-O-benzyl-α-D-
glucopyranoside (0.31 g, 0.67 mmol), according to the general
procedure for the alkylation of free hydroxyl groups. This
reaction yielded a yellow oil (0.30 g, 89%). Rf= 0.61
(EtOAc:hexane 1:2).
1H NMR (500.13 MHz; CDCl3): δ7.37−7.26 (m, 15H,
arom. H), 4.98 and 4.83 (each d, each 1H, J=−10.9 Hz, 3-
OCH2Ph), 4.87 and 4.65 (each d, each 1H, J=−11.4 Hz, 4-
OCH2Ph), 4.79 and 4.68 (each d, each 1H, J=−11.7 Hz, 2-
OCH2Ph), 4.60 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.19 (dd, 1H,
JCH2a,CH =−2.4, JCH2a,CH2b =−16.0 Hz, 6-OCH2aCCH),
4.13 (dd, 1H, JCH2a,CH =−2.4 Hz, 6-OCH2bCCH), 3.98 (dd,
1H, J3,4 = 9.0, J3,2 = 9.7 Hz, H-3), 3.84 (dd, 1H, J6a,5 = 3.5, J6a,6b
=−10.4 Hz, H-6a), 3.76 (ddd, 1H, J5,6b = 2.1, J5,4 = 10.1 Hz,
H-5), 3.66 (dd, 1H, H-6b), 3.61 (dd, 1H, H-4), 3.54 (dd, 1H,
H-2), 3.37 (s, 3H, 1-OCH3) and 2.37 (dd, 1H, 6-OCH2C
CH) ppm.
13C NMR (125.76 MHz; CDCl3): δ139.6−127.7 (arom.
C), 98.5 (C-1), 82.2 (C-3), 79.9 (C-2), 79.6 (6-OCH2C
CH), 77.6 (C-4), 75.9 (3-OCH2Ph), 75.2 (4-OCH2Ph), 75.1
(6-OCH2CCH), 73.6 (2-OCH2Ph), 70.0 (C-5), 68.2 (C-6),
58.7 (6-OCH2CCH) and 55.4 (1-OCH3) ppm.
HRMS: m/zcalcd. for C31H34O6Na [M + Na]+525.2253;
found 525.2258.
Methyl 2,3,4-Tri-O-benzyl-6-O-(o-carboranylmethyl)-α-D-
glucopyranoside (9). Synthesized from methyl 2,3,4-tri-O-
benzyl-6-O-propargyl-α-D-glucopyranoside (0.37 g, 0.73
mmol), according to the general procedure for coupling with
decaborane. This reaction yielded a white solid (0.27 g, 5 3%).
Rf= 0.33 (EtOAc:hexane 1:3).
1H NMR (500.13 MHz; CDCl3): δ7.38−7.25 (m, 15H,
arom. H), 4.99 and 4.80 (each d, each 1H, J=−10.8 Hz, 3-
OCH2Ph), 4.90 and 4.55 (each d, each 1H, J=−11.2 Hz, 4-
OCH2Ph), 4.81 and 4.67 (each d, each 1H, J=−12.0 Hz, 2-
OCH2Ph), 4.55 (d, 1H, J1,2 = 3.5 Hz, H-1), 3.97 (dd, 1H, J3,4 =
8.8, J3,2 = 9.6 Hz, H-3), 3.89 and 3.80 (each d, each 1H, J=
−10.5 Hz, 6-OCH2-carborane), 3.81 (br s, carborane-CH),
3.67 (ddd, 1H, J5,6b = 1.8, J5,6a = 4.4, J5,4 = 9.7, Hz, H-5), 3.65
(dd, 1H, J6a,6b =−10.9 Hz, H-6a), 3.55 (dd, 1H, H-6b), 3.48
(dd, 1H, H-2), 3.39 (dd, 1H, H-4), 3.35 (s, 3H, 1-OCH3) and
2.62−1.61 (br m, 10H, carborane-BH) ppm.
13C NMR (125.76 MHz; CDCl3): δ139.8−127.8 (arom.
C), 98.2 (C-1), 82.1 (C-3), 80.1 (C-2), 77.2 (C-4), 76.1 (3-
OCH2Ph), 75.1 (4-OCH2Ph), 73.6 (2-OCH2Ph), 72.9
(carborane-C), 71.0 (C-6), 70.3 (C-5), 60.5 (6-OCH2-
carborane), 57.6 (carborane-CH) and 55.4 (1-OCH3) ppm.
11B NMR (160.46 MHz; CDCl3): δ−2.74, −4.74, −8.94,
−11.52 and −13.06 ppm.
HRMS: m/zcalcd. for C31H44B10O6Na [M + Na]+
645.3966; found 645.3975.
Methyl 6-O-(o-Carboranylmethyl)-α-D-glucopyranoside
(2). Synthesized from methyl 2,3,4-tri-O-benzyl-6-O-(o-carbor-
anylmethyl)-α-D-glucopyranoside (0.15 g, 0.25 mmol), accord-
ing to the general procedure for the deprotection of benzyl
groups. This reaction yielded a white solid (0.06 g, 72%). Rf=
0.47 (DCM:MeOH 7:1).
1H NMR (500.13 MHz; CD3OD): δ4.66 (d, 1H, J1,2 = 3.8
Hz, H-1), 4.57 (br s, 1H, carborane-CH), 4.04 and 4.01 (each
d, each 1H, J=−11.0 Hz, 6-OCH2-carborane), 3.76 (dd, 1H,
J6a,5 = 1.8, J6a,6b =−11.3 Hz, H-6a), 3.71 (dd, 1H, J6b,5 = 5.3
Hz, H-6b), 3.60 (ddd, 1H, J5,4 = 9.7 Hz, H-5), 3.59 (dd, 1H,
J3,4 = 9.2, J3,2 = 9.7 Hz, H-3), 3.40 (s, 3H, 1-OCH3), 3.37 (d,
1H, H-2), 3.26 (d, 1H, H-4) and 2.56−1.59 (br m, 10H,
carborane-BH) ppm.
13C NMR (125.76 MHz; CD3OD): δ101.3 (C-1), 75.2
(carborane-C), 75.0 (C-3), 74.0 (6-OCH2-carborane), 73.5
(C-2), 72.7 (C-5), 72.3 (C-6), 71.6 (C-4), 60.7 (carborane-
CH) and 55.6 (1-OCH3) ppm.
11B NMR (160.46 MHz; CDCl3): δ−1.94, −3.92, −8.30, −
10.44 and −12.05 ppm.
HRMS: m/zcalcd. for C10H26B10O6Na [M + Na]+
375.2558; found 375.2519.
Methyl 6-O-tert-Butyldimethylsilyl-β-D-glucopyranoside.
Synthesized from methyl β-D-glucopyranoside (0.50 g, 2.59
mmol), according to the general procedure for selective
silylation of the 6-OH group in glucopyranosides. This
reaction yielded a white solid (0.66 g, 83%). Rf= 0.48
(DCM:MeOH 7:1).
1H NMR (500.13 MHz; CD3OD): δ4.17 (d, 1H, J1,2 = 7.8
Hz, H-1), 3.99 (dd, 1H, J6a,5 = 2.1, J6a,6b =−11.4 Hz, H-6a),
3.83 (dd, 1H, J6b,5 = 5.3 Hz, H-6b), 3.53 (s, 3H, 1-OCH3),
3.36 (dd, 1H, J3,2 = 9.0, J3,4 = 9.2 Hz, H-3), 3.33 (dd, 1H, J4,5 =
9.7 Hz, H-4), 3.27 (ddd, 1H, H-5), 3.17 (dd, 1H, H-2), 0.90
(s, 9H, 6-OSi(CH3)2C(CH3)3) and 0.09 (s, 6H, 6-OSi-
(CH3)2C(CH3)3) ppm.
13C NMR (125.76 MHz; CD3OD): δ105.3 (C-1), 78.1 (C-
3, C-5), 75.0 (C-2), 71.4 (C-4), 64.1 (C-6), 57.2 (1-OCH3),
26.4 (6-OSi(CH2)2CH3), 19.3 (6-OSi(CH2)2CH3), −5.0 and
−5.1 (each Si(CH3)2) (each 6-OSi(CH3)2C(CH3)3) ppm.
HRMS: m/zcalcd. for C13H28O6SiNa [M + Na]+331.1553;
found 331.1531.
Methyl 2,3,4-Tri-O-benzyl-6-O-tertbutyldimethylsilyl-β-D-
glucopyranoside (10). Synthesized from methyl 6-O-tert-
butyldimethylsilyl-β-D-glucopyranoside (3.05 g, 9.89 mmol),
according to the general procedure for the alkylation of free
hydroxyl groups. This reaction yielded a white solid (4.62 g,
80%). Rf= 0.33 (EtOAc:hexane 1:8).
1H NMR (500.13 MHz; CDCl3): δ7.38−7.27 (m, 15H,
arom. H), 4.92 and 4.71 (each d, each 1H, J=−11.0 Hz, 2-
OCH2Ph), 4.91 and 4.81 (each d, each 1H, J=−10.8 Hz, 3-
OCH2Ph), 4.85 and 4.68 (each d, each 1H, J=−10.9 Hz, 4-
OCH2Ph), 4.29 (d, 1H, J1,2 = 7.7 Hz, H-1), 3.88 (dd, 1H, J6a,5
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= 1.7, J6a,6b =−11.5 Hz, H-6a), 3.83 (dd, 1H, J6b,5 = 4.3 Hz, H-
6b), 3.64 (dd, 1H, J3,4 = 9.1, J3,2 = 9.2 Hz, H-3), 3.59 (dd, 1H,
J4,5 = 9.7 Hz, H-4), 3.54 (s, 3H, 1-OCH3), 3.38 (dd, 1H, H-2),
3.28 (ddd, 1H, H-5), 0.90 (s, 9H, 6-OSi(CH3)2C(CH3)3) and
0.08 and 0.07 (each s, each 3H, 6-OSi(CH3)2C(CH3)3) ppm.
13C NMR (125.76 MHz; CDCl3): δ139.6−127.7 (arom.
C), 104.6 (C-1), 84.8 (C-3), 82.7 (C-2), 77.8 (C-4), 75.9 (3-
OCH2Ph), 75.9 (C-5), 75.2 (4-OCH2Ph), 74.9 (2-OCH2Ph),
62.4 (C-6), 56.8 (1-OCH3), 26.1 (6-OSi(CH3)2C(CH3)3),
18.5 (6-OSi(CH3)2C(CH3)3), −4.9 and −5.1 (6-OSi(CH3)2C-
(CH3)3) ppm.
HRMS: m/zcalcd. for C34H46O6SiNa [M + Na]+601.2962;
found 601.2964.
Methyl 2,3,4-Tri-O-benzyl-β-D-glucopyranoside. Synthe-
sized from methyl 2,3,4-tri-O-benzyl-6-O-tert-butyldimethylsil-
yl-β-D-glucopyranoside (2.94 g, 5.08 mmol), according to the
general procedure for deprotection of silyl protective groups.
This reaction yielded a white solid (2.09 g, 89%). Rf= 0.65
(EtOAc:hexane 1:1).
1H NMR (500.13 MHz; CDCl3): δ7.38−7.27 (m, 15H,
arom. H), 4.93 and 4.81 (each d, each 1H, J=−10.9 Hz, 3-
OCH2Ph), 4.91 and 4.71 (each d, each 1H, J=−11.0 Hz, 2-
OCH2Ph), 4.87 and 4.64 (each d, each 1H, J=−10.9 Hz, 4-
OCH2Ph), 4.36 (d, 1H, J1,2 = 7.8 Hz, H-1), 3.88 (ddd, 1H, J6a,5
= 2.8, J6a,6‑OH = 5.5, J6a,6b =−12.0 Hz, H-6a), 3.73 (ddd, 1H,
J6b,5 = 4.5, J6b,6‑OH = 7.7 Hz, H-6b), 3.67 (dd, 1H, J3,4 = 9.0, J3,2
= 9.2 Hz, H-3), 3.57 (dd, 1H, J4,5 = 9.8 Hz, H-4), 3.57 (s, 3H,
1-OCH3), 3.40 (dd, 1H, H-2), 3.37 (ddd, 1H, H-5) and 1.88
(dd, 1H, 6-OH) ppm.
13C NMR (125.76 MHz; CDCl3): δ138.7−127.1 (arom.
C), 105.0 (C-1), 84.6 (C-3), 82.5 (C-2), 77.6 (C-4), 75.8 (3-
OCH2Ph), 75.3 (C-5), 75.1 (4-OCH2Ph), 75.0 (2-OCH2Ph),
62.2 (C-6) and 57.5 (1-OCH3) ppm.
HRMS: m/zcalcd. for C28H32O6Na [M + Na]+487.2097;
found 487.2089.
Methyl 2,3,4-Tri-O-benzyl-6-O-propargyl-β-D-glucopyra-
noside (11). Synthesized from methyl 2,3,4-tri-O-benzyl-β-D-
glucopyranoside (1.42 g, 3.02 mmol), according to the general
procedure for the alkylation of free hydroxyl groups. This
reaction yielded a yellow oil (1.44 g, 95%). Rf= 0.84
(EtOAc:hexane 1:1).
1H NMR (500.13 MHz; CDCl3): δ7.35−7.26 (m, 15H,
arom. H), 4.92 and 4.80 (each d, each 1H, J=−11.0 Hz, 3-
OCH2Ph), 4.91 and 4.70 (each d, each 1H, J=−11.0 Hz, 2-
OCH2Ph), 4.85 and 4.68 (each d, each 1H, J=−10.8 Hz, 4-
OCH2Ph), 4.30 (d, 1H, J1,2 = 7.8 Hz, H-1), 4.25 (dd, 1H,
JCH2a,CH =−2.4, JCH2a,CH2b =−15.9 Hz, 6-OCH2aCCH),
3.64 (dd, 1H, JCHb,CH =−2.4 Hz, 6-OCH2bCCH), 3.83 (dd,
1H, J6a,5 = 4.4, J6a,6b =−10.7 Hz, H-6a), 3.78 (dd, 1H, J6b,5 =
2.0 Hz, H-6b), 3.64 (dd, 1H, J3,2 = 9.1, J3,4 = 9.1 Hz, H-3), 3.61
(dd, 1H, J4,5 = 10.0 Hz, H-4), 3.57 (s, 3H, 1-OCH3), 3.46
(ddd, 1H, H-5), 3.42 (dd, 1H, H-2) and 2.38 (dd, 1H, 6-
OCH2CCH) ppm.
13C NMR (125.76 MHz; CDCl3): δ128.6−127.7 (arom.
C), 104.9 (C-1), 84.7 (C-3), 82.4 (C-2), 79.8 (6-OCH2C
CH), 77.7 (C-4), 77.8 (3-OCH2Ph), 76.4 (6-OCH2CCH),
75.1 (4-OCH2Ph), 74.8 (2-OCH2Ph), 74.7 (C-5), 68.5 (C-6),
58.8 (6-OCH2CCH) and 57.3 (1-OCH3) ppm.
HRMS: m/zcalcd. for C31H34O6Na [M + Na]+525.2253;
found 525.2252.
Methyl 2,3,4-Tri-O-benzyl-6-O-(o-carboranylmethyl)-β-D-
glucopyranoside (12). Synthesized from methyl 2,3,4-tri-O-
benzyl-6-O-propargyl-β-D-glucopyranoside (1.20 g, 2.38
mmol), according to the general procedure for coupling with
decaborane. This reaction yielded a white solid (0.98 g, 66%).
Rf= 0.26 (EtOAc:hexane 1:4).
1H NMR (500.13 MHz; CDCl3): δ7.35−7.23 (m, 15H,
arom. H), 4.93 and 4.78 (each d, each 1H, J=−10.8 Hz, 3-
OCH2Ph), 4.91 and 4.71 (each d, each 1H, J=−11.1 Hz, 2-
OCH2Ph), 4.87 and 4.54 (each d, each 1H, J=−11.1 Hz, 4-
OCH2Ph), 4.28 (d, 1H, J1,2 = 7.1 Hz, H-1), 3.94 (br s,
carborane-CH), 3.94 and 3.89 (each d, each 1H, J=−10.5 Hz,
6-OCH2-carborane), 3.66 (dd, 1H, J6a,5 = 2.3, J6a,6b =−10.5
Hz, H-6a), 3.65 (dd, 1H, J6b,5 = 4.4 Hz, H-6b), 3.63 (dd, 1H,
J3,4 = 8.8, J3,2 = 9.3 Hz, H-3), 3.54 (s, 3H, 1-OCH3), 3.41 (dd,
1H, J4,5 = 10.0 Hz, H-4), 3.38 (dd, 1H, H-2), 3.37 (ddd, 1H,
H-5) and 2.64−1.77 (br m, 10H, carborane-BH) ppm.
13C NMR (125.76 MHz; CDCl3): δ138.7−127.9 (arom.
C), 104.9 (C-1), 84.6 (C-3), 82.4 (C-2), 77.4 (C-4), 75.9 (3-
OCH2Ph), 75.1 (4-OCH2Ph), 75.0 (2-OCH2Ph), 74.7 (C-5),
72.9 (6-OCH2-carborane and carborane-C), 71.1 (C-6), 57.7
(carborane-CH) and 57.3 (1-OCH3) ppm.
11B NMR (160.46 MHz; CDCl3): δ−2.83, −4.62, −8.89, −
11.58 and −13.09 ppm.
HRMS: m/zcalcd. for C31H44B10O6Na [M + Na]+
645.3966; found 645.4025.
Methyl 6-O-(o-Carboranylmethyl)-β-D-glucopyranoside
(3). Synthesized from methyl 2,3,4-tri-O-benzyl-6-O-(o-carbor-
anylmethyl)-β-D-glucopyranoside (0.15 g, 0.25 mmol), accord-
ing to the general procedure for the deprotection of benzyl
groups. This reaction yielded a white solid (0.068 g, 81%). Rf=
0.61 (DCM:MeOH 5:1).
1H NMR (500.13 MHz; CD3OD): δ4.63 (br s, 1H,
carborane-CH), 4.17 (d, 1H, J1,2 = 7.8 Hz, H-1), 4.06 (each d,
each 1H, J=−11.1 Hz, 6-OCH2-carborane), 3.84 (dd, 1H, J6a,5
= 2.1, J6a,6b =−11.3 Hz, H-6a), 3.73 (dd, 1H, J6b,5 = 5.2 Hz, H-
6b), 3.53 (s, 3H, 1-OCH3), 3.38 (ddd, 1H, J5,4 = 10.0 Hz, H-
5), 3.35 (dd, 1H, J3,2 = 9.3, J3,4 = 9.3 Hz, H-3), 3.30 (dd, 1H,
H-4), 3.16 (dd, 1H, H-2) and 2.54−1.54 (br m, 10H,
carborane-BH) ppm.
13C NMR (125.76 MHz; CD3OD): δ105.4 (C-1), 77.8 (C-
3), 76.9 (C-5), 75.2 (carborane-C), 74.9 (C-2), 73.9 (6-
OCH2-carborane), 72.2 (C-6), 71.2 (C-4), 60.5 (carborane-
CH) and 57.4 (1-OCH3) ppm.
11B NMR (160.46 MHz; CDCl3): δ−2.06, −3.97, −8.30, −
10.50 and −12.08 ppm.
HRMS: m/zcalcd. for C10H26B10O6Na [M + Na]+
375.2558; found 375.2557.
2.2. Molecular Modeling. The initial geometries of the
ligands were optimized to a local minimum at the DFT level,
using the dispersion-corrected hybrid Tao−Perdew−Scuseria−
Staroverov functional TPSSh-D3(BJ),
26−28
with the doubly
polarized triple-ζbasis set def2-TZVPP.
29
The structures of
the different ligands were aligned so that geometries would be
as similar as possible. Partial atomic charges were computed
using the restrained electrostatic potential (RESP) protocol.
30
For the RESP charge calculation, the molecule was divided
into two parts, with one part consisting of the carborane and a
linking carbon and the other part comprising the sugar. Partial
charges of hydrogens bonded to the same carbon were
constrained to be equal. The geometry optimizations were
performed with Turbomole 7.3,
31,32
and the RESP calculations
with NWChem 6.8.
33
Noncovalent interactions (NCI)
34
between the ligands and protein were computed using the
promolecular approach of NCIPLOT.
35
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Molecular docking studies were performed using AutoDock
4.2.6.
36,37
All rotatable bonds in the carborane part were set to
nonrotatable (inactive). For docking, the number of torsional
degrees of freedom for the carboranes was set to 8 (torsdof 8).
The docking studies were performed using the XylE inward-
open 4QIQ and outward-open 6N3I PDB structures. The XylE
protein structures were mutated using PyMOL, changing Gln-
415 to Asn-415. The most probable rotamer, that is, the one
with the least clashes with surrounding amino acids, as
suggested by PyMOL was used. Each protein was prepared by
removing the ligand and other superfluous small molecules
(Zn for 4QIQ), adding hydrogens, merging them, and then
computing Gasteiger partial charges. For all proteins, a grid of
size 46 ×56 ×60 was used, with a grid spacing value of 0.375.
The grid center was in the middle of the protein cavity, for the
grid box to cover the binding site. During docking, the protein
was kept rigid and only ligand torsional angles changed. For
each ligand, 6000 (3 ×2000) independent search runs, each
with max 2.5 million energy evaluations and population size of
150 with max 27000 generations, were performed using the
default settings of the Lamarckian genetic algorithm (LGA),
that is, a mutation rate of 0.02 and crossover rate of 0.8, with
one top individual surviving to the next generation.
Conformations were clustered (ranked) with a cluster RMS
2.0 Å.
Parameters for boron, missing from the standard distribution
of Autodock, were added to the parameter file: R 2.285, Rii
4.57, epsilon 0.179, vol 49.9744; other parameters were set to
their corresponding carbon values. R and epsilon were taken
from Oda et al.,
38
as reproduced by Couto et al.,
39
and were
used to calculate Rii and vol. The complete parameter
definition was thus:
atom_par B 4.57 0.179 49.9744−0.00143 0.0 0.0 0 −1−10
#Boron for Carborane
2.3. Cytotoxicity Studies. The CellTiter-Glo luminescent
cell viability assay was purchased from Promega Corporation
(Madison, WI, USA). The PierceTM BCA Protein Assay Kit
was obtained from Thermo Fisher Scientific (Waltham, MA,
USA). Human CAL 27 squamous cell carcinoma was acquired
from American Type Culture Collection (Manassas, VA,
USA). The cell culturing flasks and 96-well plates were
purchased from Corning (Corning, NY, USA). Dulbecco’s
Modified Eagle’s Medium (DMEM), Dulbecco’s phosphate
buffer saline (10 ×DPBS), fetal bovine serum (FBS), and
Penicillin-Streptomycin (10,000 U/mL) were obtained from
Gibco (Life Technologies, Carlsbad, CA, USA).
The in vitro cell cytotoxicity was carried out using a
commercial CellTiter-Glo luminescent cell viability assay. The
human epithelial CAL 27 squamous carcinoma cell line was
used as a head-and-neck cancer cell model in this experiment.
The cells were plated on a 96-well plate at 15,000 cells per well
in 100 μL DMEM supplemented with 10% FBS and 1%
Penicillin-Streptomycin, and cells were allowed to attach
overnight. The medium was removed and replaced with 100
μL of the glucoconjugates 1,2,3, or sodium borocaptate
(BSH) solution in complete cell culture medium at
concentrations of 5 μM, 25 μM, 50 μM, 125 μM, and 250
μM. Fresh medium and 1% (v/v) Triton X-100 were used as
positive and negative controls of cell viability, respectively. The
incubation time points of the compounds were set at 6 and 24
h in a temperature and humidity controlled incubator (37 °C,
95% relative humidity and 5% CO2). At predetermined time
points, the plates were equilibrated to room temperature for 30
min. The incubated solutions were discarded, and the cells
were washed twice with 1 ×PBS. Then, 50 μL of both 1 ×PBS
and CellTiter-Glo reagent were added to the wells. The plates
were protected from light with aluminum foil and placed on an
orbital shaker for 2 min before luminescence measurement
with a Varioskan LUX multimode microplate reader (Thermo
Fisher Scientific, Waltham, MA, USA). All measurements were
done in quadruplicate.
Furthermore, the total protein content in each sample was
quantified using the colorimetric bicinchoninic acid (BCA)
assay (Pierce, Thermo Fisher Scientific, Waltham, MA, USA).
The procedures were carried out according to the
manufacturer protocol. Cell lysates (25 μL each) from the
cytotoxicity assay was transferred to a new 96-well plate.
Working reagent (200 μL) was subsequently added to each
sample at a 1:8 ratio. The plates were kept in the dark with
aluminum foil and gently mixed on an orbital shaker for 30 s
before proceeding to incubate at 37 °C for 30 min. The
absorbance was read at 562 nm on a plate reader, and the
protein content was determined against a bovine serum
albumin (BSA) standard curve (0−2000 μg/mL). The total
protein content results were used to normalize the cell viability
from the CellTiter-Glo assay by dividing the luminescence
value in each sample by the total protein content (μg) in the
same sample before the percent cell viability determination.
The experiment was carried out in quadruplicate and the
statistical significance of the mean viability was determined
using an unpaired Student’st-test against the negative control
for cell viability.
2.4. GLUT1 Affinity and Cellular Uptake Studies. CAL
27 squamous cell carcinoma cells were purchased from the
American Type Culture Collection (ATCC, Manassas, VA,
USA) or supplied by the University of Helsinki. The CAL 27
cells were cultured in Dulbecco’s Modified Eagle Medium
(DMEM; Gibco, ThermoFisher Scientific, Waltham, MA,
USA) supplemented with L-glutamine (2.0 mM; ThermoFisher
Scientific, Waltham, MA, USA), heat-inactivated fetal bovine
serum (10%; Gibco, ThermoFisher Scientific, Waltham, MA,
USA), penicillin (50 U/mL), streptomycin (50 μg/mL)
solution (ThermoFisher Scientific, Waltham, MA, USA). The
CAL 27 cells (passages 7−20) were seeded at the density of 5
×105cells/wells onto 24-well plates. The cells were used in
the affinity and uptake studies 2 days after seeding. The culture
medium was removed, and the cells were washed with
prewarmed HBSS (Hank’s balance salt solution) without
glucose (pH 7.4). The cells were then incubated with HBSS at
37 °C for 10 min before the experiments. Additional
information on the experimental protocols is supplied in the
Supporting Information.
In order to determine the ability of the compounds to bind
to GLUT1 in the CAL 27 cell line, the cells were cultured,
seeded, and preincubated as described above. The HBSS was
removed, and the ability of the compounds to inhibit the
uptake of the known GLUT1 substrate, [14C]-D-glucose
(PerkinElmer, Waltham, MA, USA), was studied by incubating
the cells at rt for 5 min in a buffer with a pH 7.4 (250 μL) and
further containing 1.8 μM (0.1 mCi/ml) of [14C]-D-glucose.
The compounds were studied at concentrations ranging from
0.1−1800 μM and the HBSS was used as a blank. After
incubation, the experiment was ended by the addition of ice-
cold HBSS and the cells were washed twice with ice-cold
HBSS. The cells were then lysed with 250 μL of 0.1 M sodium
hydroxide, the lysate was mixed with 1.0 mL of Emulsifier safe
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cocktail (PerkinElmer, Waltham, MA, USA), and the radio-
activity was measured by liquid scintillation counter (MicroBe-
ta
2
counter, PerkinElmer, Waltham, MA, USA). The inhibition
of [14C]-D-glucose in the presence of the boron containing
compounds compared to the control (HBSS) was calculated as
percentages (%). See Supporting Information Figure 44.
The concentration-dependent uptake studies of the
glucoconjugates were performed by adding 10−400 μMof
the compounds in 250 μL of prewarmed HBSS buffer on the
cell layer. The incubation times for each compound were 5 and
30 min. After incubation the cells were washed and lysed as
described above. The lysate from 4 wells was combined in a
Eppendorf tube, centrifuged at 4 °C, and 800 μL of the
supernatant was collected and digested in 1.0 mL of conc.
HNO3(TraceMetal grade, Fisher Chemical) for 24 h. After
sample digestion, Milli-Q water (USF Elga Purelab Ultra) was
added in order to reach a total volume of 10 mL, and the
boron concentrations were analyzed by inductively coupled
plasma mass spectrometry (ICP-MS).
The boron concentrations were analyzed by ICP-MS using a
NeXION 350D ICP-MS instrument (PerkinElmer Inc.,
Waltham, MA, USA) and ESI PrepFAST autosampler
(Elemental Scientific, Omaha, NE, USA). For sample injection
a peristaltic pump and nebulizer were used. The instrument
was operated with an RF power of 1.6 kW and with nebulizer
gas, auxiliary gas, and plasma gas flows of 0.90, 18, and 1.2 l/
min, respectively. The sample uptake rate was 3.5 mL/min,
and dwell times were set at 100 ms per AMU. To remove
polyatomic interferences, a triple-quadrupole reaction system
operating in collision mode with kinetic energy discrimination
(KED) was used (with He as the cell gas (3.7 mL/min)). An
internal standard, 89Y, was mixed online with the samples to
compensate for matrix effects and instrument drift. Boron was
determined against a certified multielement calibration stand-
ard (TraceCERT Periodic Table Mix 1, Sigma-Aldrich) under
acid conditions (6.7% HNO3,TraceMetalgrade,Fisher
Chemical). The calibration range used for 11B was 4−400
μg/lL, and the detection limit (LOD) was 1.0 μg/L. Three
replicates were obtained for each sample. The data was
processed using the PerkinElmer Syngistix Data Analysis
Software.
3. RESULTS
3.1. Synthesis and Structural Characterization of 6-O-
Carboranylmethyl Glucoconjugates. The construction of
the targeted glucoconjugates requires insights in boron cluster
chemistry and carbohydrate chemistry. A significant amount of
progress has been achieved in both areas over a considerable
timespan, and robust reaction methodologies can be found in
the existing literature.
23,24
Yet, each new synthetic target
requires the development of a suitable strategy, and unlike the
3-O-carboranylmethyl,
40
the carboranylmethyl-glucosides,
41
and other types of glucoconjugates previously evaluated,
23,42,43
the 6-O-carboranylmethyl glucoconjugates (Figure 2) have
been explicitly designed for clinical BNCT of head and neck
cancers. The methyl glucopyranosides were included in order
to evaluate if there is a difference between the affinity and
cellular uptake of the two anomers since the hemiacetal exists
as a mixture of both. In addition, the methyl group is minimally
intrusive which is beneficial since information on the substrate
tolerance of GLUT1 is limited.
44
It was important to account for the susceptibility of
decaborane to free hydroxyl groups and the possibility for
carboranes to undergo degradation under strongly basic
conditions when planning the synthesis of the 6-O-carbor-
anylmethyl glucoconjugates. With these issues in mind, we
developed the multistep synthetic routes to the three 6-O-
carboranylmethyl glucoconjugates 1,2, and 3. The synthesis
and structural characterization discussion will herein be limited
to glucoconjugate 1(Scheme 1). The reaction routes to 2and
3are displayed in Supporting Information Scheme 1, and the
synthesis and characterization of 2and 3, and all intermediates
on these routes, were conducted in a similar fashion as
described below for 1.
In short, the synthesis commenced from D-glucose. In the
first step, the sterically least hindered primary hydroxyl group
was temporarily protected as a bulky silyl ether with
TBDMSCl in pyridine in an acceptable yield. The remaining
hydroxyl groups were benzylated, through standard alkylation
protocols,
45
using BnBr and NaH in an 88% isolated yield. The
temporary silyl protective group was removed with Olah’s
reagent in excellent yield,
46
followed by the alkylation of the
unmasked hydroxyl group with propargyl bromide and NaH in
an 87% yield. The coupling between decaborane (B10H14) and
the terminal alkyne was achieved by first forming a
decaborane−ACN complex
47
followed by a substitution
reaction with 5. In the pioneering work of Tietze et al.,
which encompassed the synthesis of carboranyl C-glycosides,
the removal of benzyl groups was reported to proceed in high
yields (61%−quant.).
48
In our first attempts, we encountered
challenges regarding the debenzylation reaction, and D-glucose
was formed in considerable amounts as a side product (>40%).
These observations were consistent regardless of the employed
transition metal catalyst. While we did not optimize the
reaction conditions fully, we did note that performing the
reaction at a lower substrate concentration (40 mg/mL vs 14
mg/mL) led to a marked increase in the isolated yields (59%
→79%). The isolated yields on the synthetic routes were high
throughout and the overall yield for the synthesis of 1was
22%.
Scheme 1. Reaction Route Leading to 1
a
a
Reagents and conditions: (i) (1) TBDMSCl, pyridine, rt, 24 h, 69%; (2) BnBr, NaH, DMF, rt, 4 h, 88%; (ii) (1) HF-pyridine, THF, rt, 18 h, 99%;
(2) propargyl bromide, NaH, DMF, rt, 15 h, 83%; (iii) (1) B10H14, acetonitrile, 60 °C, 1 h; (2) 5, toluene, 80 °C, 16 h, 55%; (iv) H2, 10% Pd/C,
EtOAc:MeOH 7:1, 3−5 bar, rt, 4−6 h, 79%.
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In order to understand how the glucoconjugates interact
with GLUT1, insights on their structural properties were
required. As a result, we performed a detailed conformational
characterization of the synthesized molecules by NMR-
spectroscopy (1H, 13C, 11B, 1D-TOCSY, DQF-COSY, ed-
HSQC, and HMBC) further coupled with spectral simulations
by quantum mechanical optimization utilizing the PERCH
peak research software. The 1H NMR spectrum of 1was
challenging to solve because the hemiacetal exists as an
anomeric mixture (59% α, 41% β) and the signals overlap in
several parts of the spectrum. In order to overcome these
challenges, 1D-TOCSY was utilized.
49,50
The well separated H-
1α(5.10 ppm) and H-1β-protons (4.46 ppm) were irradiated,
and a mixing time of 300 ms was applied in order to ensure the
transfer of magnetization throughout the spin-systems. This
experiment resulted in information on the chemical shifts of all
proton signals on both residues. By the use of standard 2D-
NMR techniques, all of the 1H and 13C NMR signals of both
anomers could be assigned. The coupling constants which
provide information on the angles between adjacent protons
and constitute the basis of a conformational characterization
could not be reliably extracted from the 1H NMR spectra
alone. By use of the PERCH-software, the 1H NMR spectrum
was simulated and the coupling constants were obtained. The
coupling constants confirmed that the glucoconjugates exist
primarily in the 4C1-conformation and that the gg and gt
rotamers (C5−C6-bond) are dominating in solution (JH‑5,H‑6a
= 1.8−2.0 Hz, JH‑5,H‑6b = 4.9−5.2 Hz and JH‑6a,H‑6b =−11.3
Hz).
51
The last step on the road to a complete NMR-spectroscopic
characterization was to confirm that the boron cluster had
remained intact. To this end, we measured decoupled 11B
NMR spectra and the signals appearing in the 0 to −30 ppm
region confirmed
52
that the carboranyl cluster was indeed
intact. In addition, we assigned all the signals (carboranyl-
methyl moiety, protective groups, and carbohydrate) in the 1H
and 13C NMR spectra of all compounds and verified their
structural identity and purity also by high resolution mass
spectrometry.
3.2. Experimental and Computational GLUT1 Affinity
Studies. We were interested in understanding how the
glucoconjugates interact with GLUT1 since this provides the
biochemical foundation for their potential use in the intended
application. We sought inspiration from the previous work of
Lippard et al. focusing on cytotoxic glucose-platinum
conjugates.
53
While the requirements for a successful
GLUT1 targeting approach are similar, there is a significant
conceptual difference between the therapeutic approaches of
using either nontoxic boron delivery agents (with a targeted
external neutron beam) or delivery agents containing cytotoxic
compounds since the glucoconjugates are likely to be
transported into all cells expressing GLUT1albeit in
different amounts. In order to study the GLUT1 affinities of
the glucoconjugates, an experimental cis-inhibition assay was
devised using the human CAL 27 cell line. The CAL 27 cell
line represents a head and neck cancer type amenable to
treatment with BNCT. The overexpression of GLUT1 in CAL
27 is responsible for the aberrant growth of these tumors,
54,55
and therefore the GLUT1 targeting approach is warranted.
Before conducting the assays, we validated the GLUT1
function of CAL 27 (see Supporting Information). The cis-
inhibition assay was devised as a competition experiment
between the glucoconjugates and [14C]-D-glucose, with D-
glucose serving as a control. This experiment accurately mimics
the situation that the delivery agents would face in a biological
context. It has been previously speculated and shown that 6-O-
substituted glucoconjugates display a higher affinity to GLUT1
than free D-glucose.
53,56
In our current study, the affinities for
the glucoconjugates 1−3were in the low μM range in contrast
to the low mM-affinity displayed by free D-glucose. The exact
GLUT1 IC50-values were determined to be 43.96 μM for 1,
262.4 μM for 2, 15.2 μM for 3, and >1 mM for free D-glucose.
The 4−67 times stronger affinity displayed by the
glucoconjugates confirm that at least glucoconjugates 1and
3are capable of targeting GLUT1 in the intended application
despite the high glucose levels found in blood (6 mM).
In order to elucidate the interactions between the
glucoconjugates and the transporters on a molecular level,
we next turned to molecular modeling. We focused on the
differences in binding to GLUT1 and studied the protein−
ligand interactions of the glucoconjugates 1−3in the outward-
and inward-open conformations of the transporter, i.e., on the
outside and inside of the cell. To set up a computational
model, an experimental structure of the transporter was
required. However, only the crystal structure of the inward-
open conformation of GLUT1 has been reported.
57
In order to
perform the docking studies on equal footing for both the
inward and outward-open conformations, we created a model
based on XylE, a D-xylose-proton symporter found in E. coli for
which crystal structures of both the inward-open (PDB ID
4QIQ),
58
and outward open (PDB ID 6N3I)
59
conformations
exist. XylE is structurally very similar to the GLUT1−4
proteins (29% sequence identity and 49% similarity).
60
Importantly, the binding site residues are identical to
GLUT1 except for the Gln415 in XylE which is Asn411 in
GLUT1.
61,62
After virtual mutation of this residue, we
estimated binding energies by molecular docking studies. In
our models, the glucoconjugates 1−3bind significantly
stronger to both the outward- and inward-open binding sites
of the transporter than D-glucose, in line with the experimental
observations above. For the outward-open conformation, the
binding free energy difference is estimated to be up to 5 kcal/
mol in favor of the glucoconjugates, corresponding to a
binding affinity increase on the order of 103(see Supporting
Information for details).
There are a few observations of importance from the BNCT
delivery agent perspective. The estimated binding affinity of
each glucoconjugate is an order of magnitude lower in the
inward-open binding pocket than in the corresponding
outward-open one; that is, the ligand binds more tightly on
the outside. This is beneficial from a functional point-of-view,
as it implies that after the conformational change of the protein
from the outward-open to the inward-open conformation, the
ligand is more readily released to the inside of the cell. For D-
glucose, this difference in binding energy between the inward-
and outward-open conformations is absent.
For the outward-open structure, two major binding poses for
the glucoconjugates were identified. In one, the sugar end of
the glucoconjugate overlaps with the most favorable binding
pose of free glucose, while the carboranyl end extends toward
the hydrophobic end of the binding pocket; see Figure 3. This
agrees with the hydrophobic nature of the carboranes,
22
as
manifested by the almost neutral partial atomic charges of both
boron and hydrogen in the B−H bonds (see Supporting
Information). In the second pose, the glucoconjugate is slightly
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rotated. This pose is estimated to bind with practically equal
affinity (see Table S1).
In general, the binding pocket seems rather accommodating
from a structural point-of-view, with enough space for the
bulky carboranylmethyl substituent at the sixth position in
addition to the sugar. In order to corroborate this, we
performed a quantum mechanical noncovalent interaction
(NCI) analysis
34
on the ligand/transporter complex. The
analysis revealed favorable interactions between ligand and
protein at both ends of the glucoconjugate (Figure 3, top).
Importantly, no repulsive intramolecular steric interactions are
identified; even the largest of the boron cluster conjugates fits
snugly to the transporter pocket. The inward-open con-
formation, on the other hand, displays greater flexibility in the
binding site, which leads to a number of binding poses for both
glucose and the glucoconjugates. This might also explain the
lower binding affinity for the glucoconjugates to this site.
3.3. Cytotoxicity and Cellular Uptake Studies. With
the biochemical foundations of the GLUT1 targeting approach
investigated, we continued by addressing the cellular uptake
and cytotoxicity of the glucoconjugates since these are essential
properties of boron delivery agents and important factors for
eventual translation into the clinics. The CAL 27 cell line was
used in these studies because of its clinical relevance. In the
cytotoxicity assays, the glucoconjugates 1,2, and 3were
incubated with the cells at concentrations of 5 μM, 25 μM, 50
μM, 125 μM, and 250 μMfor6and24h.These
concentrations were chosen based on the affinity results and
the time points were selected with the clinical perspective
related to intravenous administration of the boron carriers in
mind. In these studies, the clinically deployed BSH was used as
a reference. BPA was omitted because its IC50-value has been
previously reported to be in the low mM range.
14
The cell
viability was quantified by the detection of ATP metabolic
activity-generated luminescence from the viable cells after
incubation using a commercially available Cell-Titer Glo assay.
The glucoconjugates 1−3displayed IC50-values in the μM
range and were consistently less toxic than BSH. The IC50-
values were obtained from nonlinear regression fitting of the
cell viability data and were found to be 214.8 μM for 1, 196.1
μM for 2, 276.6 μM for 3, and 98.7 μM for BSH (see Figure
4). From a toxicity standpoint, there is therefore no objection
to their use as delivery agents in BNCT.
Our final focus in this study was to determine if the
glucoconjugates are transported into the cells through GLUT1
or if they, at the very least, remain attached to the cells. This is
within the critical range required in clinical BNCT for the
generated alpha particles to exert a cell-killing effect. This
information was obtained by determining the boron content in
the CAL 27 cell lysates after incubation with the compounds
and careful washing. The development of a functioning
protocol featuring suitable incubation times, compound
concentrations, workup protocols, and methods for the robust
analysis of boron content required an extensive number of
trials. In the end, incubation times of 5 and 30 min were
selected based on the optimal performance of [14C]-D-glucose
under these conditions, and the concentration range 10−400
μM was selected based on the GLUT1 affinity results. The
ICP-MS instrument used in determination of the boron
content was found to be somewhat insensitive, and cells from
Figure 3. Glucoconjugate 3bound to the outward-open conformation
of the transporter. Top: Green areas indicate intramolecular
noncovalent interactions between 3and the protein. Bottom: The
closest amino acids surrounding the ligands, using PDB 6N3I
numbering; hydrogen bonds shown as blue dashes, the binding pose
of β-Glc superimposed in green color over the glucoconjugate.
Figure 4. Cell cytotoxicity studies in CAL 27 cells after incubation with negative (cell culture medium) and positive (1% Triton X-100) controls,
and glucoconjugates 1,2,3, and BSH at 5 μM, 25 μM, 50 μM, 125 μM, and 250 μM for 6 and 24 h. Error bars represent the mean ±s.d. (n=4)in
comparison with the negative control. The statistical hypothesis was evaluated by unpaired Student’st-test where the significant probabilities were
set at *p< 0.05, **p< 0.01, and ***p< 0.001.
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Mol. Pharmaceutics 2020, 17, 3885−3899
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four wells were combined and digested in order to obtain
results of high reliability. The results are summarized in Figure
5.
In addition to studying the uptake of glucoconjugates 1−3,
both BSH and BPA were included as representatives of
delivery agents in clinical use. All glucoconjugates delivered a
significantly higher boron content to the CAL 27 cells than
BPA and BSH across the entire concentration range. To a
certain degree, the observations may be explained by the
different uptake mechanisms of the glucoconjugates, BPA, and
BSH. Nevertheless, according to our preliminary assessment,
targeting GLUT1 translates into a competitive strategy for
BNCToutperforming the passive transport of BSH and the
LAT1-targeting approach of BPA in the in vitro cellular uptake
model used. Glucoconjugates 1and 3were found to have the
best boron delivery capacity, with 3being slightly better at the
5 min mark and 1being considerably better at the 30 min
mark. We note that the correlation to the GLUT1 affinity
results is perhaps weaker than one would expect. The
difference is natural when considering that the two methods
provide complementary insights on two separate properties: the
ability to compete with other GLUT1 substrates for the
transporter and the ability to remain attached to the cell or
internalized.
Lastly, in order to distinguish which of our two prime
candidates, glucoconjugates 1and 3, would be better suited for
future in vivo and potential preclinical BNCT studies, we
determined their aqueous solubility. This is an important factor
from the formulation and treatment perspective, as exemplified
by BPA which is administered as a fructose complex due to the
low aqueous solubility of BPA itself. Surprisingly, glucoconju-
gate 1displayed a significantly higher aqueous solubility than 3
(1 mg/mL vs <1 mg/500 mL). When this property is further
coupled with its high GLUT1 affinity, low cytotoxicity, and
outstanding in vitro delivery capacity, we are pleased to report
that 6-O-(ortho-carboranylmethyl)-D-glucopyranose, our “Tro-
jan horse”, has considerable potential as a delivery agent for
BNCT.
4. DISCUSSION
Head and neck cancers account for up to 10% of all cancers,
and the recurrent ones are accompanied by a poor survival rate
in patients.
3
BNCT has been successfully applied to the
treatment of head and neck cancers
63,64
and is currently
attracting large investments on a global scale due to the recent
development of in-hospital neutron accelerators which is a
game-changer from a patient treatment perspective.
In this work, we have designed and synthesized molecular-
scale “Trojan horses”, i.e. conjugates of glucose and boron
clusters containing a high boron content, and studied the
biochemical foundations of a GLUT1-targeting strategy to
BNCT. In more detail, we have used a chemistry-based
approach featuring both experimental and computational
methodologies. From the onset, important factors such as
the possible interference with glucose metabolism through the
glycolysis route was accounted for. In addition to addressing
the biochemical foundations of this approach, we have
identified a hit molecule which displays good cytocompati-
bility, sufficient aqueous solubility, and high cellular uptake in
the relevant human CAL 27 head and neck cancer cell line. In
our in vitro assessment, glucoconjugate 1was able to
outperform the current delivery agents in clinical use (BPA
and BSH) in terms of boron delivery capacity while
simultaneously having a sufficiently high affinity to GLUT1
to permit competition with the high levels of glucose found in
blood. Therefore, in addition to providing a missing link on the
biochemical foundations of a GLUT1-targeting strategy to
BNCTwe have identified a potentially promising new
glucoconjugate for clinical BNCT. With the solid basis
reported herein, the development of a suitable formulation
featuring the 10B-enriched version of glucoconjugate 1with
accompanied in vivo-testing is soon to follow. The results from
these studies will be reported in due course.
■ASSOCIATED CONTENT
*
sıSupporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.molpharma-
ceut.0c00630.
Information on additional synthetic routes, NMR-
spectra of the synthesized compounds, details on the
molecular docking studies and ligand PDBQT files, as
well as additional information on the characterization of
the GLUT1 function in the CAL 27 cell line and
affinity/uptake studies (PDF)
Figure 5. Cell uptake studies in the CAL 27 cell line after incubation with glucoconjugates 1(▲), 2(■), 3(●), BPA (⧫), and BSH (▼) in the
10−400 μM range for 5 min (A; n= 3) and 30 min (B; n= 3). The Michaelis−Menten kinetic parameters for glucoconjugates when available: At 5
min incubation time (A), glucoconjugate 2:Vmax = 2.791; Km= 80.49. At 30 min incubation time (B), glucoconjugate 1:Vmax = 16.29; Km= 630.5,
glucoconjugate 2:Vmax = 6.417; Km= 488.3, glucoconjugate 3:Vmax = 16.89; Km= 894.2, and BPA: Vmax = 3.625; Km= 3737.
Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics Article
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Mol. Pharmaceutics 2020, 17, 3885−3899
3896
■AUTHOR INFORMATION
Corresponding Author
Filip S. Ekholm −Department of Chemistry, University of
Helsinki, Finland, FI-00014 Helsinki, Finland; orcid.org/
0000-0002-4461-2215; Email: filip.ekholm@helsinki.fi
Authors
Jelena Matović
−Department of Chemistry, University of
Helsinki, Finland, FI-00014 Helsinki, Finland
Juulia Järvinen −School of Pharmacy, University of Eastern
Finland, FI-70211 Kuopio, Finland
Helena C. Bland −Department of Chemistry, University of
Helsinki, Finland, FI-00014 Helsinki, Finland
Iris K. Sokka −Department of Chemistry, University of Helsinki,
Finland, FI-00014 Helsinki, Finland; orcid.org/0000-0002-
5148-4987
Surachet Imlimthan −Department of Chemistry, University of
Helsinki, Finland, FI-00014 Helsinki, Finland; orcid.org/
0000-0003-2520-2146
Ruth Mateu Ferrando −Department of Chemistry, University
of Helsinki, Finland, FI-00014 Helsinki, Finland
Kristiina M. Huttunen −School of Pharmacy, University of
Eastern Finland, FI-70211 Kuopio, Finland; orcid.org/
0000-0002-1175-8517
Juri Timonen −School of Pharmacy, University of Eastern
Finland, FI-70211 Kuopio, Finland; orcid.org/0000-0001-
7720-2215
Sirpa Peräniemi −School of Pharmacy, University of Eastern
Finland, FI-70211 Kuopio, Finland
Olli Aitio −Glykos Finland Ltd., FI-00790 Helsinki, Finland
Anu J. Airaksinen −Department of Chemistry, University of
Helsinki, Finland, FI-00014 Helsinki, Finland; Turku PET
Centre, Department of Chemistry, University of Turku, FI-
20521 Turku, Finland; orcid.org/0000-0002-5943-3105
Mirkka Sarparanta −Department of Chemistry, University of
Helsinki, Finland, FI-00014 Helsinki, Finland; orcid.org/
0000-0002-2956-4366
Mikael P. Johansson −Department of Chemistry, University of
Helsinki, Finland, FI-00014 Helsinki, Finland; Helsinki
Institute of Sustainability Science, HELSUS, FI-00014 Helsinki,
Finland; orcid.org/0000-0002-9793-8235
Jarkko Rautio −School of Pharmacy, University of Eastern
Finland, FI-70211 Kuopio, Finland
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.molpharmaceut.0c00630
Author Contributions
ϕ
J.M. and J.J. contributed equally. F.S.E. and O.A. conceived
the idea and F.S.E. designed the project and led the initiate
with input from O.A., A.A., M.P.J., M.S., and J.R. J.M., H.C.B.,
R.M.F., and F.S.E. contributed to the synthesis and structural
characterization of the glucoconjugates. M.S. and S.I. were
responsible for the cytotoxicity testing. M.P.J. and I.K.S. were
responsible for the molecular modeling aspects and analysis.
J.J., K.H., J.T., S.P., and J.R. were responsible for the
experimental affinity and cellular uptake studies. The manu-
script was written with contributions from all authors.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
Jussi Kärkkäinen (University of Eastern Finland), Ronja Rättö
(University of Helsinki), and Joonas Waaramaa (University of
Helsinki) are acknowledged for laboratory assistance, and
Hé
lder A. Santos and Alexandra Correia (University of
Helsinki) are acknowledged for their contributions to the
cytotoxicity studies. CSC-The Finnish IT Center for Science
and the Finnish Grid and Cloud Infrastructure (urn:nbn:fi:r-
esearch-infras-2016072533) are acknowledged for providing
the computational resources required for this work. Further,
the authors are grateful for financial support from the Jane and
Aatos Erkko foundation, the Cancer foundation, the Swedish
Cultural foundation, the Ruth and Nils-Erik Stenbäck
foundation, the Finnish Academy of Science and Letters, the
University of Helsinki research funds, the Academy of Finland
(projects: 308329, 318422, 289179, and 319453), and the
Waldemar von Frenckell foundation.
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