Exploring the Biochemical Foundations of a Successful GLUT1-Targeting Strategy to BNCT: Chemical Synthesis and in Vitro Evaluation of the Entire Positional Isomer Library of ortho-Carboranylmethyl-Bearing Glucoconjugates

Article abstract of DOI:10.1021/acs.molpharmaceut.0c00917

Boron neutron capture therapy (BNCT) is a noninvasive binary therapeutic modality applicable to the treatment of cancers. While BNCT offers a tumor-targeting selectivity that is difficult to match by other means, the last obstacles preventing the full har

Full text of DOI:10.1021/acs.molpharmaceut.0c00917

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Article
Exploring the Biochemical Foundations of a Successful GLUT1-
Targeting Strategy to BNCT: Chemical Synthesis and In Vitro
Evaluation of the Entire Positional Isomer Library of ortho-
Carboranylmethyl-Bearing Glucoconjugates
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Jelena Matovic, Juulia Jarvinen, Iris K. Sokka, Surachet Imlimthan, Jan-Erik Raitanen,
̈
Ahmed Montaser, Hannu Maaheimo, Kristiina M. Huttunen, Sirpa Peraniemi, Anu J. Airaksinen,
Mirkka Sarparanta, Mikael P. Johansson, Jarkko Rautio, and Filip S. Ekholm*
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ABSTRACT: Boron neutron capture therapy (BNCT) is a
noninvasive binary therapeutic modality applicable to the treat-
ment of cancers. While BNCT offers a tumor-targeting selectivity
that is difficult to match by other means, the last obstacles
preventing the full harness of this potential come in the form of the
suboptimal boron delivery strategies presently used in the clinics.
To address these challenges, we have developed delivery agents
that target the glucose transporter GLUT1. Here, we present the
chemical synthesis of a number of ortho-carboranylmethyl-
substituted glucoconjugates and the biological assessment of all
positional isomers. Altogether, the study provides protocols for the
synthesis and structural characterization of such glucoconjugates and insights into their essential properties, for example, cytotoxicity,
GLUT1-affinity, metabolism, and boron delivery capacity. In addition to solidifying the biochemical foundations of a successful
GLUT1-targeting approach to BNCT, we identify the most promising modification sites in D-glucose, which are critical in order to
further develop this strategy toward clinical use.
KEYWORDS: boron neutron capture therapy, cancer therapeutics, carbohydrates, drug delivery, glucose transporters, medicinal chemistry
present in a cellular environment, and the high-energy α
particles (4He) generated in the fission reactions have a path
length of 5−9 μm in tissue, that is, the diameter of a single
cell.5,6 Previously, nuclear reactors have been used as the sole
neutron source in clinical BNCT. This has not only been
suboptimalit has been straight-out inconvenient from the
clinical practice standpoint, which is reflected in the fact that
there is not a single phase III clinical study performed to date.
To answer the needs of modern BNCT, in-hospital neutron
sources have recently begun to emerge.7,8 The time to revisit
the development of novel boron delivery agents is now at hand.
While boron delivery agents have been developed for the
better part of a century,9 only three delivery agents are in
actual clinical use, namely, boronophenylalanine (BPA),10
1. INTRODUCTION
Cancer is among the leading causes of death in modern society
and thus constitutes a significant societal burden. Despite the
continuous progress made in radio- and chemotherapies, there
are still a vast number of patients with untreatable as well as
recurring cancers. Head and neck cancers account for 10% of
all cancers with 630,000 new cases diagnosed annually on a
global scale.1,2 While a combination of surgery and chemo-
therapy remains the primary method for treatment of these
cancers, the conventional therapies face significant challenges
as the percentage of inoperable and recurrent cancers in this
category (20−40%) is exceptionally high.2,3 In these cases,
there is a clear need for novel and improved treatment options.
Among the existing treatment possibilities available, boron
neutron capture therapy (BNCT) stands out as one of the
most promising alternatives.4
Received: September 10, 2020
Revised: November 17, 2020
Accepted: November 18, 2020
BNCT is a combinatorial treatment featuring accumulation
of 10B-atoms in malignant tissue, followed by irradiation with
an external neutron beam in a narrow region (see Figure 1). In
theory, an unrivalled selectivity can be achieved because the
cross section of 10B is far greater than those of other elements
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gates have been conducted under identical conditions, thus
allowing direct comparison of the results. The need for the
study is evident as our results do not fully match those of
Lippard et al.,24 which implies that great care should be taken
when interpreting or comparing results obtained with
glucoconjugates bearing different substituents. Last, and most
importantly, every single glucoconjugate synthesized displayed
improved properties over the agents in current clinical use,
thereby indicating that a correctly designed GLUT1-targeting
approach to BNCT is a viable alternative to boron delivery
agents used clinically at present.
2. EXPERIMENTAL SECTION
Figure 1. Principle of BNCT is showcased together with the different
delivery strategies of boronophenylalanine (BPA), sodium borocap-
tate (BSH), and glucoconjugate 3. Stage 1: Boron delivery agents
enter the cancer cells. Stage 2: Irradiation by a precise neutron beam.
Stage 3: 10B captures a neutron and a fission reaction occurs. Stage 4:
4He destroys the cancer cell.
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, 13C:
125.76 MHz, 11B: 160.46 MHz). The probe temperature
during the experiments was kept at 23 °C, unless otherwise
stated. All products were characterized by utilization of the
sodium borocaptate (BSH),11 and decahydrodecaborate (GB-
10).12 None of these agents give good tumor-to-blood (T/B)
or tumor-to-normal tissue (T/N) ratios, which leads to the
conclusion that new delivering strategies may be required in
order to improve the overall treatment prospects of
BNCT.6,13−15 The development of delivery agents for BNCT
is challenging, and the requirements on the end products are
numerous: sufficient aqueous solubility, minimal systemic
toxicity, a minimum cellular uptake of 20−35 μg/g of the
tumor, high tumor/normal tissue (T/N) and tumor/blood
(T/B) ratios, and a detailed understanding on the behavior of
the delivery agents in a biological setting.9
Our approach to target all of the current deficiencies of
boron delivery agents is based on the potential embedded in
glycoconjugates which target the glucose transporter
GLUT1.16,17 The biochemical foundation of our GLUT1-
targeting strategy lies in the nature of the “Warburg effect”,18,19
that is, the increased expression of GLUT1 and uptake of D-
glucose observed in head and neck cancers, analogous to most
other tumors. This Warburg effect stems from the impaired
and inefficient aerobic glucose metabolism observed in
malignant cells, where it is closely linked to the aberrant
growth of tumors.20 In contrast to the majority of previous
studies reported on boron-glucoconjugates,21,22 our emphasis
is on critically assessing the biochemical foundations of this
approach from a BNCT point-of-view. Recently, we reported
promising results which indicated that an appropriately
designed GLUT1-targeting strategy can outperform both the
LAT1-targeting strategy of BPA and the passive transport
strategies of BSH and GB-10 (see Figure 1).16
Here, we significantly extend the initial exploratory studies
by synthesizing and studying the cytotoxicity, GLUT1 affinity,
and basis thereof, as well as cellular uptake displayed by the
entire positional isomer library of ortho-carboranylmethyl-
substituted D-glucoconjugates in the relevant human oral
adenosquamous carcinoma cell line CAL 27. In addition to
these studies, we have addressed the metabolic fate of these
glucoconjugates through an NMR-spectroscopic evaluation
and ruled out the possibility for these substrates to enter the
glycolysis or pentose phosphate metabolic pathways.23 This
study is central to laying the foundations of the GLUT1-
targeting approach to BNCT, as the preclinical in vitro
evaluations of the previously reported16 and new glucoconju-
following 1D-techniques: H, 13C, H-decoupled 11B and 1D-
TOCSY and the following 2D-techniques: Ed-HSQC, HMBC,
and DQF-COSY by using pulse sequences provided by the
instrument manufacturer. Chemical shifts are expressed on the
δ scale (in ppm) using TMS (tetramethylsilane), residual
chloroform, methanol, or 15% BF3 in CDCl3 (11B NMR) as
internal standards. Coupling constants have been obtained
through spectral simulations with the PERCH (PEak
reseaRCH) software and are given in Hz and provided only
once, when first encountered. Coupling patterns are given as s
(singlet), d (doublet), t (triplet), and so forth. High-resolution
mass spectrometry (HRMS) was 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. The substrate-specific analytical data are
1
1
1
provided below and representative H, 13C and 11B NMR
spectra are supplied in the Supporting Information. TLC was
performed on aluminium 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 (1:5) and
heating.
2.2. Experimental Procedures. The general synthetic
protocols applied to the synthesis of glucoconjugates 1−5 are
described below. The synthetic routes leading to the new
glucoconjugates 3 and 5 are presented in Scheme 1 and
discussed in detail in the Results and Discussion section, while
the slightly modified synthetic routes employed to the
synthesis of previously reported glucoconjugates 1, 2, and 4
are presented in Supporting Scheme S1 in the Supporting
Information and not discussed at all. The protocols employed
to the synthesis of 6 were discussed recently and have therefore
been omitted from the present study.16
2.2.1. General Procedure for Glycosylation of Glucose. To
a solution of D-glucose (1.0 equiv) in propargyl alcohol (5 mL/
1 g of sugar), DOWEX 50 H+-form was added (1 g/1 g of
sugar), and the resulting mixture was refluxed for 17 h at 100
°C. The reaction mixture was brought to r.t., filtered, and
B
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concentrated under reduced pressure. The crude product was
purified by column chromatography (DCM/MeOH 3:1).
2.2.2. General Procedure for Stereoselective Acetylation
of Glucose. A suspension of NaOAc (0.5 g/1 g of sugar) in
Ac2O (7 mL/1 g of sugar) was gently heated to reflux followed
by the addition of D-glucose (1.0 equiv). After the addition was
completed, the reaction mixture was heated at reflux for 30
min. The mixture was then brought to r.t., poured onto an
ice−water bath, and stirred for 2 h with external cooling. The
precipitate was filtered, giving the crude product which was
purified by recrystallization from ethanol.
2.2.3. General Procedure for Glycosylation of Peracety-
lated Glucose. To a solution of the peracetylated glucopyr-
anose (1.0 equiv) in dry DCM (9 mL/1 g of starting material),
propargyl alcohol or BnOH (4 equiv) and preactivated 4 Å
molecular sieves were added. The reaction mixture was cooled
on an ice-bath and BF3·OEt2 (6−8 equiv) was added dropwise.
The mixture was left in the ice-bath for 20 min after which it
was stirred for 17 h at r.t. The reaction mixture was then
diluted with DCM (12 mL/1 g of starting material), poured
into ice−water, and stirred, and then the aqueous layer was
decanted off. A satd. aq solution of NaHCO3 (12 mL/1 g of
starting material) was added to the mixture, the aqueous layer
was decanted off, and the organic layer was filtered through
Celite, washed with brine (12 mL/1 g of starting material),
dried over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure. The crude product was purified by column
chromatography (EtOAc/hexane 1:5).
2.2.4. General Procedure for Deacetylation. To an ice−
water-cooled solution of the corresponding glucopyranoside
(1.0 equiv) in MeOH/THF (5:1, 7 mL/1 g of starting
material), NaOMe (1.0 equiv) was added until the pH ∼ 10.
The reaction mixture was stirred for 1−17 h at r.t., followed by
the addition of Dowex 50 H+-form until the pH was neutral.
The reaction mixture was filtered and concentrated under
reduced pressure. The crude product was purified by column
chromatography (DCM/MeOH 5:1).
2.2.5. General Procedure for Alkylation of Free Hydroxyl
Groups. To an ice−water-cooled solution of the partially
protected sugar (1.0 equiv) in dry DMF (2 mL/100 mg of
starting material), NaH (2.3 equiv/free OH group) was added
portionwise. The mixture was brought to r.t., and the
corresponding alkyl bromide was added (1.8 equiv/free OH
group). The resulting mixture was stirred for 1.5 h, quenched
with MeOH (0.4 mL/1 mmol of starting material), diluted
with DCM (50 mL/1 g of starting material), and washed with
a satd. aq solution of NaHCO3 (30 mL/1 g of starting
material). The organic phase was separated, and the aqueous
phase was extracted with DCM (3 × 30 mL/1 g of starting
material). The combined organic phases were washed with
brine (30 mL/1 g of starting material), dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure.
The crude product was purified by column chromatography
(EtOAc/hexane 1:3).
brought to r.t., and concentrated under reduced pressure. The
crude product was purified by column chromatography
(EtOAc/hexane 1:3).
2.2.7. General Procedure for Deprotection of Benzyl
Groups. To a solution of the protected glucoside (1.0 equiv) in
dry EtOAc/MeOH (7:1, 1 mL/10−15 mg of starting
material), Pd/C (10% Pd, 1.0 mass equiv) was added. The
reaction mixture was stirred in an autoclave under H2-pressure
(4 bar) for 4 h and filtered through Celite. The Celite was
washed with EtOAc/MeOH (5:1, 1 mL/10−15 mg of starting
material), and the filtrate concentrated under reduced pressure.
The crude product was purified by column chromatography
(EtOAc/MeOH 5:1).
2.2.8. General Procedure for Installation of 4,6-O-
benzylidene Acetals. To a solution of the corresponding
deacetylated glucopyranoside (1 equiv) in DMF (1 mL/40 mg
starting material), PTSA (10 mol %) and benzaldehyde
dimethyl acetal (1 equiv) were added. The resulting mixture
was stirred at 60 °C and 200 mbar for 2 h and concentrated.
The crude product was purified by column chromatography
(DCM/MeOH 5:1).
2.2.9. General Procedure for Selective Ring-Opening of
the Benzylidene Acetal to Give the 4-OH/6-OBn Substrate.
To a solution containing the corresponding 4,6-O-benzylidene
acetal (1 equiv) in DCM (6 mL/0.5 g of starting material),
Et3SiH (5 equiv) was added, followed by dropwise addition of
TFA (5 equiv) at 0 °C. The resulting mixture was brought to
r.t., stirred for 2 h, diluted with EtOAc (20 mL/0.5 g), and
washed with satd. aq solution of NaHCO3 (20 mL/0.5 g) and
brine (20 mL/0.5 g). The organic phase was separated, dried
over Na2SO4, filtered, and concentrated. The crude product
was purified by column chromatography (EtOAc/hexane 2:3).
2.2.10. General Procedure for 1,2-Orthoester Formation.
To a solution containing peracetylated glucose in DCM (12
mL/g of starting material), I2 (1.4 equiv), and Et3SiH (1.4
equiv) were added, and the mixture was refluxed for 1 h. After
1 h, the mixture was brought to r.t. and 2,6-lutidine (4 equiv),
MeOH (6 equiv), and TBAI (0.25 equiv) were added. The
mixture was refluxed for 3 h, then concentrated, and dissolved
in EtOAc. The resulting mixture was extracted with 5% aq
Na2S2O3 (3 × 6 mL/g of starting material) and washed with
EtOAc (4 mL/g of starting material). The organic phase was
separated, dried over Na2SO4, filtered, and concentrated. The
crude product was purified by column chromatography
(EtOAc/hexane 1:2).
2.2.11. General Procedure for Opening of the 1,2-
Orthoester. To a solution containing the 1,2-orthoester (1
equiv) in acetone/H2O 7:3 (15 mL/g of starting material), p-
TsOH (0.4 equiv) was added at 0 °C. The mixture was
brought to r.t. and left to stir for 2 h after which it was
neutralized with TEA and concentrated under reduced
pressure. The crude product was purified by column
chromatography (EtOAc/hexane 1:2).
2.2.12. General Procedure for the Formation of an
Imidate Donor. To a solution containing the corresponding
pyranose in DCM (20 mL/g of starting material), Cl3CCN (10
equiv) and DBU (0.25 equiv) were added. The mixture was
left to stir for 1 h at r.t. after which it was diluted with DCM
(10 mL/g of starting material) and washed with brine (20 mL/
g of starting material). The organic phase was separated, dried
over Na2SO4, filtered, and concentrated. The crude product
was purified by column chromatography (EtOAc/hexane 2:3
containing 0.01% TEA).
2.2.6. General Procedure for Coupling with Decaborane.
Decaborane (B10H14, 1.7 equiv) and dry acetonitrile (5 mL/
150 mg of starting material) were stirred under reflux for 1 h at
60 °C to form the B10H12·2CH3CN adduct. Separately, the
propargylated glucoconjugate (1.0 equiv) was dissolved in dry
toluene (5 mL/150 mg of starting material) and added to the
reaction mixture, which was left to reflux at 80 °C for 17 h.
The mixture was quenched with dry MeOH (1.5 mL/150 mg
of starting material), refluxed at 80 °C for another 30 min,
C
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2.2.13. General Procedure for Glycosylation Using
TMSOTf. To a solution containing the corresponding acceptor
(1 equiv) in DCM (2.8 mL/100 mg of starting material),
preactivated 4 Å molecular sieves were added and the mixture
was cooled to −20 °C. TMSOTf (2 equiv) was added and the
mixture was stirred for 10 min. The corresponding donor (1.4
equiv) dissolved in DCM (2.8 mL/100 mg of starting
material) was added dropwise to the solution. The reaction
mixture was left to stir for 2 h at −20 °C after which it was
brought to r.t., diluted with DCM (2 mL/g of starting
material), and washed with a satd. aq solution of NaHCO3 (2
mL/g of starting material) and brine (2 mL/g of starting
material). The organic phase was separated, dried over
Na2SO4, filtered, and concentrated. The crude product was
purified by column chromatography (EtOAc/hexane 1:4).
2.2.14. General Procedure for the Installation of 1,2:4,6-
Di-O-isopropylidene Acetals. To a solution containing D-
glucose (1 equiv) in acetone (50 mL/g of starting material), I2
(0.2 equiv) was added, and the mixture was left to stir at r.t. for
3.5 h after which the mixture was quenched with 5% aq
Na2S2O3 (8.6 mL/g of starting material). Acetone was
removed under reduced pressure, and the resulting mixture
was extracted with DCM (3 × 9 mL/g of starting material).
The organic phase was further washed with a satd. aq solution
of NaHCO3 (9 mL/g of starting material), separated, dried
over Na2SO4, filtered, and concentrated. The crude product
was purified by recrystallization from petroleum ether.
2.2.15. General Procedure for 1,2:4,6-Di-O-isopropylidene
Acetal Removal. The corresponding protected sugar (1 equiv)
was dissolved in H2O/TFA 1:1 (14 mL/g of starting material)
and left to stir at r.t. for 21 h. The resulting mixture was
concentrated under reduced pressure and the crude product
was purified by column chromatography (DCM/MeOH 5:1)
to give the desired product.
for glycosylation of peracetylated glucose to give propargyl
2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside. This product was
deprotected according to the general procedure for deacety-
lation to give the title compound as a white solid (2.35 g, yield
over two steps 52%). TLC: Rf: 0.54 (DCM/MeOH 5:1).
1H NMR (500.13 MHz, CD3OD, 23 °C): δ = 4.46 (d, 1H,
J
1,2 = 7.8 Hz, H-1), 4.44 (dd, 1H, JCH ,CH = −15.6, JCH
=
2a,CH
2a
2b
−2.4 Hz, 1-OCH2aCCH), 4.41 (dd, 1H, JCH ,CH = −2.4 Hz,
2b
1-OCH2bCCH), 3.87 (dd, 1H, J6a,6b = −12.0, J5,6a = 2.1 Hz,
H-6a), 3.66 (dd, 1H, J6b,5 = 5.7 Hz, H-6b), 3.36 (dd, 1H, J3,2
=
9.3, J3,4 = 8.9 Hz, H-3), 3.28 (dd, 1H, J4,5 = 8.9 Hz, H-4), 3.27
(ddd, 1H, H-5), 3.20 (dd, 1H, H-2), and 2.86 (dd, 1H, 1-
OCH2CCH) ppm.
13C NMR (125.76 MHz, CD3OD, 23 °C): δ = 102.1 (C-1),
80.1 (1-OCH2CCH), 78.1 (C-5), 78.0 (C-3), 76.2 (1-
OCH2CCH), 74.9 (C-2), 71.6 (C-4), 62.7 (C-6), and 56.5
(1-OCH2CCH) ppm.
HRMS m/z: calcd for C9H14O6Na [M + Na]+, 241.0688;
found, 241.0720.
2.3.3. Propargyl 2,3,4,6-Tetra-O-benzyl-β-D-glucopyrano-
side. Synthesized from 15 (0.81 g, 3.7 mmol), NaH (0.62 g,
26.0 mmol), and BnBr (4.176 g, 24.0 mmol) according to the
general procedure for alkylation of free hydroxyl groups. The
title compound was obtained as a white solid (1.78 g, 82%).
TLC: Rf: 0.54 (EtOAc/hexane 1:3).
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 7.40−7.13 (m,
20H, arom. H), 4.97 and 4.69 (each d, each 1H, J = −10.8 Hz,
2-OCH2Ph), 4.93 and 4.78 (each d, each 1H, J = −10.9 Hz, 3-
OCH2Ph), 4.82 and 4.53 (each d, each 1H, J = −10.8 Hz, 4-
OCH2Ph), 4.63 (d, 1H, J1,2 = 7.8 Hz, H-1), 4.61 and 4.54
(each d, each 1H, J = −12.2 Hz, 6-OCH2Ph), 4.46 (dd, 1H,
JCH
= −15.8, JCH ,CH = −2.5 Hz, 1-OCH2aCCH), 4.41
2a,CH2b
2a
(dd, 1H, JCH ,CH = −2.4 Hz, 1-OCH2bCCH), 3.74 (dd, 1H,
2b
2.3. Substrate-Specific Analytical Data. In this section,
the substrate-specific analytical data featuring synthetic
protocol employed, reaction scale and yield, and NMR and
MS characterization data are supplied for all intermediates and
end products on the synthetic routes to 1−5.
2.3.1. 1,2,3,4,6-Penta-O-acetyl-β-D-glucopyranoside. Syn-
thesized from D-glucose (15.09 g, 84.0 mmol) and Ac2O
(113.40 g, 1.1 mol) according to the general procedure for
stereoselective acetylation of glucose. This reaction gave the
title compound as a white solid (18.52 g, 57%). TLC: Rf: 0.68
(EtOAc/hexane 1:1).
J6a,6b = −10.8, J6a,5 = 1.8 Hz, H-6a), 3.69 (dd, 1H, J6b,5 = 4.6
Hz, H-6b), 3.66 (dd, 1H, J3,2 = 9.0, J3,4 = 9.2 Hz, H-3), 3.62
(dd, 1H, J4,5 = 9.6 Hz, H-4), 3.49 (dd, 1H, H-2), 3.47 (ddd,
1H, H-5), and 2.46 (dd, 1H, 1-OCH2CCH) ppm.
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 138.7−127.7
(arom. C), 101.6 (C-1), 84.7 (C-3), 82.1 (C-2), 79.2 (1-
OCH2CCH), 77.8 (C-4), 75.9 (3-OCH2Ph), 75.1 (4-
OCH2Ph and 1-OCH2CCH), 75.0 (C-5), 74.9 (2-
OCH2Ph), 73.6 (6-OCH2Ph), 68.9 (C-6), and 56.1 (1-
OCH2CCH) ppm.
HRMS m/z: calcd for C37H38O6Na [M + Na]+, 601.2566;
found, 601.2551.
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 5.72 (d, 1H, J1,2
= 8.3 Hz, H-1), 5.25 (dd, 1H, J3,2 = 9.6, J3,4 = 9.4 Hz, H-3),
5.14 (dd, 1H, H-2), 5.13 (dd, 1H, J4,5 = 10.1 Hz, H-4), 4.29
(dd, 1H, J6a,5 = 4.5, J6a,6b = −12.5 Hz, H-6a), 4.12 (dd, 1H, J6b,5
= 2.1 Hz, H-6b), 3.84 (ddd, 1H, H-5), 2.12 (s, 3H, 1-
OCOCH3), 2.09 (s, 3H, 6-OCOCH3), 2.04 (s, 6H, 2-
OCOCH3 and 4-OCOCH3), and 2.02 (3-OCOCH3) ppm.
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 170.7 (6-
OCOCH3), 170.2 (3-OCOCH3), 169.5 (4-OCOCH3), 169.3
(2-OCOCH3), 169.0 (1-OCOCH3), 91.9 (C-1), 73.0 (C-3),
72.9 (C-5), 70.4 (C-2), 67.9 (C-4), 61.6 (C-6), 20.9 (1-
OCOCH3), 20.8 (6-OCOCH3), and 20.7 (2-OCOCH3, 3-
OCOCH3, and 4-OCOCH3) ppm.
2.3.4. (O)-Carboranylmethyl 2,3,4,6-Tetra-O-benzyl-β-D-
glucopyranoside (16). Synthesized from propargyl 2,3,4,6-
tetra-O-benzyl-β-D-glucopyranoside (0.83 g, 1.4 mmol) and
B10H14 (0.33 g, 2.7 mmol) according to the general procedure
for coupling with decaborane. The title compound was
obtained as a white solid (0.64 g, 64%). TLC: Rf: 0.49
(EtOAc/hexane 1:3).
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 7.38−7.13 (m,
20H, arom. H), 4.89 and 4.81 (each d, each 1H, J = −11.0 Hz,
3-OCH2Ph), 4.79 and 4.52 (each d, each 1H, J = −10.5 Hz, 4-
OCH2Ph), 4.77 and 4.76 (each d, each 1H, J = −11.5 Hz, 2-
OCH2Ph), 4.56 and 4.50 (each d, each 1H, J = −12.1 Hz, 6-
OCH2Ph), 4.32 (d, 1H, J1,2 = 7.7 Hz, H-1), 4.21 and 4.04
(each d, each 1H, J = −11.5 Hz, 1-OCH2−carborane), 4.00
HRMS m/z: calcd for C16H22O11Na [M + Na]+, 413.1060;
found, 413.1030.
2.3.2. Propargyl β-D-Glucopyranoside (15). Synthesized
from 1,2,3,4,6-penta-O-acetyl-β-D-glucopyranoside (8.04 g,
21.0 mmol, 1.0 equiv) according to the general procedure
(br s, 1H, carborane−CH), 3.66 (dd, 1H, J6a,6b = −10.5, J6a,5 =
1.9 Hz, H-6a), 3.63 (dd, 1H, J6b,5 = 4.6 Hz, H-6b), 3.62 (dd,
1H, J3,2 = 9.0, J3,4 = 8.9 Hz, H-3), 3.60 (dd, 1H, J4,5 = 9.6 Hz,
D
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H-4), 3.42 (dd, 1H, H-2), 3.41 (ddd, 1H, H-5), and 3.00−1.45
(br m, 10H, carborane−BH) ppm.
(each d, each 1H, J = −10.9 Hz, 3-OCH2Ph), 4.83 and 4.47
(each d, each 1H, J = −10.8 Hz, 4-OCH2Ph), 4.76 and 4.71
(each d, each 1H, J = −12.0 Hz, 2-OCH2Ph), 4.60 and 4.47
(each d, each 1H, J = −12.4 Hz, 6-OCH2Ph), 4.27 (dd, 1H,
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 138.4−127.9
(arom. C), 103.2 (C-1), 84.7 (C-3), 81.7 (C-2), 77.6 (C-4),
75.8 (3-OCH2Ph), 75.2 (2-OCH2Ph and 4-OCH2Ph), 75.0
(C-5), 73.7 (6-OCH2Ph), 72.5 (carborane−C), 70.9 (1-
OCH2−carborane), 68.6 (C-6), and 58.2 (carborane−CH)
ppm.
JCH
= −2.7, JCH
= −15.8 Hz, 1-OCH2aCCH), 4.27
2a,CH
2a,CH2b
(dd, 1H, JCH ,CH = −2.2 Hz, 1-OCH2bCCH), 3.98 (dd, 1H,
2b
J
3,2 = 9.7, J3,4 = 9.1 Hz, H-3), 3.78 (ddd, 1H, J5,4 = 10.0, J5,6a =
11B NMR (160.46 MHz, CDCl3, 23 °C): δ = −2.5, −4.4,
−9.0, −11.6 and −12.9 ppm.
3.6, J5,6b = 2.0 Hz, H-5), 3.72 (dd, 1H, J6a,6b = −10.9 Hz, H-
6a), 3.65 (dd, 1H, H-4), 3.63 (dd, 1H, H-6b), 3.61 (dd, 1H,
H-2), and 2.43 (dd, 1H, 1-OCH2CCH) ppm.
HRMS m/z: calcd for C37H48B10O6Na [M + Na]+,
721.4279; found, 721.4357.
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 138.9−128.7
(arom. C), 95.4 (C-1), 82.1 (C-3), 79.5 (C-2), 79.1 (1-
OCH2CCH), 77.6 (C-4), 75.9 (3-OCH2Ph), 75.2 (4-
OCH2Ph), 74.9 (1-OCH2CCH), 73.6 (6-OCH2Ph), 73.1
(2-OCH2Ph), 70.9 (C-5), 68.5 (C-6), and 54.5 (1-OCH2C
CH) ppm.
2.3.5. (O)-Carboranylmethyl β-D-Glucopyranoside (1).
Synthesized from 16 (0.10 g, 0.1 mmol) and 10% Pd/C
(0.01 g, 0.1 mmol) according to the general procedure for
deprotection of benzyl groups. This reaction gave the title
compound as a white solid (0.04 g, 86%). TLC: Rf: 0.55
(EtOAc/MeOH 5:1).
HRMS m/z: calcd for C37H38O6Na [M + Na]+, 601.2566;
found, 601.2581.
1H NMR (500.13 MHz, CD3OD, 23 °C): δ = 4.78 (br s,
1H, carborane−CH), 4.35 and 4.12 (each d, each 1H, J =
−11.5 Hz, 1-OCH2−carborane), 4.26 (d, 1H, J1,2 = 7.9 Hz, H-
1), 3.86 (dd, 1H, J6a,6b = −11.8, J6a,5 = 2.0 Hz, H-6a), 3.64 (dd,
1H, J6b,5 = 5.7 Hz, H-6b), 3.32 (dd, 1H, J3,2 = 9.2, J3,4 = 8.9 Hz,
H-3), 3.26 (dd, 1H, J4,5 = 9.3 Hz, H-4), 3.26 (ddd, 1H, H-5),
3.18 (dd, 1H, H-2), and 2.98−1.46 (br m, 10H, carborane−
BH) ppm.
2.3.8. (O)-Carboranylmethyl 2,3,4,6-Tetra-O-benzyl-α-D-
glucopyranoside. Synthesized from 18 (0.63 g, 1.1 mmol) and
B10H14 (0.25 g, 2.1 mmol) according to the general procedure
for coupling with decaborane. This reaction gave the title
compound as a colorless oil (0.45 g, 59%). TLC: Rf: 0.44
(EtOAc/hexane 1:3).
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 7.36−7.12 (m,
20H, arom. H), 4.93 and 4.84 (each d, each 1H, J = −10.9 Hz,
3-OCH2Ph), 4.81 and 4.47 (each d, each 1H, J = −10.7 Hz, 4-
OCH2Ph), 4.77 and 4.55 (each d, each 1H, J = −11.6 Hz, 2-
OCH2Ph), 4.67 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.56 and 4.47
(each d, each 1H, J = −12.1 Hz, 6-OCH2Ph), 4.19 (br s, 1H,
carborane−CH), 4.04 and 3.78 (each d, each 1H, J = −11.2
Hz, 1-OCH2−carborane), 3.84 (dd, 1H, J3,2 = 9.6, J3,4 = 9.1
Hz, H-3), 3.67 (dd, 1H, J6a,6b = −10.7, J6a,5 = 3.8 Hz, H-6a),
3.63 (ddd, 1H, J5,4 = 9.9, J5,6b = 1.8 Hz, H-5), 3.61 (dd, 1H, H-
4), 3.60 (dd, 1H, H-6b), 3.54 (dd, 1H, H-2), and 2.87−1.44
(br m, 10H, carborane−BH) ppm.
13C NMR (125.76 MHz, CD3OD, 23 °C): δ = 104.5 (C-1),
78.2 (C-5), 77.8 (C-3), 74.8 (C-2), 74.6 (carborane−C), 71.6
(1-OCH2−carborane), 71.4 (C-4), 62.6 (C-6), and 60.4
(carborane−CH) ppm.
11B NMR (160.46 MHz, CD3OD, 23 °C): δ = −2.3, −4.0,
−8.5, −10.7 and −12.2 ppm.
HRMS m/z: calcd for C9H24B10O6Na [M + Na]+, 361.2401;
found, 361.2381.
2.3.6. Propargyl α-D-Glucopyranoside (17). Synthesized
from D-glucose (0.50 g, 2.8 mmol), propargyl alcohol (2.41 g,
43.0 mmol), and DOWEX 50 H+-form (0.50 g) according to
the general procedure for glycosylation of glucose. This
reaction gave the title compound as an off-white oil (0.21 g,
34%). TLC: Rf: 0.38 (DCM/MeOH 5:1).
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 138.6−127.9
(arom. C), 98.7 (C-1), 81.7 (C-3), 80.0 (C-2), 77.5 (C-4),
75.8 (3-OCH2Ph), 75.4 (4-OCH2Ph), 74.1 (2-OCH2Ph), 73.7
(6-OCH2Ph), 72.4 (carborane−C), 71.4 (C-5), 69.4 (1-
OCH2−carborane), 68.3 (C-6), and 57.9 (carborane−CH)
ppm.
1H NMR (500.13 MHz, CD3OD, 23 °C): δ = 5.00 (d, 1H,
J1,2 = 3.8 Hz, H-1), 4.31 (dd, 1H, JCH
= −15.8, JCH ,CH =
2a,CH2b
2a
−2.5 Hz, 1-OCH2aCCH), 4.30 (dd, 1H, JCH ,CH = −2.3 Hz,
2b
11B NMR (160.46 MHz, CDCl3, 23 °C): δ = −0.9, −3.2,
−4.8, −8.8 and −13.2 ppm.
1-OCH2bCCH), 3.81 (dd, 1H, J6a,6b = −11.9, J6a,5 = 2.3 Hz,
H-6a), 3.68 (dd, 1H, J6b,5 = 5.5 Hz, H-6b), 3.63 (dd, 1H, J3,2
=
HRMS m/z: calcd for C37H48B10O6Na [M + Na]+,
721.4279; found, 721.4316.
9.7, J3,4 = 8.9 Hz, H-3), 3.57 (ddd, 1H, J5,4 = 10.0 Hz, H-5),
3.42 (dd, 1H, H-2), 3.31 (dd, 1H, H-4), and 2.85 (dd, 1H, 1-
OCH2CCH) ppm.
2.3.9. (O)-Carboranylmethyl α-D-Glucopyranoside (2).
Synthesized from (O)-carboranylmethyl 2,3,4,6-tetra-O-ben-
zyl-α-D-glucopyranoside (0.30 g, 0.4 mmol) and 10% Pd/C
(0.05 g, 0.4 mmol) according to the general procedure for
removal of benzyl groups. This reaction gave the title
compound as an off-white oil (0.12 g, 81%). TLC: Rf: 0.53
(EtOAc/MeOH 5:1).
13C NMR (125.76 MHz, CD3OD, 23 °C): δ = 98.6 (C-1),
80.1 (1-OCH2CCH), 76.0 (1-OCH2CCH), 75.0 (C-3),
74.1 (C-5), 73.3 (C-2), 71.7 (C-4), 62.5 (C-6), and 55.2 (1-
OCH2CCH) ppm.
HRMS m/z: calcd for C9H14O6Na [M + Na]+, 241.0688;
found, 241.0678.
1H NMR (500.13 MHz, CD3OD, 23 °C): δ = 4.84 (br s,
1H, carborane−CH), 4.80 (d, 1H, J1,2 = 3.7 Hz, H-1), 4.22 and
3.98 (each d, each 1H, J = −11.2 Hz, 1-OCH2−carborane),
3.81 (dd, 1H, J6a,6b = −11.9, J6a,5 = 2.2 Hz, H-6a), 3.63 (dd,
1H, J6b,5 = 6.1 Hz, H-6b), 3.59 (dd, 1H, J3,2 = 9.8, J3,4 = 8.9 Hz,
H-3), 3.49 (ddd, 1H, J4,5 = 10.0 Hz, H-5), 3.42 (dd, 1H, H-2),
3.25 (dd, 1H, H-4), and 3.04−1.42 (br m, 10H, carborane−
BH) ppm.
2.3.7. Propargyl 2,3,4,6-Tetra-O-benzyl-α-D-glucopyrano-
side (18). Synthesized from 17 (0.44 g, 2.0 mmol), NaH (0.34
g, 14.1 mmol), and BnBr (2.23 g, 13.0 mmol) according to the
general procedure for alkylation of free hydroxyl groups. This
reaction gave the title compound as a white solid (0.96 g,
82%). TLC: Rf: 0.41 (EtOAc/hexane 1:3).
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 7.40−7.11 (m,
20H, arom. H), 5.08 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.98 and 4.81
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13C NMR (125.76 MHz, CD3OD, 23 °C): δ = 100.6 (C-1),
74.8 (C-5), 74.7 (C-3), 74.5 (carborane−C), 73.3 (C-2), 71.6
(C-4), 69.9 (1-OCH2−carborane), 62.6 (C-6), and 60.5
(carborane−CH) ppm.
opening of 1,2-orthoester, and then a further reaction of 2-O-
acetyl-3,4,6-tri-O-benzyl-α-D-glucopyranose (1.00 g, 2.03
mmol) with Cl3CCN (2.93 g, 20.3 mmol) and DBU (0.12 g,
0.50 mmol) according to the general procedure for the
formation of an imidate donor. This reaction gave the title
compound as a colorless oil (1.17 g, 91%). TLC: Rf: 0.51
(EtOAc/hexane 1:4).
11B NMR (160.46 MHz, CD3OD, 23 °C): δ = −2.4, −4.2,
−8.5, −11.1 and −12.3 ppm.
HRMS m/z: calcd for C9H24B10O6Na [M + Na]+, 361.2401;
found, 361.2395.
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 8.56 (s, 1H,
NH), 7.35−7.16 (m, 15H, arom. H), 6.52 (d, 1H, J1,2 = 3.6 Hz,
H-1), 5.07 (dd, 1H, J2,3 = 10.0 Hz, H-2), 4.85 and 4.77 (each
d, each 1H, J = −11.4 Hz, 3-OCH2Ph), 4.83 and 4.57 (each d,
each 1H, J = −10.6 Hz, 4-OCH2Ph), 4.63 and 4.50 (each d,
each 1H, J = −12.0 Hz, 6-OCH2Ph), 4.09 (dd, 1H, J3,4 = 9.3
Hz, H-3), 4.01 (ddd, J5,4 = 10.1, J5,6a = 3.3, J5,6b = 1.9 Hz, H-5),
3.88 (dd, 1H, H-4), 3.81 (dd, 1H, J6a,6b = −11.1 Hz, H-6a),
3.70 (dd, 1H, H-6b), and 1.92 (s, 3H, 2-OCOCH3) ppm.
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 170.2 (2-
OCOCH3), 161.2 (imidate CNH), 138.4−127.9 (arom. C),
94.2 (C-1), 91.2 (imidate CCl3), 79.6 (C-3), 77.2 (C-4), 75.6
(4-OCH2Ph), 75.5 (3-OCH2Ph), 73.7 (6-OCH2Ph), 73.6 (C-
5), 72.5 (C-2), 68.0 (C-6), and 20.7 (2-OCOCH3) ppm.
HRMS m/z: calcd for C31H32Cl3NO7Na [M + Na]+,
658.1142; found, 658.1487.
2.3.10. 1,2-O-(1-Methoxyethylidene)-3,4,6-tri-O-acetyl-α-
D-glucopyranose. Synthesized from peracetylated D-
glucopyranose(11.99 g, 30.7 mmol), I2 (10.93 g, 43.1
mmol), Et3SiH (5.01 g, 43.1 mmol), 2,6-lutidine (13.18 g,
0.123 mol), TBAI (2.84 g, 7.69 mmol) according to the
general procedure for the formation of 1,2-orthoester. This
reaction gave the title compound as a colorless oil (10.1 g,
91%). TLC: Rf: 0.25 (EtOAc/hexane 1:3).
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 5.72 (d, 1H, J1,2
= 5.2 Hz, H-1), 5.20 (dd, 1H, J3,2 = 3.0, J3,4 = 2.7 Hz, H-3),
4.90 (ddd, 1H, J4,2 = −1.0, J4,5 = 9.6 Hz, H-4), 4.32 (ddd, 1H,
H-2), 4.21 (dd, 1H, J6a,5 = 5.6, J6a,6b = −12.2 Hz, H-6a), 4.19
(dd, 1H, J6b,5 = 2.6 Hz, H-6b), 3.95 (ddd, 1H, H-5), 3.29 (s,
3H, C(CH3)OCH3), 2.11 (s, 3H, 3-OCOCH3), 2.10 (s, 3H, 6-
OCOCH3), 2.09 (4-OCOCH3), and 2.71 (s, 3H, C(CH3)-
OCH3) ppm.
2.3.13. Benzyl 2-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyr-
anoside (9). Synthesized from 8 (1.11 g, 1.75 mmol), BnOH
(0.13 g, 1.25 mmol), and TMSOTf (0.56 g, 2.5 mmol)
according to the general procedure for glycosylation using
TMSOTf. The reaction gave the title compound as a white
solid (0.70 g, 96%). TLC: Rf: 0.68 (EtOAc/hexane 1:2).
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 7.37−7.16 (m,
20H, arom. H), 5.08 (dd, 1H, J1,2 = 7.9, J2,3 = 9.5 Hz, H-2),
4.89 and 4.61 (each d, each 1H, J = −12.4 Hz, 1-OCH2Ph),
4.79 and 4.56 (each d, each 1H, J = −10.8 Hz, 4-OCH2Ph),
4.77 and 4.65 (each d, each 1H, J = −11.4 Hz, 3-OCH2Ph),
4.64 and 4.57 (each d, each 1H, J = −12.2 Hz, 6-OCH2Ph),
4.42 (d, 1H, H-1), 3.76 (dd, 1H, J6a,5 = 1.9, J6a,6b = −10.9 Hz,
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 170.8 (6-
OCOCH3), 169.8 (4-OCOCH3), 169.3 (3-OCOCH3), 121.7
(C(CH3)OCH3), 97.1 (C-1), 73.2 (C-2), 70.3 (C-3), 68.3 (C-
4), 67.1 (C-5), 63.2 (C-6), 51.2 (C(CH3)OCH3), 20.9 (3-
OCOCH3, 4-OCOCH3 and 6-OCOCH3), and 20.2 (C(CH3)-
OCH3) ppm.
HRMS m/z: calcd for C15H22O10Na [M + Na]+, 385.1111;
found, 385.1121.
2.3.11. 1,2-O-(1-Methoxyethylidene)-3,4,6-tri-O-benzyl-α-
D-glucopyranose (7). Synthesized over two steps, starting from
1,2-O-(1-methoxyethylidene)-3,4,6-tri-O-acetyl-α-D-glucopyra-
nose and NaOMe according to the general procedure for
deacetylation and then a further reaction of 1,2-O-(1-
methoxyethylidene)-α-D-glucopyranose (2.10 g, 6.89 mmol)
with NaH (1.15 g, 48.01 mmol) and BnBr (7.30 g, 42.67
mmol) according to the general procedure for alkylation of free
hydroxyl groups. This reaction gave the title compound as a
colorless oil (2.28 g, 51%). TLC: Rf: 0.66 (EtOAc/hexane
1:2).
H-6a), 3.72 (dd, 1H, J6b,5 = 4.9 Hz, H-6b), 3.71 (dd, 1H, J4,3
=
9.0, J4,5 = 9.8 Hz, H-4), 3.64 (dd, 1H, H-3), 3.48 (ddd, 1H, H-
5), and 1.93 (s, 3H, 2-OCOCH3) ppm.
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 169.6 (2-
OCOCH3), 138.3−127.7 (arom. C), 99.8 (C-1), 83.1 (C-3),
78.1 (C-4), 75.3 (C-5), 75.2 (4-OCH2Ph and 3-OCH2Ph),
73.7 (6-OCH2Ph), 73.3 (C-2), 70.4 (1-OCH2Ph), 68.9 (C-6),
and 21.0 (2-OCOCH3) ppm.
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 7.37−7.16 (m,
15H, arom. H), 5.78 (d, 1H, J1,2 = 5.3 Hz, H-1), 4.71 and 4.60
(each d, each 1H, J = −11.9 Hz, 3-OCH2Ph), 4.60 and 4.38
(each d, each 1H, J = −11.4 Hz, 4-OCH2Ph), 4.57 and 4.50
(each d, each 1H, J = −12.1 Hz, 6-OCH2Ph), 4.42 (ddd, 1H,
HRMS m/z: calcd for C36H38O7Na [M + Na]+, 605.2516;
found, 605.2524.
2.3.14. Benzyl 3,4,6-Tri-O-benzyl-β-D-glucopyranoside.
Synthesized from 9 (0.68 g, 1.17 mmol) and NaOMe (0.09
g, 1.75 mmol) according to the general procedure for
deacetylation. This reaction gave the title compound as a
white solid (0.57 g, 91%). TLC: Rf: 0.64 (EtOAc/hexane 1:2).
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 7.39−7.16 (m,
20H, arom. H), 4.95 and 4.63 (each d, each 1H, J = −11.7 Hz,
1-OCH2Ph), 4.92 and 4.82 (each d, each 1H, J = −11.3 Hz, 3-
OCH2Ph), 4.84 and 4.55 (each d, each 1H, J = −10.8 Hz, 4-
OCH2Ph), 4.63 and 4.56 (each d, each 1H, J = −12.2 Hz, 6-
OCH2Ph), 4.36 (d, 1H, J1,2 = 7.7 Hz, H-1), 3.76 (dd, 1H, J6a,5
= 1.9, J6a,6b = −10.8 Hz, H-6a), 3.72 (dd, 1H, J6b,5 = 4.8 Hz, H-
6b), 3.63 (dd, 1H, J4,3 = 8.9, J4,5 = 9.8 Hz, H-4), 3.62 (ddd, 1H,
J2,2‑OH = 2.2, J2,3 = 9.2 Hz, H-2), 3.58 (dd, 1H, H-3), 3.49
(ddd, 1H, H-5), and 2.30 (d, 1H, 2-OH) ppm.
J2,3 = 3.6, J2,4 = −0.8 Hz, H-2), 3.87 (dd, 1H, J3,4 = 4.6 Hz, H-
3), 3.79 (ddd, 1H, J5,4 = 9.5, J5,6a = 2.2, J5,6b = 4.3 Hz, H-5),
3.71 (ddd, 1H, H-4), 3.66 (dd, 1H, J6a,6b = −10.8 Hz, H-6a),
3.64 (dd, 1H, H-6b), 3.28 (s, 3H, C(CH3)OCH3), and 1.65 (s,
3H, C(CH3)OCH3) ppm.
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 138.2−127.7
(arom. C), 121.4 (C(CH3)OCH3), 97.9 (C-1), 78.9 (C-3),
76.0 (C-2), 75.0 (C-4), 73.5 (6-OCH2Ph), 73.0 (4-OCH2Ph),
72.0 (3-OCH2Ph), 70.6 (C-5), 69.2 (C-6), 50.7 (C(CH3)-
OCH3), and 21.4 (C(CH3)OCH3) ppm.
HRMS m/z: calcd for C30H34O7Na [M + K]+, 545.1905;
found, 545.1883.
2.3.12. 2-O-Acetyl-3,4,6-tri-O-benzyl-α-D-glucopyranose
Trichloroacetimidate (8). Synthesized over two steps, starting
from 7 and p-TsOH according to the general procedure for
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 138.8−127.8
(arom. C), 101.8 (C-1), 84.7 (C-3), 77.7 (C-4), 75.4 (C-5),
F
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75.3 (3-OCH2Ph), 75.1 (4-OCH2Ph), 74.9 (C-2), 73.7 (6-
OCH2Ph) 71.2 (1-OCH2Ph), and 69.0 (C-6) ppm.
g, 1.0 mmol) according to the general procedure for
hydrogenolysis. This reaction gave the title compound as a
colorless oil (0.04 g, 77%, α/β 33:67). TLC: Rf: 0.51 (EtOAc/
MeOH 5:1).
HRMS m/z: calcd for C34H36O6Na [M + Na]+, 563.2410;
found, 563.2405.
1
2.3.15. Benzyl 3,4,6-Tri-O-benzyl-2-O-propargyl-β-D-glu-
copyranoside (10). Synthesized from Benzyl 3,4,6-tri-O-
benzyl-β-D-glucopyranoside (0.54 g, 1.0 mmol), NaH (0.05
g, 1.9 mmol), and propargyl bromide (0.22 g, 1.5 mmol)
according to the general procedure for alkylation of free
hydroxyl groups. This reaction gave the title compound as a
yellow oil (0.49 g, 85%). TLC: Rf: 0.72 (EtOAc/hexane 1:2).
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 7.41−7.15 (m,
20H, arom. H), 5.01 and 4.76 (each d, each 1H, J = −10.8 Hz,
3-OCH2Ph), 4.95 and 4.65 (each d, each 1H, J = −11.9 Hz, 1-
OCH2Ph), 4.84 and 4.53 (each d, each 1H, J = −10.8 Hz, 4-
OCH2Ph), 4.62 and 4.55 (each d, each 1H, J = −12.2 Hz, 6-
OCH2Ph), 4.51 (dd, 1H, JCH ,CH = −2.4, JCH ,CH = −15.3 Hz,
α anomer: H NMR (500.13 MHz, CD3OD, 23 °C): δ =
5.20 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.76 (br s, 1H, carborane−
CH), 4.21 and 4.13 (each d, each 1H, J = −10.8 Hz, 2-OCH2−
carborane), 3.76 (dd, 1H, J6a,6b = −11.8, J6a,5 = 2.4 Hz, H-6a),
3.73 (dd, 1H, J3,2 = 9.7, J3,4 = 9.0 Hz, H-3), 3.72 (ddd, 1H, J5,4
= 10.0, J5,6b = 5.1 Hz, H-5), 3.68 (dd, 1H, H-6b), 3.29 (dd, 1H,
H-4), 3.23 (dd, 1H, H-2), and 2.83−1.42 (br m, 10H,
carborane−BH) ppm.
13C NMR (125.76 MHz, CD3OD, 23 °C): δ = 91.6 (C-1),
82.7 (C-2), 75.0 (carborane−C), 73.8 (C-3), 72.8 (C-5), 72.6
(2-OCH2−carborane), 71.8 (C-4), 62.5 (C-6), and 60.1
(carborane−CH) ppm.
1
β anomer: H NMR (500.13 MHz, CD3OD, 23 °C): δ =
2a
a
2b
4.81 (br s, 1H, carborane−CH), 4.51 (d, 1H, J1,2 = 7.8 Hz, H-
1), 4.33 and 4.19 (each d, each 1H, J = −11.2 Hz, 2-OCH2−
carborane), 3.83 (dd, 1H, J6a,5 = 2.1, J6a,6b = −11.9 Hz, H-6a),
3.63 (dd, 1H, J6b,5 = 5.7 Hz, H-6b), 3.38 (dd, 1H, J3,2 = 9.2, J3,4
= 8.7 Hz, H-3), 3.72 (ddd, 1H, J5,4 = 9.7 Hz, H-5), 3.25 (dd,
1H, H-4), 2.92 (dd, 1H, H-2), and 2.83−1.42 (br m, 10H,
carborane−BH) ppm.
2-OCH2aCCH), 4.45 (d, 1H, J1,2 = 7.8 Hz, H-1), 4.43 (dd,
1H, JCH
= −2.4 Hz, 2-OCH2bCCH), 3.75 (dd, 1H, J6a,5
2b,CH
= 1.9, J6a,6b = −10.8 Hz, H-6a), 3.69 (dd, 1H, J6b,5 = 4.9 Hz, H-
6b), 3.60 (dd, 1H, J3,2 = 9.1, J3,4 = 8.7 Hz, H-3), 3.59 (dd, 1H,
J4,5 = 9.6 Hz, H-4), 3.50 (dd, 1H, H-2), 3.45 (ddd, 1H, H-5),
and 2.44 (dd, 1H, 2-OCH2CCH) ppm.
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 138.7−127.7
(arom. C), 102.4 (C-1), 84.4 (C-3), 81.8 (C-2), 80.2 (2-
OCH2CCH), 77.8 (C-4), 75.9 (3-OCH2Ph), 75.2 (4-
OCH2Ph), 75.1 (C-5), 74.4 (2-OCH2CCH), 73.6 (6-
OCH2Ph), 71.2 (1-OCH2Ph), 69.0 (C-6), and 59.8 (2-
OCH2CCH) ppm.
13C NMR (125.76 MHz, CD3OD, 23 °C): δ = 97.6 (C-1),
85.9 (C-2), 77.8 (C-3), 77.3 (C-4), 75.3 (carborane−C), 74.3
(2-OCH2−carborane), 71.8 (C-5), 62.7 (C-6), and 59.9
(carborane−CH) ppm.
11B NMR (160.46 MHz, CD3OD, 23 °C): δ = −2.68, −4.40,
−8.62, −10.93 and −12.50 ppm.
HRMS m/z: calcd for C37H38O6Na [M + Na]+, 601.2566;
found, 601.2569.
HRMS m/z: calcd for C9H24B10O6Na [M + Na]+, 361.2401;
found, 361.2396.
2.3.16. Benzyl 3,4,6-Tri-O-benzyl-2-O-carboranylmethyl-
β-D-glucopyranoside. Synthesized from 10 (0.47 g, 0.8 mmol)
and B10H14 (0.10 g, 1.4 mmol) according to the general
procedure for coupling with decaborane. This reaction gave
the title compound as an off-white oil (0.29 g, 51%). TLC: Rf:
0.47 (EtOAc/hexane 1:3).
2.3.18. 1,2:5,6-Bis-O-(isopropylidene)-α-D-glucofuranose
(19). Synthesized from D-glucose (7.11 g, 39.5 mmol) and I2
(2.00 g, 7.88 mmol) according to the general procedure for
installation of 1,2:4,6-di-O-ispropylidene acetals. This reaction
gave the title compound as a white solid (4.55 g, 47%). TLC:
Rf: 0.56 (EtOAc/hexane 1:1).
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 7.40−7.12 (m,
20H, arom. H), 4.90 and 4.55 (each d, each 1H, J = −11.5 Hz,
1-OCH2Ph), 4.84 and 4.70 (each d, each 1H, J = −11.1 Hz, 3-
OCH2Ph), 4.77 and 4.55 (each d, each 1H, J = −10.8 Hz, 4-
OCH2Ph), 4.63 and 4.55 (each d, each 1H, J = −12.2 Hz, 6-
OCH2Ph), 4.35 (d, 1H, J1,2 = 7.8 Hz, H-1), 4.19 and 4.01
(each d, each 1H, J = −10.8 Hz, 2-OCH2−carborane), 3.73
(dd, 1H, J6a,5 = 1.9, J6a,6b = −10.9 Hz, H-6a), 3.70 (dd, 1H, J6b,5
= 4.5 Hz, H-6b), 3.7 (br s, 1H, carborane−CH), 3.62 (dd, 1H,
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 5.94 (d, 1H, J1,2
= 3.6 Hz, H-1), 4.34 (ddd, 1H, J5,4 = 7.8, J5,6a = 6.2, J5,6b = 5.3
Hz, H-5), 4.32 (ddd, J3,2 = 0.7, J3,4 = 2.8, J3,3‑OH = 3.9 Hz, 1H,
H-3), 4.17 (dd, 1H, J6a,6b = −8.7 Hz, H-6a), 4.07 (dd, 1H, H-
4), 4.00 (dd, 1H, H-6b), 2.76 (d, 1H, 3-OH), 1.50 (s, 3H, 1,2-
CH3′), 1.45 (s, 3H, 5,6-CH3′), 1.37 (s, 3H, 5,6-CH3″), and 1.32
(s, 3H, 1,2-CH″3 ) ppm.
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 111.9
(1,2-qC), 109.8 (5,6-qC), 105.4 (C-1), 85.2 (C-2), 81.2 (C-4),
75.2 (C-3), 73.5 (C-5), 67.7 (C-6), 26.9 (1,2-CH′3 and 5,6-
CH3′), 26.3 (1,2-CH3″), and 25.2 (5,6-CH3″) ppm.
HRMS m/z: calcd for C12H20O6Na [M + Na]+, 283.1158;
found, 283.1164.
J4,3 = 9.0, J4,5 = 9.8 Hz, H-4), 3.52 (dd, 1H, J3,2 = 9.3 Hz, H-3),
3.41 (ddd, 1H, H-5), 3.22 (dd, 1H, H-2), and 2.86−1.36 (br
m, 10H, carborane−BH) ppm.
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 138.1−127.7
(arom. C), 101.3 (C-1), 84.2 (C-3), 82.6 (C-2), 78.2 (C-4),
75.8 (3-OCH2Ph), 75.1 (C-5), 75.0 (4-OCH2Ph), 73.7 (6-
OCH2Ph), 73.2 (2-OCH2−carborane), 72.9 (carborane−C),
71.3 (1-OCH2Ph), 68.6 (C-6), and 57.8 (carborane−CH)
ppm.
2.3.19. 1,2:5,6-Bis-O-(isopropylidene)-3-O-propargyl-α-D-
glucofuranose. Synthesized from 19 (3.45 g, 13.3 mmol),
NaH (0.61 g, 25.2 mmol), and propargyl bromide (2.37 g, 19.9
mmol) according to the general procedure for alkylation of free
hydroxyl groups. This reaction gave the title compound as a
yellow oil (3.55 g, 90%). TLC: Rf: 0.35 (EtOAc/hexane 1:3).
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 5.89 (d, 1H, J1,2
= 3.7 Hz, H-1), 4.64 (dd, 1H, J2,3 = 0.03 Hz, H-2), 4.30 (dd,
11B NMR (160.46 MHz, CDCl3, 23 °C): δ = −3.07, −4.97,
−9.36, −11.81 and −13.35 ppm.
HRMS m/z: calcd for C37H48B10O6K [M + K]+, 737.4018;
found, 737.4057.
1H, JCH
= −15.9, JCH
= −2.2 Hz, 3-OCH2aCCH),
2a,CH2b
2a,CH
2.3.17. 2-O-Carboranylmethyl-D-glucopyranose (3). Syn-
thesized from Benzyl 3,4,6-tri-O-benzyl-2-O-carboranylmethyl-
β-D-glucopyranoside (0.11 g, 0.2 mmol) and 10% Pd/C (0.11
4.28 (dd, 1H, JCH
(ddd, 1H, J5,4 = 7.9, J5,6a = 6.5, J5,6b = 5.4 Hz, H-5), 4.14 (dd,
= −2.4 Hz, 3-OCH2bCCH), 4.28
2b,CH
G
https://dx.doi.org/10.1021/acs.molpharmaceut.0c00917
Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics
pubs.acs.org/molecularpharmaceutics
Article
1H, J4,3 = 2.7 Hz, H-4), 4.10 (dd, 1H, H-3), 4.09 (dd, 1H, J6a,6b
= −8.8 Hz, H-6a), 4.00 (dd, 1H, H-6b), 2.49 (dd, 1H, 3-
OCH2CCH), 1.50 (s, 3H, 1,2-CH3′), 1.43 (s, 3H, 5,6-CH3′),
1.35 (s, 3H, 5,6-CH″3 ), and 1.32 (s, 3H, 1,2-CH3″) ppm.
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 112.0
(1,2-qC), 109.2 (5,6-qC), 105.4 (C-1), 83.0 (C-2), 81.7 (C-3),
81.2 (C-4), 79.4 (3-OCH2CCH), 75.0 (3-OCH2CCH),
72.7 (C-5), 67.4 (C-4), 58.3 (3-OCH2CCH), 27.0 (1,2-
CH3′and 5,6-CH3′), 26.4 (1,2-CH3″), and 25.5 (5,6-CH3″) ppm.
HRMS m/z: calcd for C15H22O6Na [M + Na]+, 321.1314;
found, 321.1342.
2.3.20. 1,2:5,6-Bis-O-(isopropylidene)-3-O-carboranyl-
methyl-α-D-glucofuranose (20). Synthesized from 1,2:5,6-
bis-O-(isopropylidene)-3-O-propargyl-α-D-glucofuranose (1.72
g, 5.77 mmol) and B10H14 (1.34 g, 11.0 mmol) according to
the general procedure for coupling with decaborane. This
reaction gave the title compound as a clear oil (1.08 g, 45%).
TLC: Rf: 0.58 (EtOAc/hexane 1:3).
(3-OCH2−carborane), 71.2 (C-4), 62.5 (C-6), and 59.8
(carborane−CH) ppm.
11B NMR (160.46 MHz, CD3OD, 23 °C): δ = −2.87, −4.59,
−8.83, −11.03, −11.90 and −12.65 ppm.
HRMS m/z: calcd for C9H24B10O6Na [M + Na]+, 361.2401;
found, 361.2395.
2.3.22. Benzyl 2,3,4,6-Tetra-O-acetyl-β-D-glucopyranoside
(11). Synthesized from 1,2,3,4,6-penta-O-acetyl-D-glucopyra-
nose (8.70 g, 22.3 mmol), BnOH (9.64 g, 89.2 mmol), and
BF3·OEt2 (17.92 g, 126.0 mmol) according to the general
procedure for glycosylation of peracetylated glucose. This
reaction gave the title compound as a white solid (4.17 g,
43%). TLC: Rf: 0.39 (EtOAc/hexane 1:3).
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 7.38−7.25 (m,
5H, arom. H), 5.17 (dd, 1H, J3,2 = 9.7, J3,4 = 9.4 Hz, H-3), 5.11
(dd, 1H, J4,5 = 10.1 Hz, H-4), 5.07 (dd, 1H, J2,1 = 8.0 Hz, H-2),
4.90 and 4.62 (each d, each 1H, J = −12.3 Hz, 1-OCH2Ph),
4.55 (d, 1H, H-1), 4.27 (dd, 1H, J6a,5 = 4.7, J6a,6b = −12.3 Hz,
H-6a), 4.17 (dd, 1H, J6b,5 = 2.4 Hz, H-6b), 3.67 (ddd, 1H, H-
5), 2.11 (s, 3H, 6-OCOCH3), 2.02 (s, 3H, 4-OCOCH3), 2.00
(s, 3H, 2-OCOCH3), and 1.99 (s, 3H, 3-OCOCH3) ppm.
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 170.8 (6-
OCOCH3), 170.4 (3-OCOCH3), 169.5 (2-OCOCH3), 169.4
(4-OCOCH3), 136.8−127.9 (arom. C), 99.4 (C-1), 73.0 (C-
3), 72.0 (C-5), 71.4 (C-2), 70.9 (1-OCH2Ph), 68.5 (C-4), 62.1
(C-6), 20.9−20.8 (6-OCOCH3 and 2-OCOCH3), and 20.7 (3-
OCOCH3 and 4-OCOCH3) ppm.
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 5.86 (d, 1H, J1,2
= 3.7 Hz, H-1), 4.46 (dd, 1H, J2,3 = 0.03 Hz, H-2), 4.28 (br s,
1H, carborane−CH), 4.15 (dd, 1H, J6a,5 = 6.1, J6a,6b = −8.9 Hz,
H-6a), 4.14 (ddd, 1H, J5,4 = 9.0, J5,6b = 5.5 Hz, H-5), 4.12 and
4.04 (each d, each 1H, J = −10.8 Hz, 3-OCH2−carborane),
4.01 (dd, 1H, J4,3 = 3.2 Hz, H-4), 3.95 (dd, 1H, H-3), 2.94−
1.55 (br m, 10H, carborane−BH), 1.48 (s, 3H, 1,2-CH′3), 1.41
(s, 3H, 5,6-CH′3), 1.34 (s, 3H, 1,2-CH″3 ), and 1.31 (s, 3H, 5,6-
CH3″) ppm.
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 112.3
(1,2-qC), 109.6 (5,6-qC), 105.3 (C-1), 84.2 (C-3), 82.4 (C-2),
81.3 (C-4), 72.4 (carborane−C), 72.2 (C-5), 71.6 (3-OCH2−
carborane), 68.1 (C-6), 57.8 (carborane−CH), 26.8 (1,2-CH3′
and 5,6-CH′3), 26.2 (1,2-CH3″), and 25.2 (5,6-CH3″) ppm.
11B NMR (160.46 MHz, CDCl3, 23 °C): δ = −3.11, −4.69,
−8.77, −9.00, −11.13, −12.24, −13.08 and −13.41 ppm.
HRMS m/z: calcd for C15H32B10O6Na [M + Na]+,
441.3027; found, 441.3048.
HRMS m/z: calcd for C21H26O10Na [M + Na]+, 461.1424;
found, 461.1398.
2.3.23. Benzyl β-D-glucopyranoside. Synthesized from 11
(4.94 g, 11.3 mmol) according to the general procedure for
deacetylation. This reaction gave the title compound as a white
solid (3.01 g, 99%). TLC: Rf: 0.46 (DCM/MeOH 7:1).
1H NMR (500.13 MHz, CD3OD, 23 °C): δ = 7.43−7.24
(m, 5H, arom. H), 4.92 and 4.66 (each d, each 1H, J = −11.8
Hz, 1-OCH2Ph), 4.34 (d, 1H, J1,2 = 7.8 Hz, H-1), 3.89 (dd,
2.3.21. 3-O-Carboranylmethyl-D-glucopyranose (4). Syn-
thesized from 20 (0.40 g, 0.96 mmol) according to the general
procedure for 1,2:4,6-di-O-isopropylidene acetal removal. This
reaction gave the title compound as a white solid (0.29 g, 90%,
α/β 30:70). TLC: Rf: 0.61 (DCM/MeOH 5:1).
1H, J6a,5 = 2.6, J6a,6b = −11.9 Hz, H-6a), 3.68 (dd, 1H, J6b,5
=
5.9 Hz, H-6b), 3.33 (dd, 1H, J3,2 = 8.8, J3,4 = 9.3 Hz, H-3), 3.29
(dd, 1H, J4,5 = 9.7 Hz, H-4), 3.25 (ddd, 1H, H-5), and 3.24
(dd, 1H, H-2) ppm.
1
13C NMR (125.76 MHz, CD3OD, 23 °C): δ = 139.1−128.7
(arom. C), 103.3 (C-1), 78.1 (C-3), 78.0 (C-5), 75.1 (C-2),
71.7 (C-5), 71.7 (1-OCH2Ph), and 62.8 (C-6) ppm.
HRMS m/z: calcd for C13H18O6Na [M + Na]+, 293.1001;
found, 239.1009.
α anomer: H NMR (500.13 MHz, CD3OD, 23 °C): δ =
5.05 (d, 1H, J1,2 = 3.7 Hz, H-1), 4.84 (br s, 1H, carborane−
CH), 4.30 and 4.29 (each d, each 1H, J = −10.8 Hz, 3-OCH2−
carborane), 3.75 (dd, 1H, J6a,5 = 2.3, J6a,6b = −11.8 Hz, H-6a),
3.74 (ddd, 1H, J5,4 = 9.9, J5,6b = 5.0 Hz, H-5), 3.68 (dd, 1H, H-
6b), 3.48 (dd, 1H, J3,2 = 9.5, J3,4 = 9.0 Hz, H-3), 3.41 (dd, 1H,
H-2), 3.38 (dd, 1H, H-4), and 3.05−1.42 (br m, 10H,
carborane−BH) ppm.
2.3.24. Benzyl 4,6-O-Benzylidene-β-D-glucopyranoside
(12). Synthesized from benzyl β-D-glucopyranoside (2.98 g,
11.03 mmol), C6H5CH(OCH3)2 (2.68 g, 17.6 mmol), and p-
TsOH (0.19 g, 1.10 mmol) according to the general procedure
for installation of 4,6-O-benzylidene acetals. This reaction gave
the title compound as a white solid (3.95 g, 85%). TLC: Rf:
0.66 (DCM/MeOH 10:1).
13C NMR (125.76 MHz, CD3OD, 23 °C): δ = 94.0 (C-1),
85.6 (C-3), 75.6 (carborane−C), 74.8 (3-OCH2−carborane),
73.6 (C-2), 72.9 (C-5), 71.2 (C-4), 62.4 (C-6), and 59.7
(carborane−CH) ppm.
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 7.51−7.30 (m,
10H, arom. H), 5.54 (s, 1H, CHPh), 4.94 and 4.64 (each d,
each 1H, J = −11.6 Hz, 1-OCH2Ph), 4.51 (d, 1H, J1,2 = 7.7 Hz,
H-1), 4.37 (dd, 1H, J6a,5 = 5.0, J6a,6b = −10.6 Hz, H-6a), 3.81
(dd, 1H, J6b,5 = 10.0 Hz, H-6b), 3.81 (ddd, 1H, J3,2 = 9.0,
1
β anomer: H NMR (500.13 MHz, CD3OD, 23 °C): δ =
4.84 (br s, 1H, carborane−CH), 4.44 (d, 1H, J1,2 = 7.8 Hz, H-
1), 4.32 and 4.29 (each d, each 1H, J = −11.0 Hz, 3-OCH2−
carborane), 3.82 (dd, 1H, J6a,5 = 2.3, J6a,6b = −11.9 Hz, H-6a),
3.64 (dd, 1H, J6b,5 = 5.7 Hz, H-6b), 3.36 (dd, 1H, J4,3 = 8.7 Hz,
J3,3‑OH = 2.3, J3,4 = 9.3 Hz, H-3), 3.58 (dd, 1H, J4,5 = 9.4 Hz, H-
J4,5 = 9.8 Hz, H-4), 3.24 (ddd, 1H, H-5), 3.18 (dd, 1H, J3,2
=
4), 3.56 (ddd, 1H, J2,2‑OH = 2.6 Hz, H-2), 3.46 (ddd, 1H, H-5),
9.0 Hz, H-3), 3.17 (dd, 1H, H-2), and 3.05−1.42 (br m, 10H,
2.84 (d, 1H, 3-OH), and 2.67 (d, 1H, 2-OH) ppm.
carborane−BH) ppm.
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 137.1−126.4
(arom. C), 102.2 (C-1), 102.1 (CHPh), 80.7 (C-3), 74.7 (C-
13C NMR (125.76 MHz, CD3OD, 23 °C): δ = 98.1 (C-1),
88.2 (C-3), 77.7 (C-5), 76.1 (C-2), 75.5 (carborane−C), 74.7
H
https://dx.doi.org/10.1021/acs.molpharmaceut.0c00917
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4), 73.3 (C-2), 71.7 (1-OCH2Ph), 68.8 (C-6), and 66.6 (C-5)
ppm.
J6a,5 = 1.8, J6a,6b = −11.0 Hz, H-6a), 3.74 (dd, 1H, J6b,5 = 5.2
Hz, H-6b), 3.60 (dd, 1H, J3,2 = 9.2, J3,4 = 9.0 Hz, H-3), 3.51
(dd, 1H, J4,5 = 9.8 Hz, H-4), 3.48 (dd, 1H, H-2), 3.44 (ddd,
1H, H-5), and 2.40 (dd, 1H, 4-OCH2CCH) ppm.
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 138.5−127.7
(arom. C), 102.7 (C-1), 84.6 (C-3), 82.3 (C-2), 80.0 (4-
OCH2CCH), 77.5 (C-4), 75.9 (3-OCH2Ph), 75.0 (2-
OCH2Ph), 74.7 (C-5), 74.6 (4-OCH2CCH), 73.6 (6-
OCH2Ph), 71.3 (1-OCH2Ph), 69.3 (C-6), and 60.0 (4-
OCH2CCH) ppm.
HRMS m/z: calcd for C20H22O6Na [M + Na]+, 381.1314;
found, 381.1307.
2.3.25. Benzyl 2,3-Di-O-benzyl-4,6-O-benzylidene-β-D-glu-
copyranoside. Synthesized from 12 (1.00 g, 2.8 mmol), BnBr
(1.58 g, 8.9 mmol), and NaH (0.25 g, 10.6 mmol) according to
the general procedure for alkylation of free hydroxyl groups.
This reaction gave the title compound as a white solid (1.21 g,
80%). TLC: Rf: 0.35 (EtOAc/hexane 1:6).
HRMS m/z: calcd for C37H38O6Na [M + Na]+, 601.2566;
found, 601.2551.
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 7.51−7.23 (m,
20H, arom. H), 5.58 (s, 1H, CHPh), 4.95 and 4.68 (each d,
each 1H, J = −11.6 Hz, 1-OCH2Ph), 4.90 and 4.79 (each d,
each 1H, J = −11.6 Hz, 3-OCH2Ph), 4.90 and 4.77 (each d,
each 1H, J = −11.6 Hz, 2-OCH2Ph), 4.63 (d, 1H, J1,2 = 7.7 Hz,
H-1), 4.38 (dd, 1H, J6a,5 = 5.0, J6a,6b = −10.5 Hz, H-6a), 3.82
2.3.28. Benzyl 2,3,6-Tri-O-benzyl-4-O-carboranylmethyl-
β-D-glucopyranoside. Synthesized from 14 (0.75 g, 1.30
mmol) and B10H14 (0.30 g, 2.47 mmol) according to the
general procedure for coupling with decaborane. This reaction
gave the title compound as a clear oil (0.59 g, 65%). TLC: Rf:
0.59 (EtOAc/hexane 1:3).
(dd, 1H, J6b,5 = 10.0 Hz, H-6b), 3.75 (dd, 1H, J3,2 = 8.8, J3,4
=
9.4 Hz, H-3), 3.71 (dd, 1H, J4,5 = 9.4 Hz, H-4), 3.53 (dd, 1H,
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 7.39−7.21 (m,
20H, arom. H), 4.96 and 4.68 (each d, each 1H, J = −11.0 Hz,
2-OCH2Ph), 4.95 and 4.66 (each d, each 1H, J = −11.9 Hz, 1-
OCH2Ph), 4.93 and 4.57 (each d, each 1H, J = −11.0 Hz, 3-
OCH2Ph), 4.65 and 4.52 (each d, each 1H, J = −12.1 Hz, 6-
OCH2Ph), 4.46 (d, 1H, J1,2 = 7.8 Hz, H-1), 4.15 and 3.80
(each d, each 1H, J = −10.2 Hz, 4-OCH2−carborane), 3.66
(dd, 1H, J6a,5 = 1.9, J6a,6b = −10.9 Hz, H-6a), 3.65 (dd, 1H, J6b,5
= 3.9 Hz, H-6b), 3.52 (dd, 1H, J3,2 = 9.1, J3,4 = 9.0 Hz, H-3),
3.48 (dd, 1H, H-2), 3.45 (dd, 1H, J4,5 = 9.7 Hz, H-4), 3.42 (br
s, 1H, carborane−CH), 3.35 (ddd, 1H, H-5), and 2.78−1.44
(br m, 10H, carborane−BH) ppm.
H-2), and 3.42 (ddd, 1H, H-5) ppm.
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 138.6−126.1
(arom. C), 103.2 (C-1), 101.3 (CHPh), 82.3 (C-2), 81.7 (C-
4), 81.1 (C-3), 75.6 (2-OCH2Ph), 75.3 (3-OCH2Ph), 71.7 (1-
OCH2Ph), 69.0 (C-6), and 66.2 (C-5) ppm.
HRMS m/z: calcd for C34H34O6Na [M + Na]+, 561.2253;
found, 561.2279.
2.3.26. Benzyl 2,3,6-Tri-O-benzyl-β-D-glucopyranoside
(13). Synthesized from benzyl 2,3-di-O-benzyl-4,6-O-benzyli-
dene-β-D-glucopyranoside (1.19 g, 2.2 mmol) according to the
general procedure for selective ring−opening of the
benzylidene acetal to give the 4-OH/6-OBn substrate. This
reaction gave the title compound as a white solid (0.85 g,
72%). TLC: Rf: 0.61 (EtOAc/hexane 1:2).
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 138.2−127.9
(arom. C), 102.5 (C-1), 84.1 (C-3), 82.4 (C-2), 77.8 (C-4),
75.6 (3-OCH2Ph), 74.8 (2-OCH2Ph), 74.2 (C-5), 73.8 (6-
OCH2Ph), 73.3 (4-OCH2−carborane), 72.5 (carborane−C),
71.3 (1-OCH2Ph), 68.7 (C-6), and 58.1 (carborane−CH)
ppm.
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 7.39−7.25 (m,
20H, arom. H), 4.96 and 4.66 (each d, each 1H, J = −11.9 Hz,
1-OCH2Ph), 4.96 and 4.72 (each d, each 1H, J = −10.9 Hz, 2-
OCH2Ph), 4.93 and 4.72 (each d, each 1H, J = −11.4 Hz, 3-
OCH2Ph), 4.62 and 4.59 (each d, each 1H, J = −12.1 Hz, 6-
OCH2Ph), 4.53 (d, 1H, J1,2 = 7.8 Hz, H-1), 3.79 (dd, 1H, J6a,5
= 3.7, J6a,6b = −10.4 Hz, H-6a), 3.73 (dd, 1H, J6b,5 = 5.4 Hz, H-
6b), 3.61 (ddd, 1H, J4,3 = 8.9, J4,4‑OH = 2.2, J4,5 = 9.7 Hz, H-4),
3.49 (dd, 1H, J2,3 = 9.2 Hz, H-2), 3.45 (dd, 1H, H-3), 3.45
(ddd, 1H, H-5), and 2.50 (d, 1H, 4-OH) ppm.
11B NMR (160.46 MHz, CDCl3, 23 °C): δ = −2.85, −4.83,
−9.29, −11.78 and −13.34 ppm.
HRMS m/z: calcd for C37H48B10O6Na [M + Na]+,
721.4279; found, 721.4411.
2.3.29. 4-O-Carboranylmethyl-D-glucopyranose (5). Syn-
thesized from benzyl 2,3,6-tri-O-benzyl-4-O-carboranylmethyl-
β-D-glucopyranoside (0.12 g, 0.18 mmol) and 10% Pd/C (0.12
g, 1.0 mmol) according to the general procedure for the
removal of benzyl groups. This reaction gave the title
compound as a white solid (0.05 g, 77%, α/β 50:50). TLC:
Rf: 0.47 (DCM/MeOH 5:1).
13C NMR (125.76 MHz, CDCl3, 23 °C): δ = 138.7−127.8
(arom. C), 102.7 (C-1), 84.2 (C-3), 81.9 (C-2), 75.4 (3-
OCH2Ph), 74.9 (2-OCH2Ph), 74.2 (C-5), 73.8 (6-OCH2Ph),
71.7 (C-4), 71.3 (1-OCH2Ph), and 70.4 (C-6) ppm.
HRMS m/z: calcd for C34H36O6Na [M + Na]+, 563.2410;
found, 563.2453.
1
α anomer: H NMR (500.13 MHz, CD3OD, 23 °C): δ =
5.08 (d, 1H, J1,2 = 3.7 Hz, H-1), 4.68 (br s, 1H, carborane−
2.3.27. Benzyl 2,3,6-Tri-O-benzyl-4-O-propargyl-β-D-glu-
copyranoside (14). Synthesized from 13 (0.80 g, 1.49 mmol),
NaH (0.07 g, 2.83 mmol) and propargyl bromide (0.39 g, 2.38
mmol) according to the general procedure for alkylation of free
hydroxyl groups. This reaction gave the title compound as a
white solid (0.79 g, 91%). TLC: Rf: 0.68 (EtOAc/hexane 1:2).
1H NMR (500.13 MHz, CDCl3, 23 °C): δ = 7.39−7.25 (m,
20H, arom. H), 4.97 and 4.66 (each d, each 1H, J = −11.9 Hz,
1-OCH2Ph), 4.94 and 4.70 (each d, each 1H, J = −10.9 Hz, 2-
OCH2Ph), 4.89 and 4.77 (each d, each 1H, J = −10.8 Hz, 3-
OCH2Ph), 4.65 and 4.60 (each d, each 1H, J = −12.1 Hz, 6-
OCH2Ph), 4.49 (d, 1H, J1,2 = 7.8 Hz, H-1), 4.37 (dd, 1H,
JCH ,CH = −15.3, JCH ,CH = −2.4 Hz, 4-OCH2aCCH), 4.26
CH), 4.43 and 4.11 (each d, each 1H,, J = −10.6 Hz, 4-
OCH2−carborane), 3.79 (ddd, 1H, J5,4 = 9.9, J5,6a = 2.1, J5,6b
=
3.9 Hz, H-5), 3.78 (dd, 1H, J3,2 = 9.5, J3,4 = 9.0 Hz, H-3), 3.72
(dd, 1H, J6a,6b = −12.0 Hz, H-6a), 3.68 (dd, 1H, H-6b), 3.32
(dd, 1H, H-2), 3.29 (dd, 1H, H-4), and 2.96−1.46 (br m, 10H,
carborane−BH) ppm.
13C NMR (125.76 MHz, CD3OD, 23 °C): δ = 93.8 (C-1),
79.8 (C-4), 76.4 (C-2), 74.9 (C-3), 75.1 (carborane−C), 74.2
(4-OCH2−carborane), 71.5 (C-5), 62.1 (C-6), and 60.5
(carborane−CH) ppm.
1
β anomer: H NMR (500.13 MHz, CD3OD, 23 °C): δ =
4.68 (br s, 1H, carborane−CH), 4.45 (d, 1H, J1,2 = 7.8 Hz, H-
1), 4.42 and 4.11 (each d, each 1H, J = −10.6 Hz, 4-OCH2−
carborane), 3.79 (dd, 1H, J6a,5 = 1.8, J6a,6b = −12.0 Hz, H-6a),
2a
2b
2a
(dd, 1H, JCH ,CH = −2.4 Hz, 4-OCH2bCCH), 3.85 (dd, 1H,
2b
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3.66 (dd, 1H, J6b,5 = 4.3 Hz, H-6b), 3.49 (dd, 1H, J3,2 = 9.2, J3,4
= 9.0 Hz, H-3), 3.32 (ddd, 1H, J5,4 = 9.5 Hz, H-5), 3.30 (dd,
1H, H-4), 3.09 (dd, 1H, H-2), and 2.96−1.46 (br m, 10H,
carborane−BH) ppm.
Analyst “cavity computation” to recognize the binding sites
and for estimating binding site volumes.36
2.5. Cytotoxicity. Human epithelial CAL 27 squamous
carcinoma cell line (ATCC CRL-2095) was acquired from
American Type Culture Collection (Manassas, VA, USA). TC-
treated cell culturing flasks and 96-well plates were obtained
from Corning (Corning, NY, USA). Dulbecco’s modified
Eagle’s medium (DMEM), Dulbecco’s phosphate buffer saline
(10× DPBS), Hank’s balanced salt solution (1× HBSS), fetal
bovine serum (FBS), GlutaMax (100×), and Penicillin−
Streptomycin (10,000 U/mL) were purchased from Gibco
(Life Technologies, Carlsbad, CA, USA). A CellTiter-Glo
luminescent cell viability kit was obtained from Promega
Corporation (Madison, WI, USA). The Pierce BCA Protein
assay was purchased from Thermo Fisher Scientific (Waltham,
MA, USA).
13C NMR (125.76 MHz, CD3OD, 23 °C): δ = 98.1 (C-1),
80.0 (C-4), 78.0 (C-3), 76.6 (C-2), 75.1 (carborane−C), 74.2
(4-OCH2−carborane), 74.0 (C-5), 62.1 (C-6), and 60.5
(carborane−CH) ppm.
11B NMR (160.46 MHz, CD3OD, 23 °C): δ = −2.08, −3.96,
−8.43, −10.61 and −12.09 ppm.
HRMS m/z: calcd for C9H24B10O6Na [M + Na]+, 361.2401;
found, 361.2384.
2.4. 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),25−27 with the doubly
polarized triple-zeta basis set def2-TZVPP.28 The structures of
the different ligands were aligned so that geometries would be
as similar as possible. The restrained electrostatic potential
(RESP) protocol was used to compute partial atomic
charges.29 Each ligand molecule was divided into two parts
for the RESP charge calculation, 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 7.21,30,31 and
the RESP calculations were performed with NWChem 6.8.32
Molecular docking studies were performed using AutoDock
4.2.6.33,34 All rotatable bonds in the carborane part of the
ligands were set to nonrotatable (inactive). For docking, the
number of torsional degrees of freedom for the carboranes was
set to 8 (torsdof 8). The protein structures used for docking
studies were 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 suggested by PyMOL, that is, the one
with least clashes with surrounding amino acids, 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. A
grid of size 46 × 56 × 60, with a grid spacing value of 0.375,
was used for both protein structures. 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. Two thousand
independent search runs, each with max 2.5 million energy
evaluations and population size of 150 with max 27,000
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. A cluster RMS of 2.0 Å was
used to cluster (rank) the conformations.
The commercial CellTiter-Glo luminescent cell viability
assay was used for the determination of the in vitro cytotoxicity
of glucoconjugates (1−5) and borocaptate (BSH). The human
epithelial CAL 27 squamous carcinoma cell line was used as a
head and neck cancer cell model. Cells were seeded in 100 μL
of cell culture medium supplemented with 1× GlutaMax, 1%
Penicillin−Streptomycin, and 10% FBS at the density of 5000
cells per well and allowed to adhere overnight on a 96-well
plate. Once the cells had attached, the medium was removed
and exchanged with 100 μL of complete cell culture medium
containing either 1, 2, 3, 4, 5, or BSH at 5 μM, 25 μM, 50 μM,
125 μM, and 250 μM concentration. The cells were incubated
for 6 and 24 h in a temperature- and humidity-controlled
incubator (37 °C, 95% relative humidity and 5% CO2). Fresh
cell culture medium and 1% (v/v) Triton X-100 were used as
negative and positive controls of cell viability, respectively. At
the predetermined time points, the plates were equilibrated to
r.t. for 30 min. The test solutions were discarded, and the cells
were washed with 100 μL of 1× DPBS twice. For the viability
assay, 50 μL of 1× HBSS and CellTiter-Glo cocktail were
added to each well. The plates were immediately protected
from light with aluminum foil and gently shaken on an orbital
shaker for 2 min at r.t. The ATP-generated luminescence was
measured using a Varioskan LUX multimode microplate reader
(Thermo Fisher Scientific, Waltham, MA, USA). The total
protein content was quantified using the colorimetric
bicinchoninic acid (BCA) protein assay. Each sample was
used to normalize the cell viability results. The BCA assay
procedure was carried out according to the manufacturer’s
protocol. Briefly, 25 μL of cell lysates from the Cell-TiterGlo
samples was pipetted to a 96-well clear bottom UV-transparent
microplate. Then, 200 μL of the working reagent was added to
each well (1:8 ratio). The plates were wrapped with an
aluminum foil and mixed on an orbital shaker for 30 s before
further incubation at 37 °C for 30 min. The absorbance was
read at 562 nm, and the protein content was calculated using a
bovine serum albumin (BSA) standard curve (0−2000 μg/
mL). All experiments were carried out in quadruplicate. The
statistical significance of mean cell viability was determined
using unpaired Student’s t-test and compared to the negative
control (untreated cells).
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 Couto et al.35 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 0-1-1 0
For the binding pose analysis, clusters with less than 10
conformations, clusters at unphysical location, and clusters
with positive binding energies were removed. We used CAVER
2.6. GLUT1 Protein Quantification in CAL27 Cells.
CAL27 squamous cell carcinoma cells (ATCC CRL-2095)
were purchased from the American Type Culture Collection
(ATCC, Manassas, VA, USA). CAL27 cells were cultured in
DMEM (Gibco, ThermoFisher Scientific, Waltham, MA,
USA) supplemented with 2 mM L-glutamine (ThermoFisher
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Scientific, Waltham, MA, USA), 10% heat-inactivated FBS
(Gibco, ThermoFisher Scientific, Waltham, MA, USA), and
penicillin (50 U/mL)−streptomycin (50 μg/mL) solution
(ThermoFisher Scientific, Waltham, MA, USA) at 37 °C in a
humidified incubator with 5% CO2. The CAL27 cells were
seeded onto 24-well plates at the density of 5 × 105 cells/wells.
The cells were used in further studies 2 days after seeding,
while the passage number of the cells was in the range of 8−20.
The culture medium was removed, and the cells were washed
and preincubated at 37 °C for 10 min with prewarmed HBSS
without glucose (including 125 mM NaCl, 4.8 mM KCl, 1.2
mM MgSO4, 1.3 mM KH2PO4, 1.3 mM CaCl2 and 25 mM
HEPES adjusted to pH 7.4) before the experiments.
source operated in the positive mode (Agilent Technologies,
Santa Clara, CA, USA). Initially, the peptides were separated
on a 2.1 × 250 mm, 2.7 μm column (Agilent Technologies,
Santa Clara, CA, USA) and eluted by a gradient of 0.1% formic
acid in water (A) and acetonitrile (B). A constant flow rate of
0.3 mL/min was utilized, and the gradient was shifted in the
following way: 2−7% B for 2 min, followed by 7−30% B for 48
min, 30−45% B for 3 min, and 45−80% B for 2.5 min before
re-equilibrating the column for 4.5 min. The data were
acquired using the Agilent MassHunter Workstation Acquis-
ition and processed using the Skyline software 20.1. The
GLUT1 and Na+K+ATPase proteins were quantified based on
the ratio between the light and heavy peptides.
2.7. GLUT1 Affinity and Cellular Uptake Studies.
Determination of the GLUT1 affinity was conducted as
described recently by our team.16 In more detail, the CAL27
cells were cultured, seeded, and preincubated as described
above. The GLUT1 affinity of the glucoconjugates was studied
through a cis-inhibition assay using a known radiolabeled
GLUT1 substrate, [14C]-D-glucose (PerkinElmer, Waltham,
MA, USA). Briefly, the preincubated cells were further
incubated at r.t. for 5 min with the glucoconjugates (0.1−
1800 μM) containing 1.8 μM (0.1 mCi/mL) of [14C]-D-
glucose in HBSS (250 μL), with HBSS as a control. The
reaction was quenched by the addition of ice-cold HBSS, the
cells were washed twice with ice-cold HBSS and lysed with 250
μL of 0.1 M NaOH, and the lysate was mixed with 1.0 mL of
Emulsifier safe cocktail (PerkinElmer, Waltham, MA, USA).
The radioactivity was measured by a liquid scintillation counter
(MicroBeta2 counter, PerkinElmer, Waltham, MA, USA). The
inhibition of [14C]-D-glucose uptake in the presence of the
boron cluster glucoconjugates was compared to the control
(HBSS) and calculated as percentages (%) (see Supporting
Information, Figure S75 and Table S5).
The cellular uptake of the glucoconjugates was studied by
adding the compounds in concentrations ranging from 10 to
400 μM [in prewarmed HBSS buffer (250 μL)] onto the cell
layer, followed by incubation at r.t. for 5 and 30 min,
respectively. After incubation, the reaction was quenched with
ice-cold HBSS, and the cells were washed and lysed as
described previously. The lysate used in the determination of
cellular uptake was collected from four wells into Eppendorf
tubes and centrifuged at 4 °C. From each sample supernatant,
800 μL was collected and digested in 1.0 mL of conc. HNO3
for 24 h. After sample digestion, Milli-Q water was then added
in order to reach a total volume of 10 mL. The cellular uptake
was then determined by analyzing the boron concentrations by
inductively coupled plasma mass spectrometry (ICP-MS).
The NeXION 350D ICP-MS instrument (PerkinElmer Inc.,
Waltham, MA, USA) equipped with an ESI PrepFAST
autosampler (Elemental Scientific, Omaha, NE, USA) was
used. A peristaltic pump and a nebulizer were used for sample
injection. The radio frequency (RF) was used at a power of 1.6
kW during the operations and the nebulizer gas, auxiliary gas,
and plasma gas flows were 0.90, 18, and 1.2 L min−1,
respectively. The sample uptake rate was 3.5 mL min−1 and the
dwell time was set to 100 ms per AMU. To remove polyatomic
interferences, a triple−quadrupole reaction system was used in
the collision mode with kinetic energy discrimination (KED).
Helium was used as the cell gas (3.7 mL min−1). Yttrium-89
was used as an internal standard and mixed online with the
samples to compensate for matrix effects and instrument drift.
The boron concentration was determined against the certified
The preparation of crude membrane fractions of CAL27
cells was accomplished in the following way. The cells were
washed twice with ice-cold phosphate-buffered saline (PBS),
and 2 mL of PBS was added. The cells were scraped off the
dishes and centrifuged at 250g, +4 °C, 10 min. The
supernatant was removed, and the cell pellet was snap-frozen
and stored at −80 °C before sample preparation. The
Membrane Protein Extraction Kit (BioVision Incorporated,
Milpitas, CA, USA) was used for extraction of crude
membrane fractions from the cell pellet according to the
manufacturer’s protocol. The protein concentration was
measured using a Bio-Rad Protein Assay (EnVision,
PerkinElmer, Inc., Waltham, MA, USA), and 50 μg of protein
from each sample (n = 3) was taken for further analysis.
These samples were further processed in order to prepare
them for protein quantification studies. In more detail, a total
of 50 μg of protein was dissolved in 7 M guanidine
hydrochloride, 0.5 M Tris-HCl [pH 8.5], and 10 mM
EDTA-Na [pH 8.0] in Milli-Q water. The samples were
then reduced by dithiothreitol (1:50, w/w) for 60 min at r.t.
and alkylated by iodoacetamide (1:20, w/w) (Sigma-Aldrich,
St. Louis, MO, USA) for 60 min at r.t. in the dark. The
proteins were then precipitated with cold methanol and
chloroform. The pellet (alkylated proteins) was resuspended in
a 6 M urea solution and mixed for 10 min at r.t. The alkylated
proteins were dissolved completely by intermittent sonication
(Branson 3510, Danbury, CT, USA) after diluting the samples
with 0.1 M Tris-HCl to a final concentration of 1.2 M urea.
The dissolved proteins were first digested with LysC (1/100,
w/w) (Sigma-Aldrich, St. Louis, MO, USA) and 0.05%
ProteaseMax (Promega Biotech AB, Nacka, Sweden) for 3 h
at r.t. and then spiked with 10 μL (60 fmol) of the labeled
JPT’s peptides for absolute quantification (JPT Peptide
Technologies GmbH, Berlin, Germany) (see Supporting
Information, Table S4) and were further digested with (1/
100, w/w) TPCK-Trypsin (Promega Biotech AB, Nacka,
Sweden) for 18 h at 37 °C. The digestion was stopped by
adding 40 μL of 5% formic acid, and the diluted samples were
centrifuged at 18,000g × 5 min at 4 °C. The supernatants were
transferred to HPLC vials for quantification studies.
The absolute quantity of GLUT1 was determined by a LC−
MS/MS-SRM setup. In more detail, the quantification of
GLUT1 and the membrane marker Na+K+ATPase was based
on three selected reaction monitoring (SRM) transitions of
precursor and product ions from both the light and heavy
peptide chains (see Supporting Information, Table S4 and
Figures S69−S74), as previously described.37 A total of 20 μL
of the digested peptides (10 μg) was injected into an Agilent
1290 LC system coupled with an Agilent 6495 triple
quadrupole mass spectrometer with an electrospray ionization
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multielement calibration standard, TraceCERT Periodic Table
Mix 1 (Sigma-Aldrich) in acid conditions (6.7% HNO3,
TraceMetal grade, Fisher Chemical). The calibration range
used for 11B was 4−400 μg L−1. The limit of detection (LOD)
was 1.0 μg L−1. Three replicates were analyzed from each
concentration. The data were analyzed with the PerkinElmer
Syngistix Data Analysis Software.
which could have a detrimental effect on the transport
process.40−42 In addition, a glucoconjugate bearing a
carboranylmethyl-substituent delivers 10 times the amount of
boron atoms per molecule compared to BPA, has a small
degree of rotational freedom which may be important for
passage through the transporter, and retains a minimally
intrusive nature from a steric point-of-view. The targeted
glucoconjugates are thus a seemingly ideal solution for BNCT.
From a medicinal chemistry perspective, the importance of
comparing the properties of the complete set of positional
isomers under identical conditions cannot be sufficiently
stressed: such studies are mandatory in order to identify the
most promising modification sites. In the literature, the
synthesis of 1, 2, and 4 has been reported a few decades ago
and the synthesis of 6 was recently described by our
team.16,43,44 While the previous work has left several questions
unanswered regarding the potential of glucoconjugates as
delivery agents for BNCT, the work has nevertheless been of
importance in establishing functioning reaction protocols for
the construction of boron-glucoconjugates. One observable
limitation in some of the previous reports is the lack of 11B
NMR spectra as part of the reported structural characterization
data. Here, we bridge this gap as all of the structures containing
boron clusters were fully characterized by NMR spectroscopy
2.8. NMR Spectroscopic Hexokinase Studies. The
activity of human hexokinase 1 on each of the glucoconjugates
was studied by incubating each compound with the enzyme in
1
an NMR tube at 30 °C and recording a time series of H and
31P NMR spectra overnight. The reaction mixture contained 2
mM glucoconjugate, 5 mM ATP, and 5 mM MgCl2 in 50 mM
Tris-HCl buffer, pH 7.5, containing 10% of D2O (EurisoTop).
A 2 mM solution of D-glucose was used as a positive control.
The experiments were performed on a 600 MHz Bruker
AVANCE III NMR spectrometer equipped with a QCI H-P/
C/N-D cryoprobe and SampleJet automated sample changer.
After the initial screening of both the 1H and 31P NMR
experiments, 5 μL (10 mU) of recombinant human hexokinase
1 (Abcam) was added to the NMR tube and recording of the
time series of the spectra was initiated. The preheater block of
the SampleJet was used as a sample incubator during the
experiments. This enabled automated monitoring of several
reactions in parallel.
1
including H-decoupled 11B NMR. Focus will next be placed
on the synthesis of the new glucoconjugates 3 and 5, which
have not been reported previously in the literature. Both
synthetic routes are depicted in Scheme 1.
3. RESULTS AND DISCUSSION
3.1. Synthesis and Structural Characterization of the
Positional Isomer Library. Thirty-three chemical reactions
(without accounting for method development) are required in
order to access the entire library of positional isomers depicted
in Figure 2. Because of our recent work on the synthesis of 6,16
For the synthesis of 3, an 11-step synthetic route was
originally devised starting from commercially available
peracetylated D-glucose. From a synthetic perspective, it was
important to consider the susceptibility of decaborane to
nucleophiles,45 that is, protecting groups would be a necessity.
The requirements for the protecting group strategy were
twofold. First, in order to access boron cluster derivative 3, a
conjugation reaction between an alkyne and a decaborane-
ACN complex was envisioned. This required the preinstalla-
tion of a propargyl group at the 2nd position. Second, ortho-
carboranes are known to degrade under strongly basic
conditions and therefore protecting groups requiring such
deprotection conditions were avoided.45
With these premises in mind, we started by converting
peracetylated D-glucose into ortho-ester 7 via a four-step
protocol. First, the anomeric acetate was displaced by an iodine
with I2 and Et3SiH, followed by conversion into an 1,2-ortho-
ester species with 2,6-lutidine, TBAI, and MeOH in a 91%
yield (over two steps).46
The acetyl groups were next exchanged to benzyl groups
through a two-step deacetylation47−benzylation48 protocol
with an overall yield of 51%. The selective ring-opening of the
ortho-ester was performed with p-TsOH in acetone/H2O 7:3
in order to obtain the 1-OH/2-OAc derivative. While the
overall yield was 88%, the formation of an inseparable and
unwanted 1-OAc/2-OH regioisomer was also noted in a 7:1
ratio in favor of the desired regioisomer. The mixture was
converted to imidate donor 8 with DBU and Cl3CCN in DCM
in a 91% yield.46,49 It should be noted that this yield would not
be possible to obtain from the mixture as such and therefore, it
is likely that acyl migration takes place under the basic
conditions employed, which increases the amount of the 1-
OH/2-OAc isomer in situ.50
Figure 2. Molecular structures of all positional isomers of ortho-
carboranylmethyl substituted D-glucose. All glucoconjugates were
prepared in the present study. The ones for which synthetic protocols
have not been previously reported in the literature are shaded with a
blue glow (3 and 5). In the ortho-carboranylmethyl moiety, the blue
dots represent boron atoms and the gray dots represent carbon atoms
(hydrogen atoms omitted).
and the α and β methyl glycosides thereof, we had already
obtained the required knowledge in both boron cluster
chemistry and carbohydrate chemistry to plan appropriate
synthetic pathways.
From a design perspective, it is important to note that the
chosen boron cluster contains 10 boron nuclei per delivery
agent in a charge-neutral way. While the carboranes exhibit
three-dimensional aromaticity which could afford them some
added stability,38,39 we opted to use a neutral hydrophobic
boron cluster in order to avoid undesirable interactions
between charged boron clusters and amino acid residues,
Following a previously reported robust TMSOTf-promoted
glycosylation protocol,51,52 but instead employing BnOH as
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a
Scheme 1. Synthetic Routes and Reaction Conditions Leading to 3 and 5
a
(i) (1) I2, Et3SiH, DCM, reflux, 1 h; (2) 2,6-lutidine, TBAI, MeOH, reflux, 3 h, 91% over two steps; (3) NaOMe, MeOH, RT, 1.5 h, quant.; (4)
NaH, BnBr, DMF, 2 h, 51%; (ii) (1) p-TsOH, acetone:H2O 7:3, RT, 2 h, 88%; (2) Cl3CCN, DBU, RT, 1 h, 91%; (iii) BnOH, TMSOTf, DCM,
−20 °C, 2 h, 96%; (iv) (1) NaOMe, MeOH, THF, RT, 24 h, 91%; (2) NaH, propargyl bromide, DMF, RT, 2 h, 85%; (v) (1) B10H14, ACN, 60 °C,
1 h; (2) 10, toluene, 80 °C, 17 h, 51%; (3) H2, 10% Pd/C, EtOAc:MeOH 7:1, 5 bar, 4 h, 77%; (vi) BnOH, BF3·OEt2, DCM, 0 °C → RT, 56%;
(vii) (1) NaOMe, MeOH/THF, RT, 17 h, 99%; (2) C6H5CH(OCH3)2, p-TsOH, DMF, 60 °C, 0.2 bar, 2 h, 85%; (viii) (1) Bu2SnO, toluene; 120
°C, 3 h; (2) TBAB, CsF, BnBr, 120 °C, 80% (selectivity 2:1); (ix) (1) NaH, BnBr, DMF, RT, 2.5 h, 80%; (2) Et3SiH, TFA, DCM, RT, 3 h, 72%;
(x) NaH, propargyl bromide, DMF, RT, 2 h, 91%; (xi) (1) B10H14, ACN, 60 °C, 1 h; (2) 14, toluene, 80 °C, 20 h, 65%; (3) H2, 10% Pd/C,
EtOAc:MeOH 7:1, 5 bar, 4 h, 77%.
Figure 3. Left: An overlapping Ed-HSQC (CH/CH3 in blue and CH2 in green) and HMBC-spectrum (red) with the most important HMBC-
correlations highlighted with gray circles. Top right: The molecular structures of the two anomers together with the colors used for visualization of
the different signals in the NMR spectra. Bottom right: the 5.2−3.2 ppm region of the 1H NMR spectrum highlighting the accuracy of the spectral
simulations with the PERCH software (measured spectrum in blue, simulated spectrum in red). The 5.0−4.6 ppm region has been removed, and
the methanol and CD3OD peaks have not been included in the simulation.
the acceptor, compound 9 could be isolated in excellent yield.
In order to convert 9 into building block 10 which was
required for the conjugation reaction with decaborane, a high-
yielding two-step deacetylation−propargylation protocol was
employed. At this stage, all hydroxyl groups were protected as
benzyl ethers, except the 2nd position, which contained a
propargyl-substituent. The benzyl ether has been found to be a
suitable protecting group during the synthesis of boron cluster
glucoconjugates of the chosen type.16,53 The conjugation with
decaborane (B10H14) was conducted in two stages following
the typical conditions reported in the literature.45 Decaborane
was first refluxed in dry ACN under inert conditions in order
to form the reactive B10H12·2ACN species, which was then
allowed to react with the alkyne functionality in 10 at elevated
temperatures overnight. The reaction was quenched by the
addition of dry MeOH before further work-up and purification.
The moderate yield obtained after isolation of the end product
is in the typically observed literature range.45 In the last step,
the benzyl protective groups were removed in a hydrogenolysis
reaction following our previously described protocol and in
good yield. While this synthetic route to 3 is rather lengthy, a
satisfactory overall yield of 11% could nevertheless be
achieved.
In principle, both glucoconjugates 3 and 5 could be
prepared through a shared synthetic route. This would shorten
the overall synthetic routes significantly. Therefore, while
shifting focus to the synthesis of 5, we did address the
possibility of using one of the intermediates for the generation
of 3 as well. Starting from commercially available peracetylated
D-glucose, we performed a BF3·OEt2 promoted glycosylation
with BnOH according to literature protocols and in similar
yields.54 Deacetylation under Zemplen conditions47 followed
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by the formation of a 4,6-O-benzylidene acetal with p-TsOH
gave 12 in an 84% yield over two steps.55,56 In order to
synthesize 3 by a shorter reaction route, we attempted a
stereoselective benzylation of the 3-OH group of 12 through
the use of a dibutylstannylene intermediate.57,58 While the
yield was high (80%) and the selectivity decent (2:1 in favor of
the desired regioisomer), we were not able to separate the two
regioisomers at this stage or at any other stage for that matter,
although we did complete the synthesis of 3 and 4 as a mixture
through this route as well. Unfortunately, the lengthy route
therefore seems to be required in order to access 3 in its pure
form. Continuing on the synthesis of 5, the hydroxyl groups in
12 were benzylated and the benzylidene acetal was selectively
ring-opened with Et3SiH and TFA59 to give the 4-OH/6-OBn
substrate in high yield. From this stage onward, similar reaction
conditions as described above were employed. In short, the
free hydroxyl group was propargylated in excellent yield,
followed by a conjugation reaction with the preformed B10H12·
2ACN species and cleavage of the benzyl groups through
hydrogenolysis. Altogether, glucoconjugate 5 was prepared in
eight synthetic steps with an overall yield of 12%.
and may alter their behavior. Therefore, these aspects have
become the focus of our GLUT1-targeting approach to the
development of improved delivery agents for BNCT. GLUT1
is an interesting transporter that is well-suited for BNCT.
Because of the “Warburg effect”, a wide range of cancer cells
overexpress GLUT1.18,20 This stems from the inefficient
glucose metabolism in cancer cells further coupled with a
greater demand for energy consumption in cell proliferation.
The head and neck cancers are no exception and GLUT1 has
been reported to be responsible for the aberrant growth of the
human CAL 27 carcinoma cell line, which is used as our model
in the in vitro studies.64,65 To gain insight into the GLUT1
expression level in the CAL 27 cells, we initially determined
the GLUT1 amount by LC−MS/MS-SRM and found it to be
4.7 0.97 fmol/μg of protein. For comparison, the GLUT1
expression in, for example, human embryonic kidney cells
(HEK 293) was found to be significantly less pronounced, only
0.37 0.09 fmol/μg of protein. The expression of GLUT1 in
the CAL 27 cancer cells is therefore 8−20 times higher than
that in the control cell line used. Even when these results were
further normalized to Na+/K+-ATPase, the GLUT1 expression
was still roughly 2.5-times higher in the CAL 27 cells. Although
these factors indicate that there is significant potential in
targeting GLUT1, this approach has previously been viewed as
suboptimal from a BNCT perspective because of open
questions regarding the possibility of modified glucoconjugates
to compete for the transporter with the high levels (6 mM) of
D-glucose found in blood.24,66 In our previous work,16 we
showed that glucoconjugate 6 has a significantly higher affinity
toward the transporter thereby proving that such doubts were
unfounded. In order to investigate if the 6th position was
unique in this matter or if this is a general feature of these types
of glucoconjugates, we performed a similar cis-inhibition assay
in the CAL 27 cell line with the entire positional isomer library
in this work (the GLUT1-function and expression in the CAL
27 cell line had been validated). In short, the cis-inhibition
assay was conducted as a competition assay between the
individual glucoconjugates and radiolabeled [14C]-D-glucose
with a control experiment performed with D-glucose. This
experimental setup is an accurate representation of the
situation that the glucoconjugates would face in the intended
application. The experimentally determined relative GLUT1
IC50-values for glucoconjugates 1−5 were in the low μM-
range: 84.6 μM for 1, 107.6 μM for 2, 122.6 μM for 3, 86.2 μM
for 4, and 32.07 μM for 5. Compared to the >1 mM IC50-value
displayed by D-glucose, the transporter-targeting capability of
the glucoconjugates is at least 8−31 times higher. From a
practical point-of-view, the glucoconjugates are therefore
expected to be able to compete for the transporter with the
high D-glucose levels found in blood. While care should be
taken in drawing conclusions based on affinity results alone,
the experimental IC50-values determined herein, and previously
for 6 (44.0 μM), suggest that modification at positions 4 or 6
lead to the highest GLUT1-affinities.
On any given synthetic route, the results obtained are only
reliable if the reported structural characterization data are
accurate. In this work, high-resolution mass spectrometry
(HRMS) and NMR spectroscopy were employed in
verification of the structural features and purity of all products
synthesizedwith emphasis on the latter technique. NMR
spectroscopy is generally referred to as the number one tool for
structural elucidation studies of organic molecules. Herein, we
used a solid selection of 1D- and 2D-NMR experiments (1H,
1
13C, H-decoupled 11B, 1D-TOCSY, DQF-COSY, Ed-HSQC,
HMBC) with further processing of the 1H NMR spectra in the
PEak reseaRCH (PERCH) software60 in order to obtain
accurate coupling constants (excerpts of representative spectra
are displayed in Figure 3).
The PERCH software applies quantum mechanical opti-
mization in the spectral simulations and the accurate coupling
constants are required in order to verify the conformation of
the glucoconjugates, which is important for understanding
their interactions with GLUT1. Altogether, we were able to
assign all signals (carboranylmethyl moiety, protective groups
1
and carbohydrates) in the H and 13C NMR spectra of all
products. While the characterization flow of anomeric mixtures
was addressed in detail in our recent study16 and will not be
repeated here, it is important to note that the carboranyl-
methyl-bearing glucoconjugates exist primarily in the 4C1-chair
conformation.
Finally, the purity of glucoconjugates 1−5 was assessed by
absolute qNMR using maleic acid as an internal calibrant in
accordance with the protocol reported by Pauli et al.61 These
experiments confirmed that the purity of all glucoconjugates
evaluated in the in vitro studies exceeded 95%.
3.2. Investigating the Interactions with the GLUT1
Transporter. While a number of boron-glucoconjugates have
been reported to date,62,63 studies on the molecular level
recognition and interaction with their intended transporters,
the very essence of their biochemical functioning principle, are
lacking. These foundations need to be addressed, not only
from a functioning principle point-of-view but also from a
medicinal chemistry and drug development perspective. Efforts
in this direction have been reported for other classes of
glucoconjugates recently.24 However, a change in the
substituents inflicts a change in the character of the molecules
In order to address the molecular level basis for the observed
affinities, we used the molecular models constructed and
validated previously.16 These models are based on XylE, a D-
xylose-proton symporter found in Escherichia coli which is
structurally similar to the GLUT1−4 proteins.67 Most
importantly, the required crystal structures for both the
outward- (PDB ID 6N3I)68 and inward-open (PDB ID
4QIQ)69 conformations have been published, thus resulting
in the possibility for assessing the interactions of the
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Figure 4. Top: the volumes of the binding pockets as recognized by CAVER Analyst36 are highlighted; top left: the outward-open structure (cyan),
top right: the inward-open structure (light green). Blue string = outward-open XylE, green string = inward-open XylE, natural ligand binding site
amino acids as sticks, color scheme CAVER. Bottom: The β-anomer of 5 displayed in the binding site with the amino acids binding the natural
ligand marked. The figures highlight the changes in the positions of especially GLN168 and TRP392 during the conformational shift from (bottom
left) the outward-open structure (blue) to (bottom right) the inward-open structure (green).
glucoconjugates with the transporter on the outside and inside
of the cell. Following virtual mutation of the Gln415 in XylE to
Asn to match Asn411 in GLUT1, an identical carbohydrate
binding site can be established.70,71 Using the same model for
the inward- and outward-open conformations is crucial as it
provides directly interconnectable information on the binding
modes displayed by the glucoconjugates on the outside and
inside of the cells. As a result, the use of a XylE-based model is
warranted in our case. For GLUT1, only the inward-open
crystal structure has been reported,72 thus impeding
comparison to the observed experimental affinities, where the
outward-open structure is more relevant.
Through the use of our previously established protocols, we
determined the binding energies of glucoconjugates 1−5 by
molecular docking studies. It should be noted that molecules
3−6 exist as an anomeric mixture of α and β anomers (α/β 1:3
for 3; 1:3 for 2; 1:1 for 4 and 3:2 for 6). While the properties
of α and β anomers are indistinguishable in experimental
studies, the individual anomers can be, and were, separately
studied in the ligand-transporter modeling assay. In line with
the observations made earlier for 6,16 and the experimental
affinity values determined in the cis-inhibition assays,
glucoconjugates 1−5 displayed significantly stronger binding
affinities to both the outward- and inward-open conformation
than the natural ligand D-glucose. In the outward-open
conformation, the binding free-energy difference was found
to be ca. 5 kcal/mol in favor of the glucoconjugates compared
to glucose. This corresponds to a remarkable binding affinity
increase on the order of 103 (see the Supporting Information
for details). The energy differences between the individual
glucoconjugates, on the other hand, were marginal.
binding process. Measured experimental values are naturally a
more fitting representation of a true biological system. Both
the experimental and computational studies are in line, thereby
indicating that the affinity differences between the carbor-
anylmethyl-substituted glucoconjugates are minimal. These
results are quite surprising and prompt a couple of questions.
Is there no substantial difference between the modification
site? How could such observations be explained?
We sought to address these questions by analyzing
thoroughly the results obtained from the docking studies in
both the outward- and inward-open conformations. In the
outward-open conformation, two distinct binding poses can be
identified for the glucoconjugates. In one, the carbohydrate
part overlaps with the most favorable binding pose of free D-
glucose, as identified by the docking study, while in the other,
the glucoconjugates are slightly rotated. The amino acid
residues involved in the recognition process are Gln168,
Gln288, Asn294, Trp392, and Asn415.67 In order to piece the
puzzle together, it was important to address the binding poses
and amino acid residues participating in the glucoconjugate/
protein complex also in the inward-open conformation and
search for similarities/differences. In the inward-open con-
formation, the glucoconjugates display significantly lower
binding affinities across the board. This may be beneficial
from a functional point-of-view as it implies that the
glucoconjugates are less tightly bound to the transporter on
the inside of the cell and thus more prone to be releaseda
seemingly ideal feature of a delivery agent. The reduced
binding affinity to the inward-open structure could be due to
the “wider nature” of the inward-open protein cavity; the
binding site volume in the inward-open structure is three times
larger than that in the outward-open structure (see Figure 4
and Supporting Information, Tables S1−S3). The wider
We note that the computational values are by necessity and
construction based on an approximate description of the
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binding site offers greater flexibility in terms of binding poses
than the outward-open one.
The most noteworthy of the binding poses for the different
glucoconjugates binding to the inward-open protein structure
are the ones in which the hydrophobic boron cluster is located
close to the amino acids identified to bind the natural ligand,
that is, Asn415, Gln288, and Trp392 (and Asn294), while the
glucose-moiety points toward Gln168. The binding poses of
the glucoconjugates in the outward-open structure are
supportive of this inward-open ligand/transporter complex.
The main amino acid residues involved in the glucoconjugate−
binding are the same as in the outward-open conformation. In
the eventual outward-open to inward-open conformational
change process, the Gln168 amino acid is expected to move
away as the entire N-domain moves away from the C-
domain.70 Therefore, the glucoconjugates would be expected
to be capable of undergoing the necessary transformation from
both binding poses in the outward-open state to the binding
poses C−F in the inward-open state (data not shown). It is
encouraging that the glucoconjugates interact with the
transporter in a similar fashion in the top-candidate computa-
tional binding poses identified for both the outward- and
inward-open structures. There is thus no need for the ligand to
undergo any considerable conformational changes inside the
narrow transporter channel, a process that would be expected
to significantly hinder the cellular uptake.
Figure 5. Cell cytotoxicity studies in the human CAL 27 cell line after
incubation with cell culture medium (negative control), 1% Triton X-
100 (positive control), glucoconjugates (1−5), and BSH at the
concentrations of 5, 25, 50, 125, and 250 μM for 6 h and 24 h. Results
are represented as the mean s.d. (n = 4) in comparison with the
negative control. The statistical significance was analyzed using an
unpaired Student’s t-test, where the significance was set at *p < 0.05,
**p < 0.01 and ***p < 0.001.
3.3. Cytotoxicity and Boron Delivery Capacity. With a
concise view of the GLUT1−glucoconjugate interactions
occurring on a molecular level obtained, we continued by
addressing topics of interest from a translational medicine
perspective. The most essential properties were deemed to be
cytotoxicity, boron delivery capacity, and metabolic fate. All of
these studies were conducted in vitro. In this section, the focus
will be on the cytotoxicity and boron delivery capacity with the
metabolic fate addressed in the next section.
BNCT. Based on cytotoxicity and affinity results; 4, 5, and 6
seemed to have the highest potential at this stage.
The GLUT1-targeting approach differs from the LAT1-
targeting approach of BPA and the passive transport
mechanisms of BSH and GB-10 (the delivery agents used in
the clinics).10−12,74 In cellular uptake studies, we therefore
included both BSH and BPA in order to be able to compare
the boron delivery capacity to the approaches and agents in
clinical use. It should be noted that while the term “cellular
uptake” is used, the protocol employed does not differentiate
between intracellular components and components trapped on
the cell membrane. Regardless of if the compounds are trapped
on the cell membrane or internalized, the boron nuclei are
within the range required to exert a cell-killing effect because
the α particles travel a distance of 5−9 μm in tissue.73
Therefore, from a BNCT perspective, the results obtained are
still equally valid.
In the cellular uptake studies, we used our previously
developed protocol.16 Shortly, the boron content in the CAL
27 cell lysates was determined after incubation with the
delivery agents and careful washing to remove unattached
species from the matrix. A concentration range of 10−400 μM
was selected based on the GLUT1-affinity studies, and
incubation times of 5 and 30 min were selected based on
the optimal performance of [14C]-D-glucose under these
conditions (see the Supporting Information). Before ICP-MS
analysis of the boron contents, the lysates from four wells were
combined and digested. The results are summarized in Figure
6. Based on the results it is clear that the GLUT1-targeting
approach bears a considerable potential because the boron
delivery capacity of the glucoconjugates is significantly higher
than those of the agents in current clinical use. While the
alternate delivery strategy is the main cause for the
observations, it should be noted that the glucoconjugates do
The cytotoxicity and cellular uptake studies were conducted
in the human CAL 27 carcinoma cell line, the same cell line
used in the affinity studies because it is a representative head
and neck cancer cell line of clinical relevance and the GLUT1-
function and expression had been validated.64,65 In order for a
boron delivery agent to be of potential interest, it should not
impair the cellular viability of the cancer cells prior to BNCT
irradiation.73 In the cytotoxicity assays, the glucoconjugates 1−
5 and BSH were incubated with the CAL 27 cells at
concentrations of 5, 25, 50, 125, and 250 μM for 6 h and 24
h. BSH served as a control of a delivery agent in clinical use,
and BPA was omitted because its IC50-value has been reported
to be in the low mM range.10 Apart from this, the
concentration range was selected based on the GLUT1-affinity
results, and the incubation times are representative of time
frames used in clinical BNCT following intravenous admin-
istration of delivery agents. The results obtained from the
cytotoxicity assays are summarized in Figure 5. The cell
viability was quantified by a commercial CellTiter-Glo assay,
and the IC50-values were obtained from nonlinear regression
fitting of the cell viability data. The IC50-values were
determined to be 181 μM for 1, 101 μM for 2, 42 μM for
3, 203 μM for 4, 201 μM for 5, and 220 μM for BSH. The
IC50-value previously reported for 6 in the CAL 27 cell line was
215 μM.16 It is important to note that the modest
cytotoxicities displayed by the glucoconjugates, especially 4,
5, and 6, would not hamper their use as delivery agents in
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3.4. Outruling Potential Metabolic Pathways. The
metabolic fate of glucoconjugates has remained an open
question. Especially concerns regarding the possible incorpo-
ration of glucoconjugates into other biomolecules through
metabolic routes has been raised. Reminiscent to the previous
speculations regarding the competition with D-glucose for the
transporters, scientific studies addressing this topic are lacking.
The main metabolic pathways for D-glucose metabolism are
the glycolysis route and the pentose phosphate pathway (PPP).
In both pathways, the first step is the phosphorylation of the
6th position by the enzyme hexokinase.75 While this position is
blocked in glucoconjugate 6, the possibility of entering these
metabolic routes is in theory possible for glucoconjugates 1−5.
In order to determine if glucoconjugates 1−6 are substrates for
hexokinase, we used an in situ NMR spectroscopic approach to
analyzing this important step. In this protocol, the individual
glucoconjugates 1−6 and D-glucose (a positive control) were
dissolved in a TRIS-buffered aqueous solution (2 mM
concentration of the glucoconjugates) containing an excess
of ATP and Mg2+ ions. Recombinant human hexokinase 1 was
added to the mixture, and the NMR experiments were
1
performed at 30 °C by recording H and 31P NMR spectra
1
continuously. The H and 31P NMR spectra are well suited to
follow the transformation as the natural abundance of these
NMR-active nuclei is high, thereby leading to a sufficient
sensitivity.76 In a typical experiment, the chemical shift changes
accompanied by the transformation of ATP to ADP and the
eventual effect on the chemical shifts of the carbohydrate
signals were carefully monitored. In our methodology
validating experiment with D-glucose, we were able to observe
the expected phosphorylation resulting in glucose-6-phosphate.
The observed chemical shift changes in the 31P NMR spectra
were a decrease in the ATP signals appearing at −7.2, −11.3,
and −22.3 ppm with a corresponding increase in the ADP
Figure 6. Cellular uptake results in the CAL27 cell line after
incubation with glucoconjugates 1 (yellow), 2 (red), 3 (blue), 4
(green), 5 (purple), 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 1: Vmax = 2.654; Km = 616.8: 3:
●
○
1
signals appearing at −6.7 and −11.8 ppm. Changes in the H
Vmax = 28.65; Km = 3859 and 4: Vmax = 2.208; Km = 544.3. At 30 min
chemical shifts of D-glucose were likewise noted (e.g., H-2
shifted from 3.19 ppm to 3.22 ppm) as a result of the
phosphorylation.
incubation time (B), glucoconjugate 1: Vmax = 3.290; Km = 450.8; 2:
Vmax = 4.816; Km = 511.2; 3: Vmax = 11.85; Km = 543.0; 4: Vmax
0.887; Km = 111.4; 5: Vmax = 2.924; Km = 145.0 and BPA: Vmax
3.625; Km = 3737.
=
=
With a functioning and validated protocol developed, we
addressed the possibility of glucoconjugates 1−6 entering the
glycolysis route or the PPP. None of the glucoconjugates were
phosphorylated at the 6th position, or any other position for
that matter, thus indicating that they are not substrates for
human hexokinase and are thereby unlikely to enter these
metabolic routes. Therefore, the previous concerns regarding
the potential disruption of regular glucose metabolism by the
glucoconjugates have been unfounded. From a BNCT
perspective, the findings seem rather ideal. The glucoconju-
gates deliver significant boron contents to the cells but do not
disturb the general glucose metabolism. Thus, they do not fulfil
the energy requirements of the dividing cells, and additional D-
glucose uptake is probable. This may lead to further
accumulation of glucoconjugates and explain in part the
significant boron delivery capacity noted in the cellular uptake
studies.
contain 10 times the amount of boron nuclei/delivery agent
compared to BPA. Surprisingly, the boron delivery capacity of
glucoconjugate 3 is considerably better than that for the rest of
the glucoconjugates. While this does not correlate with the
affinity results obtained, a direct correlation is not expected as
the affinity and uptake studies provide insights into two
separate properties: the ability to compete for the transporter
with the natural substrate and the ability to remain attached or
internalized in the cells. The results obtained with
glucoconjugates 1−5 in the present study are in line with
those reported earlier by Lippard et al. with other types of
glucoconjugates.24 While modification at the second position
was found to result in the best functioning molecule in the
current work, and in the work by Lippard et al., glucoconjugate
6, which was the focus of our previous study,16 displays
improved overall qualities: higher GLUT1-affinity, lower
cytotoxicity, and higher boron delivery capacity. Therefore,
while on a substrate-specific level glucoconjugate 6 remains the
hit compound, the results obtained with the entire positional
isomer library solidify the foundations of the GLUT1-targeting
strategy and showcase the significant potential embedded in
this approach.
4. DISCUSSION
Understanding the biochemical foundations of any drug
development or medicinal chemistry campaign provides a
decisive advantage and the means for improving the selected
approach, whichever it may be. In this study, we have
concentrated on studying the biochemical foundations of a
successful GLUT1-targeting approach to BNCT. Our
Q
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Molecular Pharmaceutics
pubs.acs.org/molecularpharmaceutics
Article
approach represents an alternative boron delivery strategy to
those presently in clinical use. The GLUT1-targeting approach
has been long neglected because of a number of doubts
concerning its suitability for BNCT. Here, we have addressed
all of these and shown that there is no need for concern. On
the contrary, the glucoconjugates exhibit superior properties as
measured by key indicators.
Ahmed Montaser − School of Pharmacy, University of Eastern
Finland, FI-70211 Kuopio, Finland; orcid.org/0000-
0003-4511-467X
Hannu Maaheimo − VTT Technical Research Centre of
Finland Ltd., FI-02044 VTT, Finland
Kristiina M. Huttunen − School of Pharmacy, University of
Eastern Finland, FI-70211 Kuopio, Finland; orcid.org/
0000-0002-1175-8517
In more detail, we have synthesized the entire positional
isomer library of ortho-carboranylmethyl-bearing glucoconju-
gates and assessed their properties through a comprehensive in
vitro evaluation featuring computational/experimental
GLUT1-affinity, cytotoxicity, cellular uptake and metabolic
fate studies. The glucoconjugates are (1) capable of competing
for GLUT1 with the natural substrate, (2) display acceptable
cytotoxicity, (3) have a significantly boosted boron delivery
capacity compared to the delivery agents in clinical use, and
(4) do not enter the common metabolic routes of D-glucose.
We have conducted all of the in vitro studies in the CAL 27
cancer cell line, a tumor type amenable to the treatment with
BNCT and a solid model for head and neck cancers. Head and
neck cancers have been targeted as they are suitable for BNCT
and 20−40% of the cancers in this category are either
inoperable or recurrent and require nonconventional cancer
treatments. While still too early to draw definite conclusions
regarding the potential of the glucoconjugates in vivo, our
findings firmly support and lay the foundations for progressing
to the in vivo stage. To this end, we are currently working on
the synthesis of 10B-enriched species and in vivo biodistribution
and tumor accumulation studies. The revitalization of BNCT is
in full force.
̈
Sirpa Peraniemi − School of Pharmacy, University of Eastern
Finland, FI-70211 Kuopio, Finland
Anu J. Airaksinen − Department of Chemistry, University of
Helsinki, 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, FI-00014 Helsinki, Finland; orcid.org/0000-
0002-2956-4366
Mikael P. Johansson − Department of Chemistry, University
of Helsinki, FI-00014 Helsinki, Finland; Helsinki Institute of
Sustainability Science, HELSUS, FI-00014 Helsinki, Finland;
CSC−IT Center for Science Ltd., FI-02101 Espoo, 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.0c00917
Author Contributions
¶J.M., J.J., and I.K.S. contributed equally.
Notes
The authors declare no competing financial interest.
ASSOCIATED CONTENT
■
ACKNOWLEDGMENTS
sı
■
* Supporting Information
The authors would like to thank MSc. Helena Bland
(University of Helsinki) and laboratory technician Tarja
Ihalainen (University of Eastern Finland) for laboratory
assistance, Olli Aitio (Glykos Finland Ltd.) and PhD. Juri
Timonen (University of Eastern Finland) for fruitful
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.molpharma-
ceut.0c00917.
Additional reaction routes, NMR spectra of all
compounds prepared and additional details on molec-
ular modeling, and GLUT1-studies (PDF)
́
discussions, and Prof. Helder A. Santos and Alexandra Correia
(University of Helsinki) for the kind permission to use their
cell culturing facilities and microplate reader in the cytotoxicity
assays. CSCThe Finnish IT Center for Science is acknowl-
edged for providing ample computing resources. Financial
support from the Cancer foundation in Finland, the Jane and
Aatos Erkko Foundation, the Swedish Cultural Foundation,
AUTHOR INFORMATION
■
Corresponding Author
Filip S. Ekholm − Department of Chemistry, University of
Helsinki, FI-00014 Helsinki, Finland; orcid.org/0000-
0002-4461-2215; Email: filip.ekholm@helsinki.fi
̈
the Ruth and Nils-Erik Stenback Foundation, the University of
Helsinki research funds, the Academy of Finland (projects:
289179, 319453, 320102, and 308329), the Waldemar von
Frenckell foundation, and the Finnish Cultural Foundation is
gratefully acknowledged.
Authors
́
Jelena Matovic − Department of Chemistry, University of
Helsinki, FI-00014 Helsinki, Finland
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