Addressing the biochemical foundations of a glucose-based "trojan horse"-strategy to boron neutron capture therapy: From chemical synthesis to in vitro assessment

Article abstract of DOI:10.1021/acs.molpharmaceut.0c00630

Boron neutron capture therapy (BNCT) for cancer is on the rise worldwide due to recent developments of in-hospital neutron accelerators which are expected to revolutionize patient treatments. There is an urgent need for improved boron delivery agents, and herein we have focused on studying the biochemical foundations upon which a successful GLUT1-targeting strategy to BNCT could be based. By combining synthesis and molecular modeling with affinity and cytotoxicity studies, we unravel the mechanisms behind the considerable potential of appropriately designed glucoconjugates as boron delivery agents for BNCT. In addition to addressing the biochemical premises of the approach in detail, we report on a hit glucoconjugate which displays good cytocompatibility, aqueous solubility, high transporter affinity, and, crucially, an exceptional boron delivery capacity in the in vitro assessment thereby pointing toward the significant potential embedded in this approach.

Full text of DOI:10.1021/acs.molpharmaceut.0c00630

Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
Addressing the Biochemical Foundations of a Glucose-
based “Trojan horse”–strategy to Boron Neutron Capture
Therapy: From Chemical Synthesis to In Vitro Assessment
Jelena Matovi#, Juulia Järvinen, Helena C. Bland, Iris K Sokka, Surachet Imlimthan, Ruth
Mateu Ferrando, Kristiina M. Huttunen, Juri Timonen, Sirpa Peräniemi, Olli Aitio, Anu J.
Airaksinen, Mirkka Sarparanta, Mikael P. Johansson, Jarkko Rautio, and Filip S. Ekholm
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/
acs.molpharmaceut.0c00630 • Publication Date (Web): 11 Aug 2020
Downloaded from pubs.acs.org on August 19, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 47
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
Addressing the Biochemical Foundations of a
Glucose-based “Trojan horse”–strategy to Boron
Neutron Capture Therapy: From Chemical Synthesis
to In Vitro Assessment
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1,
2,
1
1
1
Jelena Matović, Juulia Järvinen, Helena C. Bland, Iris K. Sokka, Surachet Imlimthan, Ruth
1
2
2
2
3
Mateu Ferrando, Kristiina M. Huttunen, Juri Timonen, Sirpa Peräniemi, Olli Aitio, Anu J.
1
,4
1
1,5
2
1
Airaksinen, Mirkka Sarparanta, Mikael P. Johansson, Jarkko Rautio and Filip S. Ekholm
1
2
3
4
Department of Chemistry, University of Helsinki, Finland, P.O. Box 55, FI-00014 Helsinki, Finland
School of Pharmacy, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland
Glykos Finland Ltd., Viikinkaari 6, FI-00790 Helsinki, Finland
Turku PET Centre, Department of Chemistry, University of Turku, P.O. Box 52, FI-20521 Turku,
Finland
5
Helsinki Institute of Sustainability Science, HELSUS, FI-00014 Helsinki, Finland

Equal contributions
Contact: filip.ekholm@helsinki.fi
1
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 2 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table of content graphic
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Abstract
Boron Neutron Capture Therapy (BNCT) for cancer is on the rise worldwide due to recent
developments of in-hospital neutron accelerators which are expected to revolutionize patient
treatments. There is an urgent need for improved boron delivery agents and herein we have focused
on studying the biochemical foundations upon which a successful GLUT1-targeting strategy to
BNCT could be based. By combining synthesis and molecular modelling with affinity and
cytotoxicity studies, we unravel the mechanisms behind the considerable potential of appropriately
designed glucoconjugates as boron delivery agents for BNCT. In addition to addressing the
biochemical premises of the approach in detail, we report on a hit glucoconjugate which displays
good cytocompatibility, aqueous solubility, high transporter affinity, and, crucially, an exceptional
boron delivery capacity in the in vitro assessment thereby pointing towards the significant potential
embedded in this approach.
Keywords: boron neutron capture therapy, cancer therapeutics, carbohydrates, glucose transporters,
medicinal chemistry, drug delivery
2
ACS Paragon Plus Environment

Page 3 of 47
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
1
. Introduction
As one of the leading causes of morbidity and mortality on a global scale, cancer is a significant
societal economic burden with annual global costs of 723–930 billion euros. Head and neck cancers
account for up to 10 % of all cancers with 630 000 new cases annually diagnosed worldwide.1,2
Despite traditional treatments featuring surgery, radiation and chemotherapy, all of which are arduous
for the patients, many of these cancers recur. In head and neck cancers, the inoperable recurrent ones
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
are accompanied by a poor survival rate with a mean survival time of only a few months. A number
of novel treatment strategies have recently gained ground. These include antibody-drug conjugates,4
5
,6
7
proton therapy, and, especially, boron neutron capture therapy (BNCT). BNCT represents one of
the most promising non-invasive binary treatment modalities for head and neck cancers since it can
eradicate cancer cells while simultaneously sparing healthy cells (the basis of our approach is
8
,9
displayed in Figure 1). The selectivity in BNCT arises from a two-fold effect. First, only cells with
1
0
a sufficient concentration of B-atoms are destroyed and, second, the external neutron beam can be
applied to a narrow and highly specific area where malignant cells are present. Previously, the
applications of, and interest in, clinical BNCT have been hampered by the need for nuclear reactors,
as a neutron source, and the poor properties of clinically used boron delivery agents. In recent years,
1
0
new in-hospital neutron accelerators have emerged thus revolutionizing the clinical aspects of
patient treatments; a renewed interest in the BNCT field has been invoked – now, the final challenge
to solve is that of developing improved boron delivery agents.
3
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 4 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. The principles of our approach to BNCT. Blue dots represent boron atoms while grey dots
represent carbon atoms in the ortho-carboranylmethyl moiety.
An optimally functioning delivery agent for BNCT should display a minimal systemic toxicity, a
cellular uptake of 20–35 g/g of tumour (i.e. ppm range), and tumour/normal tissue (T/N) and
tumour/blood (T/B) ratios above 3:1, with higher ratios naturally desirable. Combining these different
aspects into one single delivery agent has proved challenging. Despite the large number of delivery
agents (amino acids, carbohydrates, porphyrins, antibody-boron conjugates, polymers, peptides,
liposomes and nanoparticles) evaluated in the literature,11–13 only three are in clinical use. These are
sodium borocaptate (BSH), boronophenylalanine (BPA and its fructose-complex) and
decahydrodecaborate (GB-10). None of them exhibit optimal properties. BPA has poor water
solubility, contains only one boron atom/delivered molecule, and gives poor T/B- and T/N-ratios.14,15
1
6
17
BSH and GB-10 lack active targeting and uptake mechanisms and have an ionic nature which may
cause undesired interactions with other biomolecules in a biological context.
4
ACS Paragon Plus Environment

Page 5 of 47
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
The intrinsic properties of carbohydrates, i.e., high aqueous solubility, low systemic toxicity and
high biocompatibility make them seemingly ideal candidates for clinical BNCT. Polysaccharide and
oligosaccharide carriers are, however, sub-optimal from a BNCT-perspective: polysaccharides are
constrained to the extracellular matrix while oligosaccharides display low lectin-binding affinities.
Thus, we have chosen to focus on monosaccharides in combination with carbohydrate transporters,
glucose transporters (GLUTs and SGLTs) in particular.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Glucose is an essential nutrient for mammalian cells. An increased expression of GLUTs and
1
8
SGLTs, especially GLUT1, has been observed in head and neck cancers. The basis for this increase
is the switch in glucose metabolism which in cancer cells proceeds by an inefficient aerobic glycolysis
route in contrast to the oxidative metabolism in healthy non-cancerous cells.19 This inefficient
metabolic pathway leads to a substantial increase in glucose uptake which allows the cancer cells to
2
0
grow rapidly and proliferate. Exploiting this “Warburg effect”, named after Nobel laureate Otto
Heinrich Warburg, provides the foundation for the development of novel glucose-based “Trojan
horses” for clinical BNCT. Before reaching the end stages of the development process (in vivo-studies
with/without neutron sources), the biochemical foundations of the approach need to be addressed in
detail. In this regard, it is important to note that concerns have been raised regarding the effects of
glucoconjugates on glucose metabolism in healthy cells, the possible incorporation of metabolic
products into other biomolecules, and the competition for the transporters with the high glucose levels
21
found in blood. Therefore, for a GLUT targeting approach to be successful, it is crucial to address
these issues already at the design stage.
5
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 6 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The current delivery agents in clinical use
SH
HOOC
The glucoconjugate "Trojan horses" studied
O
O
O
Na+
NH2
OH
O
O
O
2
HO
HO
HO
HO
HO
HO
B
OH
OMe
-2
OH
OH
HO
OH
OMe
BSH
BPA
1
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Two of the delivery agents in clinical use (left box) and the 6-O-ortho-carboranylmethyl
glucoconjugates prepared in the current study (right box). Blue dots represent boron atoms while grey
dots represent carbon atoms in the carboranyl moiety.
To this end, we have designed and synthesized three glucoconjugates bearing an ortho-
carboranylmethyl substituent. The carboranyl provides ten boron nuclei per delivery molecule in a
charge-neutral, chemically stable form, and is thus highly suitable for the purposes of BNCT. In
addition, a neutral and hydrophobic boron cluster should be advantageous when aiming for transport
through a transmembrane protein since possible unfavourable interactions between charged boron
clusters and amino acids can be avoided.22 Figure 2 shows the three 6-O-carboranylmethyl
glucoconjugates targeted; the hemiacetal and both methyl glycosides. The attachment of boron
th
clusters at the 6 position in glucose is rare in the scientific literature and conjugates with charge-
neutral boron clusters have not been prepared earlier.23,24 A modification at this site will, however,
remove the concerns regarding interference with glucose metabolism and incorporation into other
biomolecules through the glycolysis route; the 6-O-carboranylmethyl glucoconjugates are no longer
substrates for the glycolysis route in which the first transformation is a phosphorylation at the 6th
position.25
In addition to synthesizing the glucoconjugates and conducting the most detailed structural
characterization of such conjugates to date, we have addressed the biochemical foundations of the
GLUT1-targeting approach through a preliminary, yet, comprehensive in vitro evaluation study
6
ACS Paragon Plus Environment

Page 7 of 47
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
featuring cytotoxicity, computational/experimental receptor affinity, and cellular uptake experiments
in the relevant human head and neck cancer cell line CAL 27 (oral adenosquamous carcinoma cell
line). To our satisfaction, the new glucoconjugates display a significantly stronger binding affinity
to GLUT1 than glucose. This shows that the previous fear regarding their competition with the high
levels of free glucose in blood has been unfounded. Moreover, the glucoconjugates display a boron
delivery capacity 40 times higher than the best agents currently in clinical use – showing that there is
considerable potential imbedded in this alternative approach.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
. Experimental section
2.1 Synthesis and structural characterization
Reaction 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
1
spectra were recorded with a Bruker Avance III NMR spectrometer operating at 500.13 MHz ( H:
1
3
11
5
00.13 MHz, C: 125.76 MHz, B: 160.46 MHz). The probe temperature during the experiments
1
was kept at 23 ˚C. All products were characterized by utilization of the following 1D-techniques: H,
1
3
11
C, B and 1D-TOCSY and the following 2D-techniques: Ed-HSQC, HMBC and COSY by using
pulse sequences provided by the instrument manufacturer. Chemical shifts are expressed on the δ
scale (in ppm) using TMS (tetramethylsilane), residual chloroform, methanol or 15% BF in CDCl
3
3
1
1
( B NMR) as internal standards. Coupling constants have been obtained through spectral simulations
with the Perch Peak Research software, are given in Hz and provided only once, when first
encountered. Coupling patterns are given as s (singlet), d (doublet), t (triplet) etc. HRMS were
recorded using Bruker Micro Q-TOF with ESI (electrospray ionization) operated in positive mode.
The purity of the compounds were determined to be > 95 % in all cases. TLC was performed on
aluminum sheets precoated with silica gel 60 F254 (Merck). Flash chromatography was carried out
7
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 8 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
on silica gel 40. Spots were visualized by UV, followed by spraying the TLC plates with a solution
of H SO :MeOH (1:4) and heating.
2
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
General experimental procedures
General Procedure for selective silylation of the 6-OH group in glucopyranoses: Tert-
butyldimethylsilyl chloride (1.35 equiv.) was added portion-wise to a solution of D-glucose (1 equiv.)
in pyridine (10 ml/g of starting material) at 0 °C. The mixture was brought to rt and stirred for 21 h.
The solvent was removed in vacuo and the crude product was purified by column chromatography
(DCM:MeOH 7:1), the solvents were removed and the corresponding silylated glycopyranose was
dried on the vacuum line.
General Procedure for selective silylation of the 6-OH group in glucopyranosides: The
corresponding Methyl D-glucopyranoside (1 equiv.) was dissolved in dry DMF (10 ml/g of starting
material) at 0 °C under an atmosphere of argon. Imidazole (1.5 equiv.) and the tert-butyldimethylsilyl
chloride (1.35 equiv.) were added and the reaction mixture was brought to rt. The mixture was stirred
o/n. The reaction was quenched by the addition of MeOH (0.25 ml/g of starting material) and
concentrated in vacuo. The crude product was purified by column chromatography (DCM:MeOH
7
:1), the solvents were removed and the corresponding silylated glycoside was dried on the vacuum
line.
General Procedure for alkylation of free hydroxyl groups: The partially protected
glucoside/glucopyranose (1 equiv.) was dissolved in dry DMF (2 ml/100 mg of starting material)
under an atmosphere of argon. The solution was cooled on an ice bath and NaH (1.9 equiv.) was
added. The reaction mixture was stirred for 15 min., then brought to rt and stirred for a further 10
min. The corresponding bromide (1.5 equiv./free OH-group) was added and the resulting mixture was
8
ACS Paragon Plus Environment

Page 9 of 47
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
stirred for 1–4 h, quenched with MeOH (0.4 ml/mmol of starting material), diluted with DCM (4
ml/100 mg) and washed with a satd. NaHCO -solution. The organic phase was separated and the
3
aqueous phase was extracted with DCM (3 x 3 ml/100 mg). The organic phases were combined and
washed with brine (3 ml/100 mg), dried over Na SO , filtered and concentrated. The crude product
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4
was purified by column chromatography (EtOAc:Hexane 1:8), the solvents were removed and the
corresponding alkylated glycoside was dried on the vacuum line.
General procedure for deprotection of silyl protective groups: To a solution containing the
protected glycoside (1 equiv.) in dry THF (3 ml/200 mg of starting material) at 0 °C, HF-pyridine (18
μl/0.03 mmol of starting material) was added. The resulting mixture was brought to rt and stirred for
2
0 h. The reaction mixture was diluted with DCM (30 ml/0.5 g of starting material) and quenched by
the addition of a satd. NaHCO -solution (20 ml/200 mg of starting material). The aqueous phase was
3
extracted with DCM (3 x 20 ml) and the organic phases were combined and washed with brine (20
ml/500 mg of starting material). The combined organic phase was dried over Na SO , filtered and
2
4
concentrated. The crude product was purified by column chromatography (Hexane:EtOAc 2:1), the
solvents were removed and the corresponding deprotected glycoside was dried on the vacuum line.
General procedure for coupling reaction with decaborane: B H (1.8 equiv.) in dry ACN (5
1
0
14
ml/150 mg) under argon was heated to 60 °C and stirred for 1 h. Meanwhile, the propargylated
glycoside (1 equiv.) was dissolved in dry toluene (5 ml/150 mg) and added after the first hour. The
reaction mixture was stirred for 15–18 h at 80 °C. The mixture was quenched by the addition of dry
methanol (1.8 ml/200 mg starting material) and allowed to stir for 30 min. at 80 °C. The solvent was
removed and the crude product was purified by column chromatography (EtOAc:Hexane 1:3), the
solvents were removed and the corresponding carboranyl glycoside was dried on the vacuum line.
9
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 10 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
General procedure for deprotection of benzyl groups: The corresponding protected glucoside
was dissolved in EtOAc:MeOH 7:1 (1 ml/10 mg of starting material). Pd/C (10% Pd, 1 weightequiv.)
was added and the reaction mixture was stirred in an autoclave under H (3–5 bar) for 4–6 h. The
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
resulting mixture was filtered through celite, washed with EtOAc:MeOH 7:1 (3 x 10 ml) and
concentrated under vacuum. The crude product was purified by column chromatography
(
DCM:MeOH 5:1), the solvents were removed and the product was dried on the vacuum line to give
the corresponding deprotected glucoside/glucopyranose.
Substrate specific analytical data
6-O-(tert-butyldimethylsilyl)-D-glucopyranose. Synthesised from D-glucose (9.99 g/ 55.5
mmol), according to the general procedure for selective silylation of the 6-OH group in
glucopyranoses. This reaction yielded an off-white powder (10.64 g, 69 %; : 58:42). R = 0.61
f
(DCM:MeOH 5:1).
1
H NMR of the -anomer (500.13 MHz; CD OD): δ 5.08 (d, 1H, J = 3.7 Hz, H-1), 3.85 (dd, 1H,
3
1,2
J6a,5 = 2.1, J6a,6b = –11.2 Hz, H-6a), 3.84 (dd, 1H, J6b,5 = 4.6 Hz, H-6b), 3.75 (ddd, 1H, J = 9.8 Hz,
5,4
H-5), 3.67 (dd, 1H, 1H, J = 9.1, J = 9.6 Hz, H-3), 3.35 (dd, 1H, H-4), 3.33 (dd, 1H, H-2), 0.90 (s,
3
,4
3,2
9
H, 6-OSi(CH ) C(CH ) ) and 0.08 and 0.07 (each s, each 3H, 6-OSi(CH ) C(CH ) ) ppm.
3 2 3 3 3 2 3 3
1
3
C NMR of the -anomer (125.76 MHz; CD OD): δ 93.9 (C-1), 74.9 (C-3), 73.8 (C-2), 73.2 (C-5),
3
71.6 (C-4), 64.1 (C-6), 26.4 (6-OSi(CH ) C(CH ) ), 19.3 (6-OSi(CH ) C(CH ) ) and –5.0 and –5.1
3
2
3 3
3 2
3 3
(
6-OSi(CH ) C(CH ) ) ppm.
3 2 3 3
1
H NMR of the -anomer (500.13 MHz; CD OD): δ 4.44 (d, 1H, J = 7.8 Hz, H-1), 3.94 (dd, 1H,
3
1,2
J6a,5 = 2.0, J6a,6b = –11.2 Hz, H-6a), 3.78 (dd, 1H, J6b,5 = 5.4 Hz, H-6b), 3.33 (dd, 1H, 1H, J = 8.9,
3,4
J = 9.8 Hz, H-3), 3.30 (dd, 1H, J = 9.4 Hz, H-4), 3.26 (ddd, 1H, H-5), 3.11 (dd, 1H, H-2), 0.90
3
,2
4,5
(
s, 9H, 6-OSi(CH ) C(CH ) ) and 0.08 and 0.07 (each s, each 3H, 6-OSi(CH ) C(CH ) ) ppm.
3 2 3 3 3 2 3 3
1
0
ACS Paragon Plus Environment

Page 11 of 47
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
C NMR of the -anomer (125.76 MHz; CD OD): δ 98.1 (C-1), 78.2 (C-3, C-5), 76.2 (C-2), 71.5
3
(C-4), 64.3 (C-6), 26.4 (6-OSi(CH ) C(CH ) ), 19.3 (6-OSi(CH ) C(CH ) ) and –5.1 (6-
3 2 3 3 3 2 3 3
OSi(CH ) C(CH ) ) ppm.
3
2
3 3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
HRMS: m/z calcd. for C H O SiNa [M + Na] 317.1397; found 317.1385.
1
2
26
6
1,2,3,4-tetra-O-benzyl-6-O-(tert-butyldimethylsilyl)-D-glucopyranose (4). Synthesised from 6-
O-(tert-butyldimethylsilyl)-D-glucopyranose (0.95 g, 3.2 mmol), NaH (0.90 g, 37.4 mmol) and BnBr
(4.90 g, 28.6 mmol) according to the general procedure for alkylation of free hydroxyl groups to give
a white solid (1.85 g, 88 %; : 31:69). TLC: R : 0.39 (EtOAc:Hex 1:8).
f
1
H NMR of the -anomer (500.13 MHz; CDCl ): δ 7.42–7.23 (m, 20H, arom. H), 5.00 and 4.84 (each
3
d, each 1H, J = –10.6 Hz, 3-OCH Ph), 4.89 and 4.66 (each d, each 1H, J = –11.0 Hz, 4-OCH Ph),
2
2
4.83 (d, 1H, J = 3.6 Hz, H-1), 4.69 and 4.56 (each d, each 1H, J = –11.9 Hz, 1-OCH Ph), 4.66 and
1
,2
2
4
.58 (each d, each 1H, J = –11.7 Hz, 2-OCH Ph), 4.06 (dd,1H, J = 9.1, J = 9.8 Hz, H-3), 3.78
2 3,4 3,2
(dd, 1H, J6a,5 = 4.6, J6a,6b = –11.4 Hz, H-6a), 3.74 (dd, 1H, J6b,5 = 1.7 Hz, H-6b), 3.71 (ddd, 1H, J =
5,4
10.0 Hz, H-5), 3.56 (dd, 1H, H-4), 3.52 (dd, 1H, H-2), 0.90 (s, 9H, 6-OSi(CH ) C(CH ) ) and 0.06
3
2
3 3
and 0.05 (each s, each 3H, 6-OSi(CH ) C(CH ) ) ppm.
3
2
3 3
1
3
C NMR of the -anomer (125.76 MHz; CDCl ): δ 139.0–127.7 (arom. C), 95.2 (C-1), 82.3 (C-3),
3
80.5 (C-2), 78.0 (C-4), 76.0 (3-OCH Ph), 75.2 (4-OCH Ph), 73.1 (2-OCH Ph), 71.9 (C-5), 68.8 (1-
2
2
2
OCH Ph), 62.3 (C-6), 26.1 (6-OSi(CH ) C(CH ) ), 18.5 (6-OSi(CH ) C(CH ) ) and –5.0 and –5.2
2
3 2
3 3
3 2
3 3
(6-OSi(CH ) C(CH ) ) ppm.
3
2
3 3
1
H NMR of the -anomer (500.13 MHz; CDCl ): δ 7.42–7.23 (m, 20H, arom. H), 4.97 and 4.73 (each
3
d, each 1H, J = –10.8 Hz, 2-OCH Ph), 4.94 and 4.67 (each d, each 1H, J = –11.6 Hz, 1-OCH Ph),
2
2
4
.92 and 4.81 (each d, each 1H, J = –10.8 Hz, 3-OCH Ph), 4.86 and 4.69 (each d, each 1H, J = –10.8
2
Hz, 4-OCH Ph), 4.51 (d, 1H, J = 7.8 Hz, H-1), 3.90 (dd, 1H, J6a,5 = 1.7, J6a,6b = –11.4 Hz, H-6a),
2
1,2
3
.86 (dd, 1H, J6b,5 = 4.4 Hz, H-6b), 3.65 (dd, 1H, J = 8.9, J = 9.0 Hz, H-3), 3.63 (dd, 1H, J4,5 =
3,2 3,4
1
1
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 12 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
9
.5 Hz, H-4), 3.49 (dd, 1H, H-2), 3.30 (ddd, 1H, H-5), 0.93 (s, 9H, 6-OSi(CH ) C(CH ) ) and 0.12
3 2 3 3
and 0.10 (each s, each 3H, 6-OSi(CH ) C(CH ) ) ppm.
3
2
3 3
1
3
C NMR of the -anomer (125.76 MHz; CDCl ): δ 139.0–127.7 (arom. C), 102.4 (C-1), 84.9 (C-3),
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
2.7 (C-2), 77.8 (C-4), 76.0 (C-5, 3-OCH Ph), 75.2 (4-OCH Ph), 75.1 (2-OCH Ph), 71.0 (1-
2
2
2
OCH Ph), 62.4 (C-6), 26.1 (6-OSi(CH ) C(CH ) ), 18.5 (6-OSi(CH ) C(CH ) ) and –4.8 and –5.2
2
3 2
3 3
3 2
3 3
(6-OSi(CH ) C(CH ) ) ppm.
3
2
3 3
+
HRMS: m/z calcd. for C H O SiNa [M + Na] 677.3275; found 677.3300.
4
0
50
6
1
,2,3,4-tetra-O-benzyl-D-glucopyranose. Synthesised from 1,2,3,4-tetra-O-benzyl-6-O-(tert-
butyldimethylsilyl)-D-glucopyranose (1.73 g, 2.6 mmol) and HF-pyridine (1.6 ml, 17.8 mmol)
according to the general procedure for deprotection of silyl protective groups to give a white solid
(1.42 g, 99 %; : 33:67). TLC: R : 0.8 (EtOAc:Hex 1:1).
f
1
H NMR of the -anomer (500.13 MHz; CDCl ): δ 7.41–7.25 (m, 20H, arom. H), 5.01 and 4.84 (each
3
d, each 1H, J = –10.8 Hz, 3-OCH Ph), 4.89 and 4.64 (each d, each 1H, J = –11.0 Hz, 4-OCH Ph),
2
2
4.80 (d, 1H, J = 3.6 Hz, H-1), 4.68 and 4.56 (each d, each 1H, J = –11.9 Hz, 2-OCH Ph), 4.68 and
1
,2
2
4
.55 (each d, each 1H, J = –12.4 Hz, 1-OCH Ph), 4.06 (dd,1H, J = 9.2, J = 9.5 Hz, H-3), 3.71
2 3,4 3,2
(ddd, 1H, J5,6a = 2.7, J5,6b = 2.8, J = 9.5 Hz, H-5), 3.70 (ddd, 1H, J6a,6-OH = 5.7, J6a,6b = –11.1 Hz, H-
5,4
6
a), 3.68 (ddd, 1H, J6a,6-OH = 7.0 Hz, H-6b), 3.54 (dd, 1H, H-4), 3.50 (dd, 1H, H-2) and 1.57 (dd, 1H,
6-OH) ppm.
1
3
C NMR of the -anomer (125.76 MHz; CDCl ): δ 139.0–127.7 (arom. C), 95.7 (C-1), 82.1 (C-3),
3
8
0.2 (C-2), 77.6 (C-4), 75.9 (3-OCH Ph), 75.2 (4-OCH Ph), 73.2 (2-OCH Ph), 71.1 (C-5), 69.4 (1-
2 2 2
OCH Ph) and 62.0 (C-6) ppm.
2
1
H NMR of the -anomer (500.13 MHz; CDCl ): δ 7.41–7.25 (m, 20H, arom. H), 4.95 and 4.73 (each
3
d, each 1H, J = –10.9 Hz, 2-OCH Ph), 4.93 and 4.81 (each d, each 1H, J = –10.9 Hz, 3-OCH Ph),
2
2
4
.92 and 4.69 (each d, each 1H, J = –11.9 Hz, 1-OCH Ph), 4.86 and 4.64 (each d, each 1H, J = –11.0
2
1
2
ACS Paragon Plus Environment

Page 13 of 47
Molecular Pharmaceutics
1
2
3
Hz, 4-OCH Ph), 4.57 (d, 1H, J = 7.9 Hz, H-1), 3.87 (ddd, 1H, J6a,5 = 2.9, J6a,6-OH = 5.8, J6a,6b = –
2
1,2
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1.5 Hz, H-6a), 3.70 (ddd, 1H, J6b,5 = 4.9, J6b,6-OH = 7.7 Hz, H-6b), 3.67 (dd,1H, J = 9.2, J = 9.2
3,2 3,4
Hz, H-3), 3.57 (dd, 1H, J = 9.7 Hz, H-4), 3.49 (dd, 1H, H-2), 3.36 (ddd, 1H, H-5) and 1.84 (dd, 1
4
,5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H, 6-OH) ppm.
1
3
C NMR of the -anomer (125.76 MHz; CDCl ): δ 139.0–127.7 (arom. C), 103.0 (C-1), 84.7 (C-3),
3
82.5 (C-2), 77.7 (C-4), 75.9 (3-OCH Ph), 75.2 (C-5, 4-OCH Ph), 75.1 (2-OCH Ph), 71.8 (1-
2
2
2
OCH Ph) and 62.2 (C-6) ppm.
2
+
HRMS: m/z calcd. for C H O Na [M + Na] 563.2410; found 563.2395.
3
4
36
6
1,2,3,4-tetra-O-benzyl-6-O-propargyl-D-glucopyranose (5). Synthesised from 1,2,3,4-tetra-O-
benzyl-D-glucopyranose (1.42 g, 2.6 mmol, 1.0 equiv.), NaH (0.15 g, 6.0 mmol) and propargyl-
bromide (0.56 g, 4.7 mmol) according to the general procedure for alkylation of free hydroxyl groups
to give a white solid (1.26 g, 83 %; : 31:69).
1
H NMR of the -anomer (500.13 MHz; CDCl ): δ 7.42–7.23 (m, 20H, arom. H), 5.00 and 4.84 (each
3
d, each 1H, J = –10.9 Hz, 3-OCH Ph), 4.87 and 4.68 (each d, each 1H, J = –11.2 Hz, 4-OCH Ph),
2
2
4
4
.83 (d, 1H, J = 3.5 Hz, H-1), 4.68 and 4.55 (each d, each 1H, J = –12.2 Hz, 1-OCH Ph), 4.67 and
1
,2
2
.56 (each d, each 1H, J = –12.1 Hz, 2-OCH Ph), 4.20 (dd, 1H, J
= –2.4, JCH2a,CH2b = –16.0 Hz,
2
CH2a,CH
6-OCH CCH), 4.12 (dd, 1H, J
= –2.4 Hz, 6-OCH CCH), 4.04 (dd,1H, J = 9.0, J = 9.7
2
a
CH2b,CH
2b
3,4
3,2
Hz, H-3), 3.83 (dd, 1H, J6a,5 = 3.4, J6a,6b = –10.0 Hz, H-6a), 3.81 (ddd, 1H, J5,6b = 1.6, J = 10.3 Hz,
5,4
H-5), 3.63 (dd, 1H, H-4), 3.58 (dd, 1H, H-6b), 3.54 (dd, 1H, H-2) and 2.37 (dd, 1H, 6-OCH CCH)
2
ppm.
13
C NMR of the -anomer (125.76 MHz; CDCl ): δ 139.1–127.7 (arom. C), 95.9 (C-1), 82.2 (C-3),
3
7
9.9 (C-2), 79.6 (6-OCH CCH), 77.6 (C-4), 75.8 (3-OCH Ph), 75.2 (6-OCH CCH), 75.1 (4-
2
2
2
1
3
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 14 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
OCH Ph), 73.1 (2-OCH Ph), 70.3 (C-5), 69.4 (1-OCH Ph), 68.1 (C-6) and 58.7 (6-OCH CCH)
2
2
2
2
ppm.
1
H NMR of the -anomer (500.13 MHz; CDCl ): δ 7.41–7.25 (m, 20H, arom. H), 4.97 and 4.66 (each
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
d, each 1H, J = -11.9 Hz, 1-OCH Ph), 4.95 and 4.72 (each d, each 1H, J = -10.9 Hz, 2-OCH Ph),
2
2
4
.92 and 4.79 (each d, each 1H, J = -11.0 Hz, 3-OCH Ph), 4.86 and 4.68 (each d, each 1H, J = -10.8
2
Hz, 4-OCH Ph), 4.51 (d, 1H, J = 7.8 Hz, H-1), 4.26 (dd, 1H, J = –2.4, JCH2a,CH2b = –15.9 Hz,
CH2a,CH
2
1,2
6
-OCH CCH), 4.20 (dd, 1H, J
= –2.4 Hz, 6-OCH CCH), 3.83 (dd, 1H, J = 4.5, J6a,6b =
2
a
CH2b,CH
2b
6a,5
-
10.7 Hz, H-6a), 3.79 (dd, 1H, J6b,5 = 2.1 Hz, H-6b), 3.64 (dd, 1H, J = 9.0, J = 9.2 Hz, H-3), 3.62
3,4 3,2
(
dd, 1H, J = 9.6 Hz, H-4), 3.51 (dd, 1H, H-2), 3.47 (ddd, 1H, H-5) and 2.39 (dd, 1H, 6-OCH CCH)
4
,5
2
ppm.
1
3
C NMR of the -anomer (125.76 MHz; CDCl ): δ 139.0–127.7 (arom. C), 102.8 (C-1), 84.8 (C-3),
3
8
2.4 (C-2), 79.8 (6-OCH CCH), 77.7 (C-4), 75.8 (3-OCH Ph), 75.1–74.9 (C-2, 4-OCH Ph, 2-
2
2
2
OCH Ph, 6-OCH CCH), 74.8 (C-5), 71.3 (1-OCH Ph), 68.5 (C-6) and 58.8 (6-OCH CCH) ppm.
2
2
2
2
+
HRMS: m/z calcd. for C H O Na [M + Na] 601.2566; found 601.2628.
3
7
38
6
1
,2,3,4-tetra-O-benzyl-6-O-(o-carboranylmethyl)-D-glucopyranose (6). Synthesised from
1
,2,3,4-tetra-O-benzyl-6-O-propargyl-D-glucopyranose (0.91 g, 1.6 mmol) and B H (0.33 g, 2.7
1
0
14
mmol) according to the general procedure for coupling reaction of decaborane with propargylated
glucosides to give a colorless oil (0.61 g, 55.1 %; : 25:75). TLC: R : 0.55 (EtOAc:Hex 1:3).
f
1
H NMR of the -anomer (500.13 MHz; CDCl ): δ 7.39–7.23 (m, 20H, arom. H), 5.01 and 4.81 (each
3
d, each 1H, J = –10.6 Hz, 3-OCH Ph), 4.90 and 4.55 (each d, each 1H, J = –11.1 Hz, 4-OCH Ph),
2
2
4.79 (d, 1H, J = 3.7 Hz, H-1), 4.68 and 4.56 (each d, each 1H, J = –11.8 Hz, 2-OCH Ph), 4.66 and
1
,2
2
4
.53 (each d, each 1H, J = –12.2 Hz, 1-OCH Ph), 4.04 (dd, 1H, J = 9.1, J = 9.4 Hz, H-3), 3.87
2 3,4 3,2
and 3.79 (each d, each 1H, J = –10.7 Hz, 6-OCH -carborane), 3.81 (br s, 1H, carborane-CH), 3.74
2
1
4
ACS Paragon Plus Environment

Page 15 of 47
Molecular Pharmaceutics
1
2
3
(
ddd, 1H, J5,6b = 1.7, J5,6a = 4.6, J = 10.1 Hz, H-5), 3.63 (dd, 1H, J
= –11.5 Hz, H-6a), 3.49 (dd,
6a,6b
5,4
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
H, H-2), 3.46 (dd, 1H, H-6b), 3.40 (dd, 1H, H-4) and 2.99–1.50 (br m, 10H, carborane-BH) ppm.
1
3
C NMR of the -anomer (125.76 MHz; CDCl ): δ 138.7–127.9 (arom. C), 95.6 (C-1), 82.1 (C-3),
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
0.2 (C-2), 77.5 (C-4), 76.0 (3-OCH Ph), 75.1 (4-OCH Ph), 73.2 (2-OCH Ph), 72.9 (6-OCH -
2
2
2
2
carborane), 71.0 (C-6, carborane-C), 70.6 (C-5), 69.4 (1-OCH Ph) and 57.6 (carborane-CH) ppm.
2
1
H NMR of the -anomer (500.13 MHz; CDCl ): δ 7.39–7.23 (m, 20H, arom. H), 4.96 and 4.73 (each
3
d, each 1H, J = –10.8 Hz, 2-OCH Ph), 4.93 and 4.78 (each d, each 1H, J = –10.8 Hz, 3-OCH Ph),
2
2
4
.88 and 4.66 (each d, each 1H, J = –11.9 Hz, 1-OCH Ph), 4.87 and 4.55 (each d, each 1H, J = –11.2
2
Hz, 4-OCH Ph), 4.49 (d, 1H, J = 7.9 Hz, H-1), 3.93 and 3.87 (each d, each 1H, J = –10.7 Hz, 6-
2
1,2
OCH -carborane), 3.91 (br s, 1H, carborane-CH), 3.67 (dd, 1H, J = 4.8, J6a,6b = –11.4 Hz, H-6a),
2
6a,5
3
3
.64 (dd, 1H, J6b,5 = 1.9 Hz, H-6b), 3.63 (dd, 1H, J = 9.0, J = 9.2 Hz, H-3), 3.47 (dd, 1H, H-2),
3,4 3,2
.44 (dd, 1H, J = 9.9 Hz, H-4), 3.36 (ddd, 1H, H-5) and 2.99–1.50 (br m, 10H, carborane-BH) ppm.
4,5
13
C NMR of the -anomer (125.76 MHz; CDCl ): δ 138.7–127.9 (arom. C), 102.8 (C-1), 84.7 (C-3),
3
8
2.4 (C-2), 77.4 (C-4), 76.0 (3-OCH Ph), 75.1 (2-OCH Ph, 4-OCH Ph), 74.8 (C-5), 72.9 (6-OCH -
2 2 2 2
carborane), 71.5 (1-OCH Ph), 71.0 (C-6, carborane-C) and 57.7 (carborane-CH) ppm.
2
11
B NMR (160.46 MHz; CDCl ): δ –2.56, –4.38, –8.64, –11.24 and –12.91 ppm.
3
+
HRMS: m/z calcd. for C H B O Na [M + Na] 721.4258; found 721.4327.
3
7
48 10
6
6-O-(o-carboranylmethyl)-D-glucopyranose (1). Synthesised from 1,2,3,4-tetra-O-benzyl-6-O-
(o-carboranylmethyl)-D-glucopyranose (0.11 g, 0.02 mmol), and Pd/C (0.17 g, 0.02 mmol) according
to the general procedure for hydrogenolysis to give a colorless oil (0.04 g, 79 %; : 60:40). TLC:
R : 0.56 (EtOAc:MeOH 5:1).
f
1
H NMR of the -anomer (500.13 MHz; CD OD): δ 5.10 (d, 1H, J = 3.7 Hz, H-1), 4.59 (br s, 1H,
3
1,2
carborane-CH), 4.04 and 4.02 (each d, each 1H, J = –10.8 Hz, 6-OCH -carborane), 3.86 (ddd, 1H,
2
J5,6b = 1.8, J5,6a = 4.9, J = 10.1 Hz, H-5), 3.75 (dd, 1H, J
= –11.3 Hz, H-6a), 3.73 (dd, 1H, H-
6a,6b
5,4
1
5
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 16 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
b), 3.66 (dd, 1H, J = 9.0, J = 9.6 Hz, H-3), 3.35(dd, 1H, H-2), 3.29 (dd, 1H, H-4) and 3.00–1.50
3,4 3,2
(br m, 10H, carborane-BH) ppm.
1
3
C NMR of the -anomer (125.76 MHz; CD OD): δ 93.9 (C-1), 75.2 (carborane-C), 74.8 (C-3), 73.9
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(6-OCH -carborane), 73.7 (C-2), 72.5 (C-6), 72.2 (C-5), 71.6 (C-4) and 60.5 (carborane-CH) ppm.
2
1
H NMR of the -anomer (500.13 MHz; CD OD): δ 4.63 (br s, 1H, carborane-CH), 4.46 (d, 1H, J
3
1,2
= 7.8 Hz, H-1), 4.05 and 4.03 (each d, each 1H, J = –10.8 Hz, 6-OCH -carborane), 3.79 (dd, 1H, J
2
6a,5
=
2.0, J6a,6b = –11.3 Hz, H-6a), 3.72 (dd, 1H, J6b,5 = 5.2 Hz,H-6b), 3.37 (ddd, 1H, J = 10.3 Hz, H-
5,4
5
), 3.34 (dd, 1H, J = 9.0, J = 9.3 Hz, H-3), 3.30 (dd, 1H, H-4), 3.12 (dd, 1H, H-2) and 3.00–1.50
3,2 3,4
(br m, 10H, carborane-BH) ppm.
13
C NMR of the -anomer (125.76 MHz; CD OD): δ 98.2 (C-1), 78.0 (C-3), 76.9 (C-5), 76.2 (C-2),
3
7
5.2 (carborane-C), 73.9 (6-OCH -carborane), 72.2 (C-6), 71.3 (C-4) and 60.6 (carborane-CH) ppm.
2
1
1
B NMR (160.46 MHz; CD OD): δ –2.09, –4.01, –8.31, –10.48 and –12.17 ppm.
3
+
HRMS: m/z calcd. for C H B O Na [M + Na] 361.2401; found 361.2382.
9
24 10
6
Methyl 6-O-tertbutyldimethylsilyl-α-D-glucopyranoside. Synthesised from methyl-α-D-
glucopyranoside (9.91 g, 51 mmol), according to the general procedure for selective silylation of the
6
-OH group in glucopyranosides. This reaction yielded a white solid (12.61 g, 80 %). R = 0.52
f
(DCM:MeOH 7:1).
1
H NMR (500.13 MHz; CDCl ): δ 4.74 (d, 1H, J = 3.7 Hz, H-1), 3.88 (dd, 1H, J = 5.0, J6a,6b = –
3
1,2
6a,5
1
0.8 Hz, H-6a), 3.83 (dd, 1H, J6b,5 = 5.0 Hz, H-6b), 3.74 (dd, 1H, J = J = 8.9, 9.6 Hz, H-3), 3.60
3,2 3-4
(ddd, 1H, J = 9.8 Hz, H-5), 3.52 (dd, 1H, H-2), 3.52 (dd, 1H, H-4), 3.42 (s, 3H, 1-OCH ), 0.91 (s,
5
,
4
3
9H, 6-OSi(CH ) C(CH ) ) and 0.10 (s, 6H, 6-OSi(CH ) C(CH ) ) ppm.
3
2
3 3
3 2
3 3
13
C NMR (125.76 MHz; CDCl ): δ 99.3 (C-1), 74.8 (C-3), 72.4–72.3 (C-2, C-4), 70.6 (C-5), 64.3 (C-
3
6
), 55.4 (1-OCH ), 26.0 (6-OSi(CH ) C(CH ) ), 18.4 (6-OSi(CH ) C(CH ) ) and –5.3 (6-
3 3 2 3 3 3 2 3 3
OSi(CH ) C(CH ) ) ppm.
3
2
3 3
1
6
ACS Paragon Plus Environment

Page 17 of 47
Molecular Pharmaceutics
1
2
3
+
HRMS: m/z calcd. for C H O SiNa [M + Na] 331.1553; found 331.1553.
1
3
28
6
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Methyl 2,3,4-tri-O-benzyl-6-O-tertbutyldimethylsilyl-α-D-glucopyranoside (7). Synthesised
from methyl 6-O-tertbutyldimethylsilyl-α-D-glucopyranoside (0.60 g, 1.93 mmol), according to the
general procedure for the alkylation of free hydroxyl groups. This reaction yielded a white solid (0.70
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
g, 78 %). R = 0.32 (EtOAc:Hexane 1:8).
f
1
H NMR (500.13 MHz; CDCl ): δ 7.37–7.26 (m, 15H, arom. H), 4.97 and 4.82 (each d, each 1H, J =
3
–
10.7 Hz, 3-OCH Ph), 4.88 and 4.64 (each d, each 1H, J = –10.9 Hz, 4-OCH Ph), 4.79 and 4.68
2 2
(
each d, each 1H, J = –12.1 Hz, 2-OCH Ph), 4.61 (d, 1H, J = 3.6 Hz, H-1), 3.99 (dd, 1H, J = 9.0,
2 1,2 3,4
J = 9.6 Hz, H-3), 3.78 (m, 2H, H-6a and H-6b), 3.61 (ddd, 1H, J = 3.1, J5,6b = 3.1, J = 10.0 Hz,
3
,2
5,6a
5,4
H-5), 3.53 (dd, 1H, H-4), 3.50 (dd, 1H, H-2) and 3.36 (s, 3H, 1-OCH ), 0.88 (s, 9H, 6-
3
OSi(CH ) C(CH ) ) and 0.04 and 0.03 (each s, each 3H, 6-OSi(CH ) C(CH ) ) ppm.
3
2
3 3
3 2
3 3
1
3
C NMR (125.76 MHz; CDCl ): δ 139.3–127.6 (arom. C), 98.0 (C-1), 82.3 (C-3), 80.4 (C-2), 77.9
3
(
C-4), 76.0 (3-OCH Ph), 75.1 (4-OCH Ph), 73.5 (2-OCH Ph), 71.6 (C-5), 62.4 (C-6), 55.0 (1-O-
2 2 2
CH ), 26.1 (6-OSi(CH ) C(CH ) ), 18.4 (6-OSi(CH ) C(CH ) ) and –5.1 (6-OSi(CH ) C(CH ) )
3
3 2
3 3
3 2
3 3
3 2
3 3
ppm.
+
HRMS: m/z calcd. for C H O SiNa [M + Na] 601.2962; found 601.2983.
3
4
46
6
Methyl 2,3,4-tri-O-benzyl-α-D-glucopyranoside. Synthesised from methyl 2,3,4-tri-O-benzyl-6-
O-tertbutyldimethylsilyl-α-D-glucopyranoside (2.6 g, 4.49 mmol), according to the general procedure
for deprotection of silyl protective groups. This reaction yielded a white solid (1.70 g, 82 %). R =
f
0
.60 (EtOAc:Hexane 1:1).
1
H NMR (500.13 MHz; CDCl ): δ 7.37–7.28 (m, 15H, arom. H), 4.99 and 4.84 (each d, each 1H, J =
3
–
10.9 Hz, 3-OCH Ph), 4.88 and 4.64 (each d, each 1H, J = –11.0 Hz, 4-OCH Ph), 4.80 and 4.67
2 2
(
each d, each 1H, J = –12.1 Hz, 2-OCH Ph), 4.56 (d, 1H, J = 3.6 Hz, H-1), 4.00 (dd, 1H, J = 8.9,
2 1,2 3,4
1
7
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 18 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
J = 9.6 Hz, H-3), 3.76 (ddd, 1H, J = 2.7, J6a,6-OH = 5.3, J6a,6b = –11.9 Hz, H-6a), 3.69 (ddd, 1H,
3
,2
6a,5
J6b,5 = 4.1, J6b,6-OH = 7.6 Hz, H-6b), 3.65 (ddd, 1H, J = 9.9 Hz, H-5), 3.52 (dd, 1H, H-4), 3.50 (dd,
5,4
1H, H-2), 3.37 (s, 3H, 1-OCH ) and 1.62 (dd, 1H, 6-OH) ppm.
3
1
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C NMR (125.76 MHz; CDCl ): δ 139.7–127.7 (arom. C), 98.3 (C-1), 82.1 (C-3), 80.1 (C-2), 77.5
3
(
C-4), 75.9 (3-OCH Ph), 75.2 (4-OCH Ph), 73.6 (2-OCH Ph), 70.8 (C-5), 62.0 (C-6) and 55.3 (1-
2 2 2
OCH ) ppm.
3
+
HRMS: m/z calcd. for C H O Na [M + Na] 487.2097; found 487.2084.
2
8
32
6
Methyl 2,3,4-tri-O-benzyl-6-O-propargyl-α-D-glucopyranoside (8). Synthesised from methyl
2,3,4-tri-O-benzyl-α-D-glucopyranoside (0.31 g, 0.67 mmol), according to the general procedure for
the alkylation of free hydroxyl groups. This reaction yielded a yellow oil (0.30 g, 89 %). R = 0.61
f
(EtOAc:Hexane 1:2).
1
H NMR (500.13 MHz; CDCl ): δ 7.37–7.26 (m, 15H, arom. H), 4.98 and 4.83 (each d, each 1H, J =
3
–
10.9 Hz, 3-OCH Ph), 4.87 and 4.65 (each d, each 1H, J = –11.4 Hz, 4-OCH Ph), 4.79 and 4.68
2 2
(
each d, each 1H, J = –11.7 Hz, 2-OCH Ph), 4.60 (d, 1H, J = 3.6 Hz, H-1), 4.19 (dd, 1H, J
2 1,2 CH2a,CH
=
–2.4, JCH2a,CH2b = –16.0 Hz, 6-OCH CCH), 4.13 (dd, 1H, J
= –2.4 Hz, 6-OCH CCH),
2a
CH2a,CH
2b
3
.98 (dd, 1H, J3,4 = 9.0, J = 9.7 Hz, H-3), 3.84 (dd, 1H, J = 3.5, J6a,6b = –10.4 Hz, H-6a), 3.76
3,2 6a,5
(
ddd, 1H, J5,6b = 2.1, J = 10.1 Hz, H-5), 3.66 (dd, 1H, H-6b), 3.61 (dd, 1H, H-4), 3.54 (dd, 1H, H-
5,4
2), 3.37 (s, 3H, 1-OCH ) and 2.37 (dd, 1H, 6-OCH CCH) ppm.
3
2
13
C NMR (125.76 MHz; CDCl ): δ 139.6–127.7 (arom. C), 98.5 (C-1), 82.2 (C-3), 79.9 (C-2), 79.6
3
(
6-OCH C≡CH), 77.6 (C-4), 75.9 (3-OCH Ph), 75.2 (4-OCH Ph), 75.1 (6-OCH C≡CH), 73.6 (2-
2 2 2 2
OCH Ph), 70.0 (C-5), 68.2 (C-6), 58.7 (6-OCH C≡CH) and 55.4 (1-OCH ) ppm.
2
2
3
+
HRMS: m/z calcd. for C H O Na [M + Na] 525.2253; found 525.2258.
3
1
34
6
1
8
ACS Paragon Plus Environment

Page 19 of 47
Molecular Pharmaceutics
1
2
3
4
5
Methyl 2,3,4-tri-O-benzyl-6-O-(o-carboranylmethyl)-α-D-glucopyranoside (9). Synthesised
from methyl 2,3,4-tri-O-benzyl-6-O-propargyl-α-D-glucopyranoside (0.37 g, 0.73 mmol), according
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
to the general procedure for coupling with decaborane. This reaction yielded a white solid (0.27 g, 5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
%). R = 0.33 (EtOAc:Hexane 1:3).
f
1
H NMR (500.13 MHz; CDCl ): δ 7.38–7.25 (m, 15H, arom. H), 4.99 and 4.80 (each d, each 1H, J =
3
–
10.8 Hz, 3-OCH Ph), 4.90 and 4.55 (each d, each 1H, J = –11.2 Hz, 4-OCH Ph), 4.81 and 4.67
2 2
(each d, each 1H, J = –12.0 Hz, 2-OCH Ph), 4.55 (d, 1H, J = 3.5 Hz, H-1), 3.97 (dd, 1H, J = 8.8,
2
1,2
3,4
J = 9.6 Hz, H-3), 3.89 and 3.80 (each d, each 1H, J = –10.5 Hz, 6-OCH -carborane), 3.81 (br s,
3
,2
2
carborane-CH), 3.67 (ddd, 1H, J5,6b = 1.8, J5,6a = 4.4, J = 9.7, Hz, H-5), 3.65 (dd, 1H, J
= –10.9
5,4
6a,6b
Hz, H-6a), 3.55 (dd, 1H, H-6b), 3.48 (dd, 1H, H-2), 3.39 (dd, 1H, H-4), 3.35 (s, 3H, 1-OCH ) and
3
2
.62–1.61 (br m, 10H, carborane-BH) ppm.
1
3
C NMR (125.76 MHz; CDCl ): δ 139.8–127.8 (arom. C), 98.2 (C-1), 82.1 (C-3), 80.1 (C-2), 77.2
3
(
C-4), 76.1 (3-OCH Ph), 75.1 (4-OCH Ph), 73.6 (2-OCH Ph), 72.9 (carborane-C), 71.0 (C-6), 70.3
2 2 2
(
C-5), 60.5 (6-OCH -carborane), 57.6 (carborane-CH) and 55.4 (1-OCH ) ppm.
2
3
1
1
B NMR (160.46 MHz; CDCl ): δ –2.74, –4.74, –8.94, –11.52 and –13.06 ppm.
3
HRMS: m/z calcd. for C H B O Na [M + Na]+ 645.3966; found 645.3975.
31 44 10
6
Methyl 6-O-(o-carboranylmethyl)-α-D-glucopyranoside (2). Synthesised from methyl 2,3,4-tri-
O-benzyl-6-O-(o-carboranylmethyl)-α-D-glucopyranoside (0.15 g, 0.25 mmol), according to the
general procedure for the deprotection of benzyl groups. This reaction yielded a white solid (0.06 g,
7
2 %). R = 0.47 (DCM:MeOH 7:1).
f
1
H NMR (500.13 MHz; CD OD): δ 4.66 (d, 1H, J = 3.8 Hz, H-1), 4.57 (br s, 1H, carborane-CH),
3
1,2
4
.04 and 4.01 (each d, each 1H, J = –11.0 Hz, 6-OCH -carborane), 3.76 (dd, 1H, J6a,5 = 1.8, J6a,6b = –
2
1
1.3 Hz, H-6a), 3.71 (dd, 1H, J6b,5 = 5.3 Hz, H-6b), 3.60 (ddd, 1H, J = 9.7 Hz, H-5), 3.59 (dd, 1H,
5,4
1
9
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 20 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
J = 9.2, J = 9.7 Hz, H-3), 3.40 (s, 3H, 1-OCH ), 3.37 (d, 1H, H-2), 3.26 (d, 1H, H-4) and 2.56–
3
,4
3,2
3
1
.59 (br m, 10H, carborane-BH) ppm.
1
3
C NMR (125.76 MHz; CD OD): δ 101.3 (C-1), 75.2 (carborane-C), 75.0 (C-3), 74.0 (6-OCH -
3
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
carborane), 73.5 (C-2), 72.7 (C-5), 72.3 (C-6), 71.6 (C-4), 60.7 (carborane-CH) and 55.6 (1-OCH3)
ppm.
1
1
B NMR (160.46 MHz; CDCl ): δ –1.94, –3.92, –8.30, –10.44 and –12.05 ppm.
3
+
HRMS: m/z calcd. for C H B O Na [M + Na] 375.2558; found 375.2519.
1
0
26 10
6
Methyl 6-O-tertbutyldimethylsilyl-β-D-glucopyranoside. Synthesised from methyl β-D-
glucopyranoside (0.50 g, 2.59 mmol), according to the general procedure for selective silylation of
the 6-OH group in glucopyranosides. This reaction yielded a white solid (0.66 g, 83 %). R = 0.48
f
(DCM:MeOH 7:1).
1
H NMR (500.13 MHz; CD OD): δ 4.17 (d, 1H, J = 7.8 Hz, H-1), 3.99 (dd, 1H, J
6a,5
= 2.1, J6a,6b
=
3
1,2
–
11.4 Hz, H-6a), 3.83 (dd, 1H, J6b,5 = 5.3 Hz, H-6b), 3.53 (s, 3H, 1-OCH ), 3.36 (dd, 1H, J = 9.0,
3 3,2
J = 9.2 Hz, H-3), 3.33 (dd, 1H, J = 9.7 Hz, H-4), 3.27 (ddd, 1H, H-5), 3.17 (dd, 1H, H-2), 0.90
3
,4
4,5
(
s, 9H, 6-OSi(CH ) C(CH ) ) and 0.09 (s, 6H, 6- OSi(CH ) C(CH ) ) ppm.
3 2 3 3 3 2 3 3
1
3
C NMR (125.76 MHz; CD OD): δ 105.3 (C-1), 78.1 (C-3, C-5), 75.0 (C-2), 71.4 (C-4), 64.1 (C-6),
3
5
7.2 (1-OCH ), 26.4 (6-OSi(CH ) CH ), 19.3 (6-OSi(CH ) CH ), –5.0 and –5.1 (each Si(CH ) )
3
2 2
3
2 2
3
3 2
(
each 6-OSi(CH ) C(CH ) ) ppm.
3 2 3 3
+
HRMS: m/z calcd. for C H O SiNa [M + Na] 331.1553; found 331.1531.
1
3
28
6
Methyl 2,3,4-tri-O-benzyl-6-O-tertbutyldimethylsilyl-β-D-glucopyranoside (10). Synthesised
from methyl 6-O-tertbutyldimethylsilyl-β-D-glucopyranoside (3.05 g, 9.89 mmol), according to the
general procedure for the alkylation of free hydroxyl groups. This reaction yielded a white solid (4.62
g, 80 %). R = 0.33 (EtOAc:Hexane 1:8).
f
2
0
ACS Paragon Plus Environment

Page 21 of 47
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
H NMR (500.13 MHz; CDCl ): δ 7.38–7.27 (m, 15H, arom. H), 4.92 and 4.71 (each d, each 1H, J =
3
–
11.0 Hz, 2-OCH Ph), 4.91 and 4.81 (each d, each 1H, J = –10.8 Hz, 3-OCH Ph), 4.85 and 4.68
2 2
(each d, each 1H, J= –10.9 Hz, 4-OCH Ph), 4.29 (d, 1H, J = 7.7 Hz, H-1), 3.88 (dd, 1H, J = 1.7,
2
1,2
6a,5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
J6a,6b = –11.5 Hz, H-6a), 3.83 (dd, 1H, J6b,5 = 4.3 Hz, H-6b), 3.64 (dd, 1H, J3,4 = 9.1, J = 9.2 Hz,
3,2
H-3), 3.59 (dd, 1H, J = 9.7 Hz, H-4), 3.54 (s, 3H, 1-OCH ), 3.38 (dd, 1H, H-2), 3.28 (ddd, 1H, H-
4
,5
3
5
), 0.90 (s, 9H, 6-OSi(CH ) C(CH ) ) and 0.08 and 0.07 (each s, each 3H, 6-OSi(CH ) C(CH ) )
3 2 3 3 3 2 3 3
ppm.
13
C NMR (125.76 MHz; CDCl ): δ 139.6–127.7 (arom. C), 104.6 (C-1), 84.8 (C-3), 82.7 (C-2), 77.8
3
(
C-4), 75.9 (3-OCH Ph), 75.9 (C-5), 75.2 (4-OCH Ph), 74.9 (2-OCH Ph), 62.4 (C-6), 56.8 (1-
2
2
2
OCH ), 26.1 (6-OSi(CH ) C(CH ) ), 18.5 (6-OSi(CH ) C(CH ) ), –4.9 and –5.1 (6-
3
3 2
3 3
3 2
3 3
OSi(CH ) C(CH ) ) ppm.
3
2
3 3
+
HRMS: m/z calcd. for C H O SiNa [M + Na] 601.2962; found 601.2964.
3
4
46
6
Methyl 2,3,4-tri-O-benzyl-β-D-glucopyranoside. Synthesised from methyl 2,3,4-tri-O-benzyl-6-
O-tertbutyldimethylsilyl-β-D-glucopyranoside (2.94 g, 5.08 mmol), according to the general
procedure for deprotection of silyl protective groups. This reaction yielded a white solid (2.09 g, 89
%). R = 0.65 (EtOAc:Hexane 1:1).
f
1
H NMR (500.13 MHz; CDCl ): δ 7.38–7.27 (m, 15H, arom. H), 4.93 and 4.81 (each d, each 1H, J =
3
–
10.9 Hz, 3-OCH Ph), 4.91 and 4.71 (each d, each 1H, J = –11.0 Hz, 2-OCH Ph), 4.87 and 4.64
2 2
(each d, each 1H, J = –10.9 Hz, 4-OCH Ph), 4.36 (d, 1H, J = 7.8 Hz, H-1), 3.88 (ddd, 1H, J6a,5
=
2
1,2
2
.8, J6a,6-OH = 5.5, J6a,6b = –12.0 Hz, H-6a), 3.73 (ddd, 1H, J6b,5 = 4.5, J6b,6-OH = 7.7 Hz, H-6b), 3.67
(
(
dd, 1H, J = 9.0, J = 9.2 Hz, H-3), 3.57 (dd, 1H, J = 9.8 Hz, H-4), 3.57 (s, 3H, 1-OCH ), 3.40
3
,4
3,2
4,5
3
dd, 1H, H-2), 3.37 (ddd, 1H, H-5) and 1.88 (dd, 1H, 6-OH) ppm.
2
1
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 22 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
C NMR (125.76 MHz; CDCl ): δ 138.7–127.1 (arom. C), 105.0 (C-1), 84.6 (C-3), 82.5 (C-2), 77.6
3
(C-4), 75.8 (3-OCH Ph), 75.3 (C-5), 75.1 (4-OCH Ph), 75.0 (2-OCH Ph), 62.2 (C-6) and 57.5 (1-
2 2 2
OCH ) ppm.
3
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HRMS: m/z calcd. for C H O Na [M + Na] 487.2097; found 487.2089.
2
8
32
6
Methyl 2,3,4-tri-O-benzyl-6-O-propargyl-β-D-glucopyranoside (11). Synthesised from methyl
2,3,4-tri-O-benzyl-β-D-glucopyranoside (1.42 g, 3.02 mmol), according to the general procedure for
the alkylation of free hydroxyl groups. This reaction yielded a yellow oil (1.44 g, 95 %). R = 0.84
f
(EtOAc:Hexane 1:1).
1
H NMR (500.13 MHz; CDCl ): δ 7.35–7.26 (m, 15H, arom. H), 4.92 and 4.80 (each d, each 1H, J =
3
–
11.0 Hz, 3-OCH Ph), 4.91 and 4.70 (each d, each 1H, J = –11.0 Hz, 2-OCH Ph), 4.85 and 4.68
2 2
(each d, each 1H, J = –10.8 Hz, 4-OCH Ph), 4.30 (d, 1H, J = 7.8 Hz, H-1), 4.25 (dd, 1H, J
=
2
1,2
CH2a,CH
–
2.4, JCH2a,CH2b = –15.9 Hz, 6-OCH C≡CH), 3.64 (dd, 1H, J
= –2.4 Hz, 6-OCH C≡CH), 3.83
CHb,CH 2b
2a
(dd, 1H, J6a,5 = 4.4, J6a,6b = –10.7 Hz, H-6a), 3.78 (dd, 1H, J6b,5 = 2.0 Hz, H-6b), 3.64 (dd, 1H, J =
3,2
9
.1, J = 9.1 Hz, H-3), 3.61 (dd, 1H, J = 10.0 Hz, H-4), 3.57 (s, 3H, 1-OCH ), 3.46 (ddd, 1H, H-
3
,4
4,5
3
5
), 3.42 (dd, 1H, H-2) and 2.38 (dd, 1H, 6-OCH CCH) ppm.
2
13
C NMR (125.76 MHz; CDCl ): δ 128.6–127.7 (arom. C), 104.9 (C-1), 84.7 (C-3), 82.4 (C-2), 79.8
3
(6-OCH C≡CH), 77.7 (C-4), 77.8 (3-OCH Ph), 76.4 (6-OCH C≡CH), 75.1 (4-OCH Ph), 74.8 (2-
2 2 2 2
OCH Ph), 74.7 (C-5), 68.5 (C-6), 58.8 (6-OCH C≡CH) and 57.3 (1-OCH ) ppm.
2
2
3
+
HRMS: m/z calcd. for C H O Na [M + Na] 525.2253; found 525.2252.
3
1
34
6
Methyl 2,3,4-tri-O-benzyl-6-O-(o-carboranylmethyl)-β-D-glucopyranoside (12). Synthesised
from methyl 2,3,4-tri-O-benzyl-6-O-propargyl-β-D-glucopyranoside (1.20 g, 2.38 mmol), according
to the general procedure for coupling with decaborane. This reaction yielded a white solid (0.98 g,
6
6 %). R = 0.26 (EtOAc:Hexane 1:4).
f
2
2
ACS Paragon Plus Environment

Page 23 of 47
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
H NMR (500.13 MHz; CDCl ): δ 7.35–7.23 (m, 15H, arom. H), 4.93 and 4.78 (each d, each 1H, J =
3
–
10.8 Hz, 3-OCH Ph), 4.91 and 4.71 (each d, each 1H, J = –11.1 Hz, 2-OCH Ph), 4.87 and 4.54
2 2
(each d, each 1H, J = –11.1 Hz, 4-OCH Ph), 4.28 (d, 1H, J = 7.1 Hz, H-1), 3.94 (br s, carborane-
2
1,2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CH), 3.94 and 3.89 (each d, each 1H, J = –10.5 Hz, 6-OCH -carborane), 3.66 (dd, 1H, J = 2.3,
2
6a,5
J6a,6b = –10.5 Hz, H-6a), 3.65 (dd, 1H, J6b,5 = 4.4 Hz, H-6b), 3.63 (dd, 1H, J3,4 = 8.8, J = 9.3 Hz,
3,2
H-3), 3.54 (s, 3H, 1-OCH ), 3.41 (dd, 1H, J = 10.0 Hz , H-4), 3.38 (dd, 1H, H-2), 3.37 (ddd, 1H,
3
4,5
H-5) and 2.64–1.77 (br m, 10H, carborane-BH) ppm.
13
C NMR (125.76 MHz; CDCl ): δ 138.7–127.9 (arom. C), 104.9 (C-1), 84.6 (C-3), 82.4 (C-2), 77.4
3
(
C-4), 75.9 (3-OCH Ph), 75.1 (4-OCH Ph), 75.0 (2-OCH Ph), 74.7 (C-5), 72.9 (6-OCH -carborane
2 2 2 2
and carborane-C), 71.1 (C-6), 57.7 (carborane-CH) and 57.3 (1-OCH ) ppm.
3
11
B NMR (160.46 MHz; CDCl ): δ –2.83, –4.62, –8.89, –11.58 and –13.09 ppm.
3
+
HRMS: m/z calcd. for C H B O Na [M + Na] 645.3966; found 645.4025.
3
1
44 10
6
Methyl 6-O-(o-carboranylmethyl)-β-D-glucopyranoside (3). Synthesised from methyl 2,3,4-tri-
O-benzyl-6-O-(o-carboranylmethyl)-β-D-glucopyranoside (0.15 g, 0.25 mmol), according to the
general procedure for the deprotection of benzyl groups. This reaction yielded a white solid (0.068 g,
81 %). R = 0.61 (DCM:MeOH 5:1).
f
1
H NMR (500.13 MHz; CD OD): δ 4.63 (br s, 1H, carborane-CH), 4.17 (d, 1H, J = 7.8 Hz, H-1),
3
1,2
4
.06 (each d, each 1H, J = –11.1 Hz, 6-OCH -carborane), 3.84 (dd, 1H, J = 2.1, J6a,6b = –11.3 Hz,
2 6a,5
H-6a), 3.73 (dd, 1H, J6b,5 = 5.2 Hz, H-6b), 3.53 (s, 3H, 1-OCH ), 3.38 (ddd, 1H, J = 10.0 Hz, H-5),
3
5,4
3
.35 (dd, 1H, J = 9.3, J = 9.3 Hz, H-3), 3.30 (dd, 1H, H-4), 3.16 (dd, 1H, H-2) and 2.54–1.54 (br
3,2 3,4
m, 10H, carborane-BH) ppm.
1
3
C NMR (125.76 MHz; CD OD): δ 105.4 (C-1), 77.8 (C-3), 76.9 (C-5), 75.2 (carborane-C), 74.9
3
(
C-2), 73.9 (6-OCH -carborane), 72.2 (C-6), 71.2 (C-4), 60.5 (carborane-CH) and 57.4 (1-OCH )
2
3
ppm.
2
3
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 24 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
11
B NMR (160.46 MHz; CDCl ): δ –2.06, –3.97, –8.30, –10.50 and –12.08 ppm.
3
+
HRMS: m/z calcd. for C H B O Na [M + Na] 375.2558; found 375.2557.
1
0
26 10
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
.2 Molecular modelling
The initial geometries of the ligands were optimized to a local minimum at DFT level, using the
dispersion-corrected hybrid Tao-Perdew-Scuseria-Staroverov functional TPSSh-D3(BJ),26–28 with
2
9
the doubly polarized triple-zeta basis set def2-TZVPP. The structures of the different ligands were
aligned so that geometries would be as similar as possible. Partial atomic charges were computed
3
0
using the restrained electrostatic potential (RESP) protocol. For the RESP charge calculation, the
molecule was divided into two parts, with one part consisting of the carborane and a linking carbon,
and the other part comprising the sugar. Partial charges of hydrogens bonded to the same carbon were
constrained to be equal. The geometry optimizations were performed with Turbomole 7.3,31,32 and
3
3
34
the RESP calculations with NWChem 6.8. Non-covalent interactions (NCI) between the ligands
and protein were computed using the promolecular approach of NCIPLOT.35
Molecular docking studies were performed using AutoDock 4.2.6.36,37 All rotatable bonds in the
carborane part were set to nonrotatable (inactive). For docking, the number of torsional degrees of
freedom for the carboranes was set to 8 (torsdof 8). The docking studies were performed using the
XylE inward-open 4QIQ and outward-open 6N3I PDB structures. The XylE protein structures were
mutated using PyMOL, changing Gln-415 to Asn-415. The most probable rotamer, that is, the one
with least clashes with surrounding amino acids, as suggested by PyMOL was used. Each protein was
prepared by removing the ligand and other superfluous small molecules (Zn for 4QIQ), adding
hydrogens, merging them and then computing Gasteiger partial charges. For all proteins, a grid of
size 46 x 56 x 60 was used, with a grid spacing value of 0.375. The grid center was in the middle of
the protein cavity, for the grid box to cover the binding site. During docking, the protein was kept
2
4
ACS Paragon Plus Environment

Page 25 of 47
Molecular Pharmaceutics
1
2
3
rigid and only ligand torsional angles changed. For each ligand, 6000 (3  2000) independent search
runs, each with max 2.5 million energy evaluations and population size of 150 with max 27000
generations, were performed using the default settings of the Lamarckian genetic algorithm (LGA),
that is, a mutation rate of 0.02 and crossover rate of 0.8, with one top individual surviving to the next
generation. Conformations were clustered (ranked) with a cluster RMS 2.0 Å.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Parameters for boron, missing from the standard distribution of Autodock, were added to the
parameter file: R 2.285, R 4.57, epsilon 0.179, vol 49.9744; other parameters were set to their
ii
3
8
corresponding carbon values. R and epsilon were taken from Oda. et al., as reproduced by Couto et
3
9
al., and were used to calculate R and vol. The complete parameter definition was thus:
ii
atom_par B
4.57 0.179 49.9744 -0.00143 0.0 0.0 0 -1 -1 0 # Boron for Carborane
2.3. Cytotoxicity studies
The CellTiter-Glo® luminescent cell viability assay was purchased from Promega Corporation
Madison, WI, USA). PierceTM BCA Protein Assay Kit was obtained from Thermo Fisher Scientific
(
(Waltham, MA, USA). Human CAL 27 squamous cell carcinoma was acquired from American Type
Culture Collection (Manassas, VA, USA). The cell culturing flasks and 96-well plates were purchased
from Corning (Corning, NY, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), Dulbecco’s
phosphate buffer saline (10×DPBS), fetal bovine serum (FBS), and Penicillin-Streptomycin (10,000
U/ml) were obtained from Gibco (Life Technologies, Carlsbad, CA, USA).
The in vitro cell cytotoxicity was carried out using a commercial CellTiter-Glo® luminescent cell
viability assay. The human epithelial CAL 27 squamous carcinoma cell line was used as a head-and-
neck cancer cell model in this experiment. The cells were plated on a 96-well plate at 15,000 cells
per well in 100 µl DMEM supplemented with 10% FBS and 1% Penicillin-Streptomycin, and cells
2
5
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 26 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
were allowed to attach overnight. The medium was removed and replaced with 100 µl of the
glucoconjugates 1, 2, 3 or sodium borocaptate (BSH) solution in complete cell culture medium at
concentrations of 5 µM, 25 µM, 50 µM, 125 µM and 250 µM. Fresh medium and 1% (v/v) Triton X-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
00 were used as positive and negative controls of cell viability, respectively. The incubation time
points of the compounds were set at 6 h and 24 h in a temperature and humidity controlled incubator
37 °C, 95 % relative humidity and 5 % CO ). At predetermined time points, the plates were
(
2
equilibrated to room temperature for 30 min. The incubated solutions were discarded and the cells
were washed twice with 1×PBS. Then, 50 µl of both 1×PBS and CellTiter-Glo® reagent were added
to the wells. The plates were protected from light with aluminum foil and placed on an orbital shaker
for 2 min before luminescence measurement with Varioskan LUX multimode microplate reader
(Thermo Fisher Scientific, Waltham, MA, USA). All measurements were done in quadruplicate.
Furthermore, the total protein content in each sample was quantified using the colorimetric
bicinchoninic acid (BCA) assay (Pierce, Thermo Fisher Scientific, Waltham, MA, USA). The
procedures were carried out according to the manufacturer protocol. Cell lysates (25 µl each) from
the cytotoxicity assay was transferred to a new 96-well plate. Working reagent (200 µl) was
subsequently added to each sample at a 1:8 ratio. The plates were kept in dark with aluminum foil
and gently mixed on an orbital shaker for 30 seconds before proceeding to incubate at 37 °C for 30
min. The absorbance was read at 562 nm on a plate reader and the protein content was determined
against a bovine serum albumin (BSA) standard curve (0–2000 µg/ml). The total protein content
results were used to normalize the cell viability from the CellTiter-Glo® assay by dividing the
luminescence value in each sample by the total protein content (µg) in the same sample before the
percent cell viability determination. The experiment was carried out in quadruplicate and the
statistical significance of the mean viability was determined using unpaired Student’s t-test against
the negative control for cell viability.
2
6
ACS Paragon Plus Environment

Page 27 of 47
Molecular Pharmaceutics
1
2
3
4
5
6
7
2
.4 GLUT1 affinity and cellular uptake studies
CAL 27 squamous cell carcinoma cells were purchased from the American Type Culture Collection
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(ATCC, Manassas, VA, USA) or supplied by the University of Helsinki. The CAL 27 cells were
cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, ThermoFisher Scientific, Waltham,
MA, USA) supplemented with L-glutamine (2.0 mM; ThermoFisher Scientific, Waltham, MA, USA),
heat-inactivated fetal bovine serum (10 %; Gibco, ThermoFisher Scientific, Waltham, MA, USA),
penicillin (50 U/ml)- streptomycin (50 µg/ml) solution (ThermoFisher Scientific, Waltham, MA,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
USA). The CAL 27 cells (passages 7–20) were seeded at the density of 5 x 10 cells/wells onto 24-
well plates. The cells were used in the affinity and uptake studies two days after seeding. The culture
medium was removed and the cells were washed with pre-warmed HBSS (Hank’s balance salt
solution) without glucose (pH 7.4). The cells were then incubated with HBSS at 37 °C for 10 min.
before the experiments. Additional information on the experimental protocols is supplied in the
supporting information.
In order to determine the ability of the compounds to bind to GLUT1 in the CAL 27 cell line, the
cells were cultured, seeded and pre-incubated as described above. The HBSS was removed and the
1
4
ability of the compounds to inhibit the uptake of the known GLUT1 substrate, [ C]-D-glucose
(PerkinElmer, Waltham, MA, USA), was studied by incubating the cells at rt for 5 min. in a buffer
14
with a pH 7.4 (250 µl) and further containing 1.8 µM (0.1 mCi/ml) of [ C]-D-glucose. The
compounds were studied at concentrations ranging from 0.1–1800 µM and the HBSS was used as a
blank. After incubation, the experiment was ended by the addition of ice-cold HBSS and the cells
were washed twice with ice-cold HBSS. The cells were then lysed with 250 µl of 0.1 M sodium
hydroxide, the lysate was mixed with 1.0 mL of Emulsifier safe cocktail (PerkinElmer, Waltham,
2
MA, USA) and the radioactivity was measured by liquid scintillation counter (MicroBeta counter,
2
7
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 28 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
14
PerkinElmer, Waltham, MA, USA). The inhibition of [ C]-D-glucose in the presence of the boron
containing compounds compared to the control (HBSS) was calculated as percentages (%). See
Supplementary Figure 44.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The concentration-dependent uptake studies of the glucoconjugates were performed by adding 10–
4
00 µM of the compounds in 250 µl of pre-warmed HBSS buffer on the cell layer. The incubation
times for each compound were 5 min. and 30 min. After incubation the cells were washed and lysed
as described above. The lysate from 4 wells was combined in a Eppendorf tube, centrifuged at 4°C,
TM
and 800 µl of the supernatant was collected and digested in 1.0 ml of conc. HNO (TraceMetal
3
grade, Fisher Chemical) for 24 h. After sample digestion, Milli-Q water (USF Elga Purelab Ultra)
was added in order to reach a total volume of 10 ml and the boron concentrations were analyzed by
inductively-coupled plasma mass spectrometry (ICP-MS).
The boron concentrations were analyzed by ICP-MS using a NeXION 350D ICP-MS instrument
(
PerkinElmer Inc., Waltham, MA, USA) and ESI PrepFAST autosampler (Elemental Scientific,
Omaha, NE, USA). For sample injection a peristaltic pump and nebulizer were used. The instrument
was operated with an RF power of 1.6 kW and with nebulizer gas, auxiliary gas, and plasma gas flows
of 0.90, 18, and 1.2 l/min, respectively. The sample uptake rate was 3.5 ml/min and dwell times were
set at 100 ms per AMU. To remove polyatomic interferences, a triple-quadrupole reaction system
operating in collision mode with kinetic energy discrimination (KED) was used (with He as the cell
8
9
gas (3.7 ml/min)). An internal standard, Y, was mixed online with the samples to compensate for
matrix effects and instrument drift. Boron was determined against a certified multi-element
calibration standard (TraceCERT Periodic Table Mix 1, Sigma Aldrich) under acid conditions (6.7
TM
11
%
HNO , TraceMetal grade, Fisher Chemical). The calibration range used for B was 4 – 400 µg/l
3
2
8
ACS Paragon Plus Environment

Page 29 of 47
Molecular Pharmaceutics
1
2
3
and the detection limit (LOD) was 1.0 µg/l. Three replicates were obtained for each sample. The data
was processed using the PerkinElmer Syngistix Data Analysis Software™.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
. Results
.1 Synthesis and structural characterization of 6-O-carboranylmethyl glucoconjugates
The construction of the targeted glucoconjugates requires insights in boron cluster chemistry and
carbohydrate chemistry. A significant amount of progress has been achieved in both areas over a
considerable timespan and robust reaction methodologies can be found in the existing literature.23,24
Yet, each new synthetic target requires the development of a suitable strategy and, unlike the 3-O-
4
0
41
carboranylmethyl , the carboranylmethyl-glucosides and other types of glucoconjugates
previously evaluated,23,42,43 the 6-O-carboranylmethyl glucoconjugates (Figure 2) have been
explicitly designed for clinical BNCT of head and neck cancers. The methyl glucopyranosides were
included in order to evaluate if there is a difference between the affinity and cellular uptake of the
two anomers since the hemiacetal exists as a mixture of both. In addition, the methyl group is
minimally intrusive which is beneficial since information on the substrate tolerance of GLUT1 is
limited.44
It was important to account for the susceptibility of decaborane to free hydroxyl groups and the
possibility for carboranes to undergo degradation under strongly basic conditions when planning the
synthesis of the 6-O-carboranylmethyl glucoconjugates. With these issues in mind, we developed the
multistep synthetic routes to the three 6-O-carboranylmethyl glucoconjugates 1, 2 and 3. The
synthesis and structural characterization discussion will herein be limited to glucoconjugate 1
(
Scheme 1). The reaction routes to 2 and 3 are displayed in Supplementary scheme 1 and the synthesis
and characterization of 2 and 3, and all intermediates on these routes, were conducted in a similar
fashion as described below for 1.
2
9
ACS Paragon Plus Environment