A Warburg effect targeting vector designed to increase the uptake of compounds by cancer cells demonstrates glucose and hypoxia dependent uptake

Article abstract of DOI:10.1371/journal.pone.0217712

Glycoconjugation to target the Warburg effect provides the potential to enhance selective uptake of anticancer or imaging agents by cancer cells. A Warburg effect targeting group, rationally designed to facilitate uptake by glucose transporters and promote cellular accumulation due to phosphorylation by hexokinase (HK), has been synthesised. This targeting group, the C2 modified glucose analogue 2-(2-[2-(2-aminoethoxy)ethoxy]ethoxy)-D-glu-cose, has been conjugated to the fluorophore nitrobenzoxadiazole to evaluate its effect on uptake and accumulation in cancer cells. The targeting vector has demonstrated inhibition of glucose phosphorylation by HK, indicating its interaction with the enzyme and thereby confirming the potential to facilitate an intracellular trapping mechanism for compounds it is conjugated with. The cellular uptake of the fluorescent analogue is dependent on the glucose concentration and is so to a greater extent than is that of the widely used fluorescent glucose analogue, 2-NBDG. It also demonstrates selective uptake in the hypoxic regions of 3D spheroid tumour models whereas 2-NBDG is distributed primarily through the normoxic regions of the spheroid. The increased selectivity is consistent with the blocking of alternative uptake pathways.

Full text of DOI:10.1371/journal.pone.0217712

RESEARCH ARTICLE
A Warburg effect targeting vector designed to
increase the uptake of compounds by cancer
cells demonstrates glucose and hypoxia
dependent uptake
1
ID
Alexandra Glenister1, Michela I. Simone 2, Trevor W. Hambley
*
ID
1 School of Chemistry, University of Sydney, Camperdown, New South Wales, Australia, 2 Discipline of
Chemistry, Priority Research Centre for Chemical Biology & Clinical Pharmacology, University of Newcastle,
Callaghan, New South Wales, Australia
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
* trevor.hambley@sydney.edu.au
Abstract
Glycoconjugation to target the Warburg effect provides the potential to enhance selective
uptake of anticancer or imaging agents by cancer cells. A Warburg effect targeting group,
rationally designed to facilitate uptake by glucose transporters and promote cellular accu-
mulation due to phosphorylation by hexokinase (HK), has been synthesised. This targeting
group, the C2 modified glucose analogue 2-(2-[2-(2-aminoethoxy)ethoxy]ethoxy)-D-glu-
cose, has been conjugated to the fluorophore nitrobenzoxadiazole to evaluate its effect on
uptake and accumulation in cancer cells. The targeting vector has demonstrated inhibition
of glucose phosphorylation by HK, indicating its interaction with the enzyme and thereby
confirming the potential to facilitate an intracellular trapping mechanism for compounds it is
conjugated with. The cellular uptake of the fluorescent analogue is dependent on the glu-
cose concentration and is so to a greater extent than is that of the widely used fluorescent
glucose analogue, 2-NBDG. It also demonstrates selective uptake in the hypoxic regions of
3D spheroid tumour models whereas 2-NBDG is distributed primarily through the normoxic
regions of the spheroid. The increased selectivity is consistent with the blocking of alterna-
tive uptake pathways.
OPEN ACCESS
Citation: Glenister A, Simone MI, Hambley TW
(2019) A Warburg effect targeting vector designed
to increase the uptake of compounds by cancer
cells demonstrates glucose and hypoxia dependent
uptake. PLoS ONE 14(7): e0217712. https://doi.
org/10.1371/journal.pone.0217712
Editor: Masaharu Seno, Okayama University,
JAPAN
Received: February 6, 2019
Accepted: May 16, 2019
Published: July 15, 2019
Copyright: © 2019 Glenister et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files.
Introduction
Many of the currently used, clinically-approved anticancer agents have severe side effects
resulting from high systemic toxicities, due to their lack of selectivity towards cancerous cells.
[1, 2] To improve the efficacy of anticancer agents it is necessary to develop targeted treat-
ments that enable enhanced uptake of anticancer agents by cancer cells relative to normal cells.
[3] Selective targeting requires not only a carrier dependent uptake pathway, but also the
blocking of other pathways such as passive diffusion.
Funding: This work was supported by the
Australian Research Council, DP140101574, www.
arc.gov.au, to TWH. The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
The metabolic properties of malignant cells differ significantly from those of normal cells,
providing the potential to target cellular metabolism to improve the selectivity of anticancer
Competing interests: The authors have declared
that no competing interests exist.
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A Warburg effect targeting vector demonstrates glucose and hypoxia dependent uptake
Abbreviations: 2-NBDG, 2-(N-(7-nitrobenz-2-oxa-
1,3-diazol-4-yl)amino)-2-deoxy-D-glucose; ATP,
adenosine triphosphate; G6P, glucose-6-
phosphate; GLUT, facilitative-diffusion glucose
transporter; NAD, nicotinamide adenine
dinucleotide.
therapeutics.[4, 5] Many metabolic changes exhibited by cancer cells are identified as require-
ments for malignant transformation, being necessary adaptations to survive the microenviron-
ments found in solid tumours.[6] Recently strategies to target the metabolic differences of
cancer cells have been explored, including conjugation of folate, biotin or glucose to exploit
the increased consumption of these nutrients by cancer cells.[5, 7–9] These conjugates are
designed to be recognised by specific receptors and be taken up by cancer cells at a higher rate
than by normal cells.
The avid consumption of glucose by solid tumours compared to normal tissue was first
observed by Otto Warburg in the first half of the 20th century.[10] Warburg reported the
abnormal energy metabolism of cancer cells with predominantly glycolysis occurring, rather
than oxidative phosphorylation, even in the presence of oxygen.[5, 11] The Warburg effect is
characterised by increased glucose transport and rates of glucose phosphorylation, and
reduced rates of glucose-6-phosphate dephosphorylation.[12] To facilitate their elevated glu-
cose requirements increased levels of glucose transporters (GLUTs) are observed in many
malignant cells, with tumours shown to exhibit up to 12-fold higher GLUT activity than nor-
mal cells.[13, 14]
The glucose analogue 2-deoxy-2-(18F)fluoro-D-glucose (FDG) is a PET imaging agent that
exploits the Warburg effect to visualise tumours and their metastases, and is used in over 90%
of cancer related scans.[15] The literature contains many examples of glucose conjugated with
anticancer drugs, metal complexes and imaging agents to increase their cancer selective deliv-
ery, but there has been little progress of any of these compounds through clinical trials, with
many glycoconjugates demonstrating limited advantage compared to aglycones.[16, 17] Stud-
ies of various glycoconjugates illustrate that GLUT mediated uptake alone may be insufficient
to target cancer cells, although uptake of a glycoconjugate may be increased initially.[18, 19]
This may be the result of reduced GLUT activity over time as the cell recognises an increased
intracellular glucose concentration due to the presence of the glycoconjugate. Alternatively,
compounds may be removed from cells, limiting the effectiveness of the targeted uptake. Phos-
phorylation by hexokinase (HK) can promote trapping of glucose conjugates and maintain
uptake of a glucose analogue to ensure enhanced accumulation over time, as is exploited by
FDG.[20] To enable targeting, modifications to glucose must ensure retention of GLUT and
HK recognition, to promote uptake and accumulation of the glycoconjugates.
Rational design of Warburg effect targeting vector
Cellular uptake by GLUTs is substrate specific. D-glucose is one of the main sugar substrates
transported into cells by GLUT-1,[21] but it has also been demonstrated to have high affinity
with GLUT-2, GLUT-3 and GLUT-4,[13] each of which has been shown to be overexpressed
by some malignant cells.[22]
Numerous studies have shown the importance of C2 modification of D-glucose for
enhanced GLUT mediated uptake and cellular retention of glycoconjugates,[23–25] with the
addition of bulky substituents in this position generally well tolerated.[16]
To enable trapping and promote accumulation of a compound, phosphorylation of the glu-
cose analogue is required. Hexokinase inhibition studies of C2-glucosyl-linker functionalised
Re complexes with different linker lengths found long linker lengths, of 9 atoms, to result in
stronger binding with HK than did short linkers.[26] Use of long alkyl chains as linkers has
been demonstrated to reduce the solubility of glucose Re tricarbonyl complexes in aqueous
conditions.[26, 27] A poly(ethylene glycol) linker was found to enhance the affinity for HK,
compared to the alkyl derivative, and to improve water solubility.[26] It is also expected to
reduce lipophilicity and therefore uptake by passive diffusion.
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A Warburg effect targeting vector demonstrates glucose and hypoxia dependent uptake
Fig 1. Rationally designed Warburg effect targeting vector.
https://doi.org/10.1371/journal.pone.0217712.g001
To fully exploit the potential of targeting the Warburg effect, a glucose analogue rationally
designed to facilitate uptake by GLUTs, reduce uptake by other pathways, and be retained in
cells due to phosphorylation by HK, has been synthesised (Fig 1). This targeting vector has
been designed to be conjugated to various compounds, with the potential to enable increased
tumour selective delivery and accumulation of the anticancer prodrugs or imaging agents.
Experimental procedures
General
The specific optical rotation of compounds ([α]) was measured using an Optical Activity Limited
AA65 Automatic Polarimeter, analytical version (589 nm), with a path length of 1.0 dm, with
concentrations (c) quoted in g 100 ml-1. IR spectra were collected using UATR Two, PerkinElmer
Spectrum Two FT-IR spectrometer over the range 4000–400 cm-1. Low resolution ESI mass spec-
trometry (LRMS) was performed using a Bruker amaZon SL ion trap mass spectrometer. High
resolution ESI mass spectra (HRMS) were collected on a Bruker FTICR mass spectrometer. 1H
and 13C NMR spectra were obtained at 300 K on a Bruker 400 MHz or 500 MHz spectrometer.
Hexokinase inhibition assay. The ability of compounds to inhibit glucose phosphoryla-
tion by hexokinase was tested using glucose (HK) assay kits (Sigma), containing a glucose
(HK) assay reagent, reconstituted with 20 mL H2O, and 1 mg mL-1 glucose standard solution.
5 mM aqueous stock solutions of each compound to be tested were freshly prepared. The
assay reagent (280 μL) was combined with glucose standard solution (10 μL) and a volume of
compound stock solution, and the total volume made up to 700 μL with H2O in a 1 cm pathlength
700 μL quartz fluorescence cuvette (Thorlabs). For each test compound different concentrations
were examined by adding known volumes of the stock solution to the assay solution. UV-visible
spectrometry was performed on a Cary 60 UV-Visible Spectrophotometer at room temperature,
recording absorbance at 340 nm every 2 s for 3 min. Initial rates of reaction (Vi) for glucose phos-
phorylation were determined from the gradient of a plot of absorbance at 340 nm against time.
A control experiment, to calculate the initial rate of glucose phosphorylation in the absence
of the compounds, was conducted for each batch of the HK assay reagent used. The initial rate
of glucose phosphorylation in the presence of our compounds relative to the control were cal-
culated. These results were plotted against the concentration of compound to show any rela-
tionship between compound concentration and rate of HK glucose phosphorylation.
Cell culture
DLD-1 human colon carcinoma cells were maintained as monolayers in low glucose DMEM
supplemented with 10% FBS. The cells were incubated in a humidified environment at 37˚C
with 5% (v/v) CO2, and sub-cultured using trypsin to detach cells.
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A Warburg effect targeting vector demonstrates glucose and hypoxia dependent uptake
Confocal imaging of monolayer and spheroid tumour models were performed on a Leica
SP5 II microscope, and images were analysed using LAS AF Lite. A heated stage was used to
maintain the temperature at 37˚C during live cell imaging. The samples were excited with 488
nm light, and the emission wavelengths collected between 530–650 nm.
Preparation of monolayer samples for determining glucose dependent uptake of com-
pounds by confocal fluorescence microscopy. 1 x 105 cells in 2 mL supplemented low glu-
cose DMEM were plated onto glass-bottom dishes (MatTek) and allowed to adhere overnight.
The medium was replaced with glucose free DMEM supplemented with 10% FBS and 2% glu-
tamine. The required volume of glucose solution (200 g L-1), to give concentrations from 0 to
8000 mg L-1, was added and cells were treated with 50 μM compound for 2 h.
After incubation, the medium was removed and the cells were washed with PBS (2 x 1 mL).
Cells were imaged in FluoroBrite DMEM (2 mL) supplemented with 10% FBS and 2%
glutamine.
A HCX PL APO 63x 1.2 water objective was used to image monolayer cells. At least 2 dishes
for each condition were imaged on each occasion, and the experiment was repeated 2 times.
Preparation of spheroid samples for confocal fluorescence imaging. Spheroids were
formed by plating 100 μL of a 1 x 105 cells mL-1 single cell suspension into each well of an
ultra-low cluster, round bottom ultra-low attachment 96-well plate. After allowing the spher-
oids to aggregate for 3 days, 100 μL of a 100 μM solution of compound in supplemented cell
culture medium was added to each well containing a spheroid, to give a final concentration of
50 μM. Following incubation for 4 h, spheroids were collected, the medium removed and the
spheroids washed with PBS (2 x 2 mL). Spheroids were suspended in supplemented Fluoro-
Brite DMEM (2 mL), and transferred into a glass-bottom dish (MatTek).
Synthesis
2-[2-(2-azidoethoxy)ethoxy]ethanol was synthesised by a modification of the method of
Legeay et al.[28] 2-[2-(2-aminoethoxy)ethoxy]ethanol was synthesised by the procedure of Liu
et al.[29] 2-[2-(2-azidoethoxy)ethoxy]ethyl mesylate was synthesised by a modification of the
procedure of Sakamoto et al.[30] 2-NBDG was synthesised by the method of He et al.,[31] and
purified on a Sephadex LH-20 column eluting with H2O.
1,2-O-isopropylidene-α-D-glucofuranose (1). 1,2-O-isopropylidene-α-D-glucofuranose
was prepared by a modification of the method of Yadav et al.[32] Iodine (1.47 g, 5.8 mmol, 0.3
eq.) was added to a solution of 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (5.00 g, 19.2
mmol) in MeCN (270 mL). H2O (2 mL) was added, and the reaction mixture was stirred at
room temperature for 7 h. The reaction was quenched with saturated aqueous Na2S2O3 solu-
tion and extracted into EtOAc (5 x 200 mL). The organic layers were dried over anhydrous
Na2SO4, the solvent removed and the crude mixture was purified by column chromatography
(silica) using EtOAc/hexane 2:1, EtOAc and EtOAc/MeOH 1:0.01 to give the product as a
26
white powder (3.29 g, 14.9 mmol, 78% yield). Rf 0.23 (EtOAc, silica); ½a�D -18.3˚ (c 1.00, H2O)
22
(lit. ½a�D -12.0˚ (c 1.0, H2O)[33]).
3,5,6-tri-O-benzyl-1,2-O-isopropylidene-α-D-glucofuranose (2). Under N2, 1 (1.00 g,
4.5 mmol) was dissolved in anhydrous DMF (24 mL). Sodium hydride in a 60% oily dispersion
(0.90 g, 22.5 mmol, 5 eq.) was added portionwise with vigorous stirring. After stirring for 30
min, benzyl bromide (2.80 mL, 22.5 mmol, 5 eq.) was added dropwise. The reaction mixture
was stirred at room temperature, under N2 for 24 h and then treated with H2O (150 mL) and
neutralised with 1 M hydrochloric acid. The product was extracted into DCM (3 x 150 mL),
and the organic layer was dried over Na2SO4 and the solvent removed. The product was
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A Warburg effect targeting vector demonstrates glucose and hypoxia dependent uptake
purified by column chromatography (silica) with hexane/EtOAc 8:1 eluent to yield 2 as a light
yellow oil (2.01 g, 4.1 mmol, 90% yield).
26
20
Rf 0.46 (hexane/EtOAc 2:1, silica); ½a�D -33.8˚ (c 2.03, CHCl3) (lit. ½a�D -36˚ (c 1.0, CHCl3)
25
[34]; ½a�D -33˚ (c 9.3, CHCl3)[35]).
Methyl 3,5,6-tri-O-benzyl-α-D-glucofuranoside (3a) and methyl 3,5,6-tri-O-benzyl-β-
D-glucofuranoside (3b). The method of Lee and Perlin[36] was used for the synthesis of
methyl 3,5,6-tri-O-benzyl-α-D-glucofuranoside and methyl 3,5,6-tri-O-benzyl-β-D-glucofura-
noside. 2 (1.00 g, 2.04 mmol) was dissolved in MeOH (20 mL) under N2. Amberlite IR-120
(H+) ion exchange resin (10.25 g) was added, and the reaction was refluxed under N2 for 24 h.
The reaction mixture was filtered, the filtrate evaporated and the crude mixture separated by
column chromatography (silica) with hexane/EtOAc 4:1 and hexane/EtOAc 2:1 to yield 3a
and 3b as colourless oils (3a: 0.34 g, 0.73 mmol, 36% yield; 3b: 0.31 g, 0.67 mmol, 33% yield).
26
28
3a. Rf 0.53 (hexane/EtOAc 2:1, silica); ½a�D +21.7˚ (c 0.40, CHCl3) (lit. ½a�D +28.9˚ (c 0.32,
CHCl3)[37]).
26
28
3b. Rf 0.32 (hexane/EtOAc 2:1, silica); ½a�D -58.7˚ (c 0.21, CHCl3) (lit. ½a�D -54.9˚ (c 0.25,
CHCl3.
Methyl 2-(2-[2-(2-azidoethoxy)ethoxy]ethoxy)-3,5,6-tri-O-benzyl-α-D-glucofuranoside
(4a) and methyl 2-(2-[2-(2-azidoethoxy)ethoxy]ethoxy)-3,5,6-tri-O-benzyl-β-D-glucofura-
noside (4b). 3a or 3b (1.55 g, 3.34 mmol) was dissolved in anhydrous DMF (20 mL) under
N2. Sodium hydride in a 60% oily dispersion (0.31 g, 7.68 mmol, 2.3 eq.) was added portion-
wise, and the reaction mixture stirred at room temperature for 20 min. 2-[2-(2-azidoethoxy)
ethoxy]ethyl methanesulfonate (1.31 g, 5.18 mmol, 1.55 eq.) in anhydrous DMF (1 mL) was
added dropwise to the reaction mixture, which was subsequently stirred at 60˚C under N2 for
1 week. MeOH (20 mL) was added and the mixture stirred for 30 min, before the solvent was
removed. The resulting solid was dissolved in EtOAc (60 mL), washed with H2O (2 x 50 mL)
and dried over Na2SO4.
4a. The crude mixture was purified by column chromatography (silica) with hexane/
EtOAc 4:1 and EtOAc to give the product as a yellow oil (1.61 g, 2.59 mmol, 78% yield). Rf
26
0.67 (hexane/EtOAc 1:1, silica); ½a�D +33.3˚ (c 0.25, CHCl3); LRMS (ESI+): m/z calculated
644.29 ([M+Na]+), found 644.13 ([M+Na]+).
4b. The crude mixture was purified by column chromatography (silica) with hexane/
EtOAc 9:1 and hexane/EtOAc 4:1 to give the product as a pale yellow oil (1.52 g, 2.44 mmol,
26
73% yield). Rf 0.55 (hexane/EtOAc 1:1, silica); ½a�D -22.2˚ (c 0.36, CHCl3); LRMS (ESI+): m/z
calculated 644.29 ([M+Na]+), found 644.09 ([M+Na]+).
Methyl 2-(2-[2-(2-aminoethoxy)ethoxy]ethoxy)-3,5,6-tri-O-benzyl-α-D-glucofurano-
side (5a) and methyl 2-(2-[2-(2-aminoethoxy)ethoxy]ethoxy)-3,5,6-tri-O-benzyl-β-D-glu-
cofuranoside (5b). A solution of 4a or 4b (1.60 g, 2.57 mmol) in MeOH (25 mL) was stirred
under N2. Pd(10%)/C (160 mg, 10% w/w) was added and the mixture was stirred under an
atmosphere of hydrogen for 2 h at room temperature. The catalyst was removed by filtration
through celite and solvent removed from the filtrate to yield the products as a pale-yellow oils
(5a: 1.47 g, 2.47 mmol, 96% yield; 5b: 1.50 g, 2.52 mmol, 98% yield).
26
5a. ½a� +76.7˚ (c 0.20, CHCl3); LRMS (ESI+): m/z calculated 596.32 ([M+H]+), found
D
596.00 ([M+H]+).
26
5b. ½a� -20.9˚ (c 0.86, CHCl3); LRMS (ESI+): m/z calculated 596.32 ([M+H]+), found
D
596.28 ([M+H]+).
Methyl 2-(2-[2-(2-(tert-butoxycarboxamido)ethoxy)ethoxy] ethoxy)-3,5,6-tri-O-benzyl-
α-D-glucofuranoside (6a) and methyl 2-(2-[2-(2-(tert-butoxycarboxamido)ethoxy)ethoxy]
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A Warburg effect targeting vector demonstrates glucose and hypoxia dependent uptake
ethoxy)-3,5,6-tri-O-benzyl-β-D-glucofuranoside (6b). 5a or 5b (0.70 g, 1.18 mmol) was
dissolved in MeCN (17.5 mL). Et3N (0.17 mL, 1.18 mmol, 1 eq.) and di-tert-butyl dicarbonate
(0.26 g, 1.18 mmol, 1 eq.) were added and the reaction mixture stirred for 5 h at room temper-
ature. The solvent was removed and the resulting solid partitioned between H2O (10 mL) and
EtOAc (10 mL). The EtOAc layer was collected and washed with H2O (7 mL). The organic
layer was dried over Na2SO4 and the solvent removed to give the products as yellow oils (6a:
0.62 g, 0.89 mmol, 75% yield; 6b: 0.58 g, 0.83 mmol, 70% yield).
26
6a. Rf 0.85 (EtOAc, silica); ½a�D +34.2˚ (c 0.37, CHCl3); LRMS (ESI+): m/z calculated
718.36 ([M+Na]+), found 718.00 ([M+Na]+).
26
6b. Rf 0.82 (EtOAc, silica); ½a�D -17.5˚ (c 0.85, CHCl3); LRMS (ESI+): m/z calculated
718.36 ([M+Na]+), found 718.37 ([M+Na]+).
Methyl 2-(2-[2-(2-(tert-butoxycarboxamido)ethoxy)ethoxy] ethoxy)-α-D-glucofurano-
side (7a) and methyl 2-(2-[2-(2-(tert-butoxycarboxamido)ethoxy)ethoxy] ethoxy)-β-D-glu-
cofuranoside (7b). A 9 mM solution of 6a or 6b in EtOH was passed through a ThalesNano
H-Cube P flow hydrogenation reactor at 0.3 mL min-1. Benzyl ether hydrogenolysis was
achieved using a Pd(10%)/C CatCart at 80 bar and 60˚C. The product solution was evaporated
to dryness to yield the product as a colourless oil, without any further purification (quantitative
yield).
26
7a. ½a� +64.0˚ (c 0.50, H2O); LRMS (ESI+): m/z calculated 426.23 ([M+H]+), found
D
426.51 ([M+H]+).
26
7b. ½a� -33.3˚ (c 0.24, H2O); LRMS (ESI+): m/z calculated 448.22 ([M+Na]+), found
D
448.21 ([M+Na]+).
Hydrochloride salt of 2-(2-[2-(2-aminoethoxy)ethoxy]ethoxy)-D-glucose (8). 7a or 7b
(0.20 g, 0.47 mmol) was heated at reflux in 0.5 M hydrochloric acid (12 mL) for 24 h. The sol-
vent was removed, and the product was obtained as a beige solid after freeze-drying (0.15 g,
26
0.47 mmol, quantitative yield). ½a�D +43.9˚ (c 0.94, H2O); LRMS (ESI+): m/z calculated 312.17
([M+H]+) and 334.15 ([M+Na]+), found 312.14 ([M+H]+) and 334.13 ([M+Na]+); HRMS (ESI
+): m/z calculated 312.16559 ([M+H]+) for C12H26O8N, found 312.16529 ([M+H]+).
2-(2-[2-(2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl))aminoethoxy)ethoxy]ethoxy)-D-glu-
cose (9). 8 (90 mg, 0.259 mmol, 1.1 eq.) was dissolved in MeOH (3 mL) and Et3N (99 μL,
0.706 mmol, 3 eq.) was added. After stirring at 30˚C for 1 h, NBD-Cl (47 mg, 0.235 mmol, 1
eq.) was added. The reaction was stirred overnight in the dark and at 30˚C. Solvent was
removed and the resulting solid was dissolved in EtOAc (5 mL). Insoluble material was
removed by filtration, and the filtrate was dried in vacuo. The resulting solid was dissolved in
H2O (5 mL) and purified on a sephadex LH-20 column, eluting with H2O to give the product
as an orange solid (35 mg, 0.074 mmol, 29% yield).
LRMS (ESI+): m/z calculated 497.12 ([M+Na]+), found 497.15 ([M+Na]+); HRMS (ESI+):
m/z calculated 497.14903 ([M+Na]+) for C18H26N4O11Na, found 497.14954 ([M+Na]+).
((N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)aminoethoxy)ethoxy)ethanol (10). A solution
of 2-[2-(2-aminoethoxy)ethoxy]ethanol (0.41 g, 2.75 mmol, 1.1 eq.) and Et3N (660 μL, 4.75
mmol, 1.9 eq.) in methanol (15 mL) was stirred at 30˚C for 1 h. NBD-Cl (0.5 g, 2.50 mmol, 1
eq.) was added and the reaction was stirred in the dark for 16 h at 30˚C. Insoluble material was
removed by filtration, and the filtrate was dried. The resulting solid was dissolved in H2O (5
mL) and purified on a sephadex LH-20 column, eluting with H2O to give the product as an
orange solid (0.33 g, 1.05 mmol, 42% yield).
LRMS (ESI-): m/z calculated 311.10 ([M-H]-), found 311.18 ([M-H]-), (ESI+): m/z calcu-
lated 335.27 ([M+Na]+), found 335.09 ([M+Na]+); HRMS (ESI+): m/z calculated 335.09621
([M+Na]+) for C12H16N4O6Na, found 335.09650 ([M+Na]+).
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A Warburg effect targeting vector demonstrates glucose and hypoxia dependent uptake
Results and discussion
Discussion of the synthesis of the Warburg effect targeting vector
To yield the target glucose analogue 2-(2-[2-(2-aminoethoxy)ethoxy]ethoxy)-D-glucose from
1,2:5,6-di-O-isopropylidene-α-D-glucofuranose, protection of all positions other than the C2
hydroxyl group was necessary, followed by coupling of the linker at the C2 position and finally
removal of all protecting groups (Fig 2). Maintaining the stereochemistry of the starting com-
pound was essential to ensure synthesis of a D-glucose analogue, required for GLUT and HK
recognition.
Synthesis of a glucose analogue with only the C2 hydroxyl group deprotected. The ini-
tial synthetic steps involve protection of the hydroxyl groups in the C1, C3, C4, C5 and C6
positions of glucose. Using existing literature syntheses, a synthetic route to obtain D-glucose
analogues with a hydroxyl group in only the C2 position was devised. The α- and β-anomers
(compounds 3a and 3b) were isolated by silica column chromatography and used separately in
further syntheses for ease of purification and optimisation of subsequent reactions. Upon full
deprotection of the glucose analogue, both α- and β-anomers give the same product as the
compounds will interconvert in solution.
Modification at the C2 position. Conjugation of a linker at the C2 position of 3a and 3b
by ether synthesis proceeds via an SN2 reaction. SN2 reactions result in inversion of stereo-
chemistry at the carbon centre of substitution, therefore to maintain the stereochemistry of the
glucose analogue the C2 position must act as a nucleophile, formed via proton abstraction by a
strong base, and a good leaving group must be on the PEG linker. A selection of bases and
leaving groups were investigated to optimise the conjugation of a linker to 3a and 3b.
The addition of a good leaving group on 2-[2-(2-azidoethoxy)ethoxy]ethanol increases its
reactivity, required for an SN2 reaction, but also decreases the stability of the PEG compound.
The mesylate leaving group was found to be ideal, as it does not degrade before it can undergo
the SN2 reaction with the glucose analogue. 2-[2-(2-azidoethoxy)ethoxy]ethyl mesylate enabled
conjugation of PEG to glucose C2, with good yield at increased temperatures and with repro-
ducible results.
The reactivities of 3a and 3b were found to differ. It is hypothesised that the different ste-
reochemistries of the anomers affects the approach of the base for proton abstraction. Steric
hindrance present in the β-anomer prevented alkoxide formation by lithium diisopropyla-
mide, therefore the less bulky strong base NaH was used. Optimisation of the number of NaH
equivalents used, reaction time and temperature provided conditions that are applicable to
both anomers.
Fig 2. Synthetic route to novel C2 modified glucose analogue for GLUT-1 and HK recognition (compound 8). a: I2, H2O, MeCN, 78% yield; b: NaH, benzyl
bromide, DMF, 90% yield; c: Amberlite IR-120(H+), MeOH, reflux, yield–α 36% and β 33%; d: NaH, DMF, 60 ºC, yield–α 78% and β 73%; e: Pd(10%)/C, H2(g), MeOH,
yield–α 96% and β 98%; f: Boc anhydride, Et3N, MeCN, yield–α 75% and β 70%; g: Pd(10%)/C CatCart, H2(g), 80 Bar, 60˚C, quantitative yield; h: 0.5 M HCl (aq.),
reflux, quantitative yield.
https://doi.org/10.1371/journal.pone.0217712.g002
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A Warburg effect targeting vector demonstrates glucose and hypoxia dependent uptake
Deprotecting 4a and 4b. Azides are reduced under the hydrogenation conditions used
for benzyl ether cleavage, which provided the possibility for simultaneous amine formation
and the removal of benzyl ether protecting groups. However for compounds 4a and 4b this
was unsuccessful, even at increased temperature and pressure, as reduction of the azide
occurred prior to benzyl ether deprotection. Surfraz et al. have demonstrated that starting
materials with amines poison Pd/C catalysts, affecting reactivity towards OBn protecting
groups.[38] The amine produced by azide reduction of 4a and 4b poisons the catalyst, prevent-
ing cleavage of the benzyl ether groups. This conclusion is supported by the work of Sajiki
et al., who report the inhibition of Pd/C catalysed OBn deprotection for substrates containing
reducible functional groups that give amine products, including N-Cbz and NO2.[39]
To prevent poisoning of the Pd catalyst, to enable complete benzyl ether cleavage, protec-
tion of the primary amine produced by hydrogenation (compounds 5a and 5b) was required.
Tert-butyloxycarbonyl (Boc) was identified as a suitable protecting group as it withstands
hydrogenation conditions, and benzyl ether cleavage by Pd/C catalysed hydrogenolysis of mol-
ecules containing Boc functionality has been demonstrated.[39, 40] Additionally Boc can be
removed with acid,[41, 42] the same conditions required for methyl glycoside deprotection.
[43] Following Boc protection, to give 6a and 6b, complete benzyl ether cleavage proceeded
via Pd(10%)/C catalysed hydrogenolysis only at elevated temperature and pressure to give 7a
and 7b.
The final reaction to remove the Boc and methyl glycoside groups by refluxing in 0.5 M
hydrochloric acid yielded the targeting vector as the HCl salt (8). Methyl glycoside cleavage
deprotects the C1 glucose position, enabling interconversion in aqueous solution between the
α- and β-anomers. The deprotected C1 and C6 hydroxyl groups provide the ability for this
compound to adopt the six-membered pyranose ring structure, which is favoured over the
furanose form for D-glucose.[44, 45] The α- and β-anomers of 8 are observed in the 1H and
13C NMR spectra collected in D2O. The C1 protons of the compound are identifiable in the 1H
NMR spectrum, being downfield from other signals as for α,β-D-glucose,[46] and the signifi-
cant difference in chemical shift for C1 protons between the anomers of D-glucose,[47]
enabled identification of C1 anomeric protons in the 1H NMR spectrum of 8. Integration of
these peaks showed that the α- and β-anomers to be present in equal amounts in solution.
Hexokinase inhibition studies
Once glucose enters cells it is metabolised to pyruvate through the process of glycolysis. Glu-
cose is initially phosphorylated by adenosine triphosphate (ATP) to give to glucose-6-phos-
phate (G6P) in a reaction catalysed by HK. Phosphorylated C2 modified glucose analogues,
such as FDG and 2-NBDG, cannot undergo the structural rearrangement required for subse-
quent glycolysis steps and the G6P analogue is trapped intracellularly.[48] Targeting vector 8
has been designed to provide this trapping mechanism for its conjugates.
The inhibition of HK glucose phosphorylation by glucosamine hydrochloride, the aglycone
2-[2-(2-aminoethoxy)ethoxy]ethanol and 8 has been investigated using a glucose (HK) assay
reagent containing HK, ATP, NAD+ and glucose-6-phosphate dehydrogenase (G6PDH). Fol-
lowing the addition of a glucose solution, a series of enzyme catalysed reactions occur that
enable quantification of the rate of HK glucose phosphorylation by monitoring the conversion
of NAD+ to NADH by UV spectroscopy. The glucose (HK) assay reveals whether a compound
interacts with HK to inhibit its active site, but does not establish that phosphorylation of the
compound occurs.
Glucosamine is a known inhibitor of glucose phosphorylation by HK, being a substrate for
the enzyme, it is recognised by the active site and phosphorylated.[49, 50] Therefore the
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A Warburg effect targeting vector demonstrates glucose and hypoxia dependent uptake
inhibition of HK glucose phosphorylation by glucosamine hydrochloride serves as a useful
comparison to the inhibitory capacity of the novel glucose analogue synthesised.
The results (S1 Fig) demonstrate that 8 exhibits inhibition of HK activity, at a level of about
30% of that exhibited by glucosamine, and that no inhibition is observed for the aglycone.
These results indicate that the targeting vector (8) fulfils the criteria of providing HK recogni-
tion, giving rise to the potential to facilitate an intracellular trapping mechanism for its
conjugates.
The low inhibition by compound 8 compared to glucosamine is possibly due to the flexibil-
ity of the PEG linker preventing strong interactions with HK, and is as observed for glucose
analogue ligands reported by Schibli et al.[26] However for conjugation to bulky groups, a
linker of sufficient length has been demonstrated to be essential for inhibition of HK.[20, 27,
48] Inhibition of HK glucose phosphorylation by Re complexes conjugated to C2 modified
glucose analogue ligands, with 9 atom length linkers, has been demonstrated to be enhanced
compared to the free ligand.[26] It is possible that the presence of a bulky group in the outer
cavity of HK provides stabilising interactions with the enzyme surface, which provides
enhanced binding between the active site and the glucose analogue. Therefore 8 has the poten-
tial to provide HK interaction and phosphorylation to its conjugates, which is unlikely for glu-
cosamine conjugates.
Fluorescent analogues
Glucose uptake by cells in vitro can be imaged using the fluorescent glucose analogue 2-NBGD
(Fig 3). As for D-glucose, this C2 modified glucose analogue is taken into cells via GLUT trans-
porters and phosphorylated by HK.[23, 51] 2-NBDG fluorescence has been used to quantify
glucose uptake in cell monolayers, and 3-dimensional cell aggregates and spheroids.[52, 53]
Fluorescent analogues of 8 and the aglycone 2-[2-(2-aminoethoxy)ethoxy]ethanol (Fig 3) were
synthesised following modification of the synthesis of 2-NBDG from NBD-Cl,[31] to visualise
and compare uptake of these compounds in vitro and their fluorescence spectra are shown in
S2 Fig. Comparison of the uptake and distribution of 9 with 2-NBDG and the aglycone 10 will
enable investigation and evaluation of the Warburg effect targeting of 8.
Glucose dependent uptake in monolayer cell culture. Warburg effect targeting com-
pounds are expected to compete with glucose for cellular uptake via glucose transporters but
measuring the competition directly would require a knowledge of which GLUTs are involved.
Instead, we have compared the intracellular fluorescence in the presence of different glucose
concentrations to investigate the glucose dependence of the uptake of the NBD-conjugates. Fig
4 shows the relative fluorescence intensity for DLD-1 human colon carcinoma cells in cell cul-
ture media containing varying amounts of glucose, and dosed with 2-NBDG, 9 or 10 for 4 h.
The range of glucose concentrations used were selected to be similar to those present in glu-
cose-free (0 mg L-1), low glucose media (1000 mg L-1) and high glucose media (4500 mg L-1).
Intracellular fluorescence intensities were measured for a minimum of eight 30x30 μm2
regions of DLD-1 colon carcinoma cells grown in monolayer and dosed with NBD-conjugates
(50 μM) at increasing glucose concentrations. Examples are shown in S3 Fig. They are shown
relative to the fluorescence for cells dosed with each compound in the absence of glucose
which have in each case been normalised to 100. Error bars represent standard deviations.
The results reveal that the intracellular fluorescence, and therefore uptake of the compound,
decreases with increasing glucose concentration. The cellular uptake of 9 is inhibited by glu-
cose and is so to a greater extent than is the uptake of 2-NBDG. The uptake of 10 is unaffected
by glucose concentration, therefore the higher impact of glucose concentration observed for 9
is consistent with uptake via the glucose transporter. An unpaired t-test shows that the
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A Warburg effect targeting vector demonstrates glucose and hypoxia dependent uptake
Fig 3. Structures of fluorescent analogues synthesised to investigate Warburg effect targeting in vitro.
https://doi.org/10.1371/journal.pone.0217712.g003
differences between the fluorescence levels of 9 and 10 at 4000 mg L-1 and 8000 mg L-1 are sig-
nificant at a p < 0.05. This suggests that conjugates of 8 should successfully exploit overexpres-
sion of glucose transporters by cancer cells.
Whilst targeting vector 8 has been designed to target GLUT-1, this study does not enable
identification of the specific glucose transporters 2-NBDG or 9 are internalised through, and it
is possible the greater glucose dependence of the uptake of 9 is due to internalisation by other
GLUT isoforms. The greater dependence relative to that of 2-NBDG is consistent with a lower
contribution from uptake pathways which are not dependent on glucose concentration such
as passive diffusion.
Distribution through solid tumour models. Unlike monolayer models, spheroids con-
tain different regions, resembling those seen in solid tumours, as cells are at different distances
from the nutrient source.[54] The cells on the edge of the spheroid are actively proliferating,
similar to the cells of a solid tumour in close proximity to blood vessels.[55] Depending on
their size, spheroids can develop hypoxic regions, where cells are quiescent, and necrotic cores
where most of the cells are dead.[2, 56] These different regions of spheroids are expected to
have different glucose requirements, transporter expression and glucose availability that will
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A Warburg effect targeting vector demonstrates glucose and hypoxia dependent uptake
Fig 4. Intracellular fluorescence intensity for DLD-1 colon carcinoma cells dosed with NBD-conjugates at different glucose concentrations.
https://doi.org/10.1371/journal.pone.0217712.g004
impact delivery of Warburg effect targeting compounds. The expression of GLUT-1 and
GLUT-3 transporters has been demonstrated to be hypoxia-responsive, with a 10-fold increase
in GLUT-1 protein expression observed for adipocyte cells cultured in hypoxic compared to
normoxic conditions.[57, 58] To explore this, confocal images of the cross sections of spher-
oids dosed with 2-NBDG, 9 and 10 were examined (Fig 5).
Overlays of brightfield (greyscale) and NBD fluorescence (green) (top row), and confocal
images (NBD fluorescence) (bottom row) for cross sections of spheroids which were dosed
with 2-NBDG, 9 and 10 at 50 μM for 4 h.
Uptake of 10 is very low, and distribution is even throughout the spheroid cross section,
illustrating no selective uptake by cells in different regions. This is consistent with PEG inhibit-
ing cellular uptake, as has previously been reported.[59, 60] 2-NBDG fluorescence, which is
limited to the periphery of the spheroid, is expected to mimic glucose uptake by cells through-
out a spheroid and therefore be indicative of Warburg effect targeting. Spheroids dosed with 9
however, have a markedly different fluorescence distribution. The uptake of 9 is primarily
observed at a distance of approximately 80 μm from the surface of the spheroid, the distance
around which hypoxia has been shown to develop,[55] and decreases towards the necrotic
core of the spheroid. This observation is consistent with strongly glucose dependent uptake of
9, as fluorescence is observed in a region with reduced nutrient availability and therefore lower
extracellular glucose concentration. As hypoxia is associated with overexpression of some
GLUT isoforms, the difference in fluorescence distribution between 2-NBDG and 9 may be
due the glucose analogues being internalised by different transporters. It may also result from
a higher level of non-selective uptake of 2-NBDG. Consistent with this hypothesis is the obser-
vation that the intensity of the fluorescence of cells treated with 2-NBDG is substantially higher
than that seen following treatment with 9 (S1 Table) and a conclusion that might be drawn
from Fig 4 is that half or more of the uptake of NBDG is not dependent on the glucose
concentration.
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A Warburg effect targeting vector demonstrates glucose and hypoxia dependent uptake
Fig 5. Brightfield and fluorescence images of spheroids treated with 2-NBDG, 9 and 10.
https://doi.org/10.1371/journal.pone.0217712.g005
Conclusions
A Warburg effect targeting vector (8), with the capability to conjugate with many compounds,
has been successfully synthesised, conjugated with a fluorophore and evaluated. This targeting
vector has demonstrated the ability to bind to HK and thereby inhibit HK catalysed phosphor-
ylation of glucose, an indicator of the desired interaction between the glucose analogue and
the enzyme. In vitro, the novel Warburg effect targeting vector 8 provides distinct advantages
over glucosamine for exploiting the Warburg effect to increase selective uptake by cancer cells.
Results indicate cellular uptake of 8 and its fluorescent analogue (9) is through glucose trans-
porters that cancer cells overexpress. It is possible that inhibition of the cellular uptake by
PEG, while leading to lower levels of accumulation, increases the selectivity because the GLUT
dependent pathways become more dominant. Additionally, this vector may provide the poten-
tial to target the difficult to treat hypoxic regions of tumours when conjugated to anticancer
agents.
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A Warburg effect targeting vector demonstrates glucose and hypoxia dependent uptake
Supporting information
S1 Fig. Plot of the relative rate of HK catalysed glucose phosphorylation in the presence of
increasing concentration of compounds, up to 714 μM.
(PNG)
S2 Fig. Fluorescence emission spectra of 2-NBDG, 9 and 10 (50 μM) in H2O. Emission
scans were collected between 510 and 700 nm using an excitation wavelength of 488 nm.
(PNG)
S3 Fig. Confocal images of NBD fluorescence (green), brightfield (greyscale) and overlay
of images (from left to right) for monolayer DLD-1 cells in glucose-free media, dosed with
2-NBDG, 9 and 10 (50 μM for 2 h).
(PNG)
S1 Table. Raw data values of fluorescence intensities for 3 regions of DLD-1 cells in glu-
cose-free media dosed with each compound (50 μM) for 2 h. Fluorescence intensities were
determined by quantification with LAS AF Lite.
(DOCX)
S1 Dataset. Minimal underlying data set.
(DOCX)
Acknowledgments
The authors acknowledge scientific and technical input from the Australian Microscopy &
Microanalysis Research Facility (AMMRF) node at the University of Sydney. This research
was funded in part by the Australian Government through the Australian Research Council
(Project DP140101574).
Author Contributions
Conceptualization: Michela I. Simone, Trevor W. Hambley.
Data curation: Alexandra Glenister.
Formal analysis: Alexandra Glenister.
Funding acquisition: Trevor W. Hambley.
Investigation: Alexandra Glenister.
Project administration: Trevor W. Hambley.
Supervision: Michela I. Simone, Trevor W. Hambley.
Validation: Michela I. Simone.
Writing – original draft: Alexandra Glenister.
Writing – review & editing: Michela I. Simone, Trevor W. Hambley.
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