Targeting of glut5 for transporter-mediated drug-delivery is contingent upon substrate hydrophilicity

Article abstract of DOI:10.3390/ijms22105073

Specific link between high fructose uptake and cancer development and progression highlighted fructose transporters as potential means to achieve GLUT-mediated discrimination between normal and cancer cells. The gained expression of fructose-specific transporter GLUT5 in various cancers offers a possibility for developing cancer-specific imaging and bioactive agents. Herein, we explore the feasibility of delivering a bioactive agent through cancer-relevant fructose-specific transporter GLUT5. We employed specific targeting of GLUT5 by 2,5-anhydro-D-mannitol and investigated several drug conjugates for their ability to induce cancer-specific cytotoxicity. The proof-of-concept analysis was carried out for conjugates of chlorambucil (CLB) in GLUT5-positive breast cancer cells and normal breast cells. The cytotoxicity of conjugates was assessed over 24 h and 48 h, and significant dependence between cancer-selectivity and conjugate size was observed. The differences were found to relate to the loss of GLUT5-mediated uptake upon increased conjugate size and hydrophobicity. The findings provide information on the substrate tolerance of GLUT5 and highlight the importance of maintaining appropriate hydrophilicity for GLUT-mediated delivery.

Full text of DOI:10.3390/ijms22105073

International Journal of
Molecular Sciences
Article
Targeting of GLUT5 for Transporter-Mediated Drug-Delivery Is
Contingent upon Substrate Hydrophilicity
Nazanin Nahrjou 1, Avik Ghosh 1 and Marina Tanasova 1,2,
*
1
Chemistry Department, Michigan Technological University, Houghton, MI 49931, USA;
nnrahjou@mtu.edu (N.N.); avikg@mtu.edu (A.G.)
Health Research Institute, Michigan Technological University, Houghton, MI 49931, USA
Correspondence: mtanasov@mtu.edu
2
*
Abstract: Specific link between high fructose uptake and cancer development and progression
highlighted fructose transporters as potential means to achieve GLUT-mediated discrimination
between normal and cancer cells. The gained expression of fructose-specific transporter GLUT5
in various cancers offers a possibility for developing cancer-specific imaging and bioactive agents.
Herein, we explore the feasibility of delivering a bioactive agent through cancer-relevant fructose-
specific transporter GLUT5. We employed specific targeting of GLUT5 by 2,5-anhydro-D-mannitol
and investigated several drug conjugates for their ability to induce cancer-specific cytotoxicity. The
proof-of-concept analysis was carried out for conjugates of chlorambucil (CLB) in GLUT5-positive
breast cancer cells and normal breast cells. The cytotoxicity of conjugates was assessed over 24 h and
48 h, and significant dependence between cancer-selectivity and conjugate size was observed. The
differences were found to relate to the loss of GLUT5-mediated uptake upon increased conjugate
size and hydrophobicity. The findings provide information on the substrate tolerance of GLUT5 and
highlight the importance of maintaining appropriate hydrophilicity for GLUT-mediated delivery.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Nahrjou, N.; Ghosh, A.;
Tanasova, M. Targeting of GLUT5 for
Transporter-Mediated Drug-Delivery
Is Contingent upon Substrate
Hydrophilicity. Int. J. Mol. Sci. 2021,
22, 5073. https://doi.org/10.3390/
ijms22105073
Keywords: sugar transport; fructose transport; GLUT5; drug conjugates; targeted delivery; cancer
selectivity
1. Introduction
The development of targeted approaches is the ultimate goal to achieve improvements
in disease diagnostics and treatment. The chronic proliferation of cells representing the
essence of neoplasia requires rapid consumption of nutrients compared to non-transformed
tissues. Almost a century ago, the difference in the effectiveness of glycolysis and the citric
acid cycle to produce energy or adapt to alteration in glycolysis efficiency (Warburg effect)
was established as one of the characterizations that discriminate cancer cells from normal
Academic Editor: Valentina De Falco
Received: 7 April 2021
Accepted: 8 May 2021
Published: 11 May 2021
cells [1–3]. As a consequence of enhanced energy requirements, higher sugar concentrations
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published maps and institutional affil-
iations.
are also needed for anabolic reactions to continue replication. Higher sugar uptake in
cancers is reflected by the elevated activity and gained expression of facilitative sugar
transporters—GLUTs. GLUTs are not coupled with energy, and sugar translocation across
the cell membrane occurs via gradient-dependent influx and efflux of carbohydrates [4].
Individual GLUT isoforms demonstrate different tissue specificity, substrate specificity,
and kinetic characteristics. Glucose is the predominant one among various carbohydrates
that are transported by GLUTs. In addition to glucose, GLUTs supply cells with galactose,
fructose, and other sugars. The transport kinetics and affinity for sugars differ between
GLUTs, with the majority taking up more than one substrate and selected few showing
substrate specificities [5,6].
The diversity of GLUTs allows for a tissue-specific adaptation of sugar uptake via
regulation of gene expression [7]. The overall differences in GLUT composition between
cells in conjunction with higher sugar consumption in cancer cells have provided a strong
basis to view GLUTs as important therapeutic targets. Significant impact on advancing
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disease detection has been made by the development of the 18F-labeled analog of 2-fluoro-
2-deoxy-D-glucose (2-18F-FDG) to target glucose-transporting GLUTs and its use in the
clinic as a reflector of enhanced glucose uptake and predictor of tumorigenesis and tumor
aggressiveness [8–12]. However, a large number of false-positive results [13] have high-
lighted the lack of required discrimination of cancer cells when targeting glucose GLUTs.
Directing drugs or imaging agents to GLUTs is primarily done through corresponding
glycoconjugates. Glucose conjugates of various bioactive or imaging agents have been
synthesized and studied [14–16]. However, despite a large number of efforts, the exam-
ples of achieved improvements in toxicity and selectivity of bioactive agents due to the
GLUT-mediated uptake are very limited. Limited is also the understanding of factors that
contribute to a broadly observed loss of GLUT-mediated uptake upon derivatization of
sugars.
The growing understanding of GLUTs-disease connections has identified several
transporters that appear to play explicit roles in disease development and progression.
Deregulation of nonspecific glucose and fructose transporter GLUT2 were found to be
linked to obesity and diabetes [17], and high GLUT2 expression is viewed as a prognostic
factor for liver cancers [18]. The glucose transporter GLUT3 gains expression in various
types of cancer while primarily expressed strictly in the brain [7]. Fructose transporter
GLUT5 is associated with various cancers, as well as obesity, fatty liver disease, and
other metabolic deregulations [19]. The expression of nonspecific glucose and fructose
transporter GLUT12 is established for early-stage and late-stage breast cancers [20]. While
these direct links highlight the feasibility of impacting metabolically compromised cells
directly through relevant nutrient transporter(s), the approach has not been explored due
to the limited advancements in specific targeting of GLUTs.
Prior studies in our laboratory have yielded fluorescently labeled glycoconjugates
that showed high specificity and high affinity to GLUT5—fructose-specific transporter
that gains expression in various cancers while tightly regulated in healthy tissues [21].
GLUT5-targeting conjugates were constructed from the 2,5-anhydro-D-mannitol (Man) as
the locked fructofuranose mimic and various coumarins (Cou) as fluorescent moieties. The
resulting ManCou probes (ManCou-CH3 representative in Figure 1) were found useful in
specifically targeting GLUT5 for imaging applications and transport activity evaluation
in vitro in the complete cell culture media, enabling live cell analysis [22,23]. Following
this attainment, we contemplated assessing the tolerance of fructose transporter GLUT5
towards passing a bioactive moiety in a glycoconjugate form and the feasibility of achiev-
ing cancer-specific cytotoxicity. In this manuscript, we summarize our pilot studies on
exploring the feasibility of transporting a bioactive agent through GLUT5 and achieving
cancer-specific cytotoxicity due to the differences in the presence of GLUT5 in cancer vs.
normal cells.

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Figure 1. GLUT5-targeting delivery platforms amenable for late-stage functionalization with bioac-
tive moiety for specific delivery of cytotoxic agents to GLUT5-positive cancer cells.
2. Results
2.1. Synthesis of CLB Conjugates
For this proof-of-concept study, we have used chlorambucil as a bioactive target [24].
Several linkages for conjugating 2,5-anhydro-D-mannitol and CLB were assessed (Figure 2)
.
Those included an amide and ester linkages directly to 2,5-anhydro-D-mannitol as well
as analogous linkages to a ManCou conjugate that have been showing higher affinity to
GLUT5 than 2,5-anhydro-D-mannitol. The two types of linkages were explored due to
the feasibility of such transformation to be used as the latest stage functionalization in the
drug conjugate synthesis. In addition, the “click” reaction was also explored for late-stage
insertion of the bioactive agent.
Figure 2. Structures of ManCou-CH3, chlorambucil, mannitol, mannitolamine, and chloambucil glycoconjugates.

Int. J. Mol. Sci. 2021, 22, 5073
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Scheme 1 describes the synthesis of two starting sugars to target GLUT5. 2,5-Anhydro-
D-mannitol (mannitol) and 1-amino-2,5-anhydro-D-mannitol (mannitol amine) were both
synthesized from D-(+)-glucosamine according to reported synthetic procedures [22]. The
conversion to mannitol amine went over three steps with ~45% overall yield. The con-
version to mannitol went over two steps with ~72% overall yield. All compounds were
purified by silica gel column chromatography, and their structures were confirmed through
NMR analysis. MS data are provided for all new compounds. High resolution MS (HRMS)
is provided for final conjugates I–IV.
Scheme 1. Synthesis of 2,5-anhydro-D-mannitol and 1-amino-2,5-anhydro-D-mannitol.
To obtain conjugates
reaction with N,N-diisopropylethylamine (DIEA), respectively. While mannitol was used
directly to obtain a Man–Ester–CLB conjugate (Scheme 2), protection of hydroxyl group of
I and II, mannitol and mannitol amine were used in conjugation
I
mannitol amine was necessary to avoid off-site conjugation. We employed the strategy of
orthogonal protection to mask hydroxyl groups and continue using the amine functionality
for conjugation. Considering a higher reactivity of NH2 over OH, the amino group was
protected (
tection of the Boc-group revealed a reactive amine (
using DIEA to produce compound 25]. The final one-step Zemplen deacetylation [25] of
7 produced the desired amide conjugate II.
4
) in the form of a carbamate (Boc). After acetylation of all hydroxyls (
5), depro-
6) that was further conjugated to CLB
7
[
Scheme 2. Synthesis of CLB and 2,5-anhydro-D-mannitol conjugates I and II via ester (A) and amide (B) linkages.

Int. J. Mol. Sci. 2021, 22, 5073
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Conjugates
I
and II were designed to assess the cargo-carrying capacity of mannitol
as GLUT5-specific ligand. As a next step, we explored the conjugates that could provide
higher affinity compounds—compounds III and IV. Here, we based the design on using
ManCou as a carrier, considering a 150-fold higher affinity of these conjugates to GLUT5
due to the presence of an aromatic moiety [22]. To explore such conjugation, two strategies
were used: (i) to directly conjugate ManCou with CLB through ester linkage and (ii) to
explore click conjugates. Both strategies can introduce the bioactive moiety at the last
step of the chemical synthesis, allowing it potentially to be used for the conjugation of
chemically diverse bioactive compounds.
To obtain conjugate III, we explored the formation of ester linkage through a direct
displacement of a leaving group at the exocyclic methylene of coumarin. For this part, the
7-amino-4-chloromethylcoumarin was synthesized according to the reported procedure us-
ing ethyl 4-chloro-3-oxobutanoate (
of and in acidic conditions, followed by acid-mediated cleavage of N-ethyl formate from
then formed compound (10) has provided the desired 7-amino-4-chloromethylcoumarin
11) in high yield (Scheme 3). The following reductive amination resulted in the correspond-
8) and N-protected 3-aminophenol (9) [26]. Cyclization
8
9
(
ing mannitol conjugate (12) that was used for conjugation with CLB in basic conditions.
After column and HPLC purification, the resulting ManCou–Ester–CLB conjugate III was
obtained in 20% yield.
Scheme 3. Synthesis of ManCou–Ester–CLB conjugate (III).
We further explored a synthesis of conjugate IV. The orthogonal reactivity between
azide and alkyne groups has been demonstrated as a practical synthetic tool for the mod-
ification of carbohydrates, waiving the requirement for the protection of free hydroxyl
groups [27]. So, we proceeded with forming two components for the “click” reaction:
ManCou-azide (14) and alkyne ester of CLB (13) (Scheme 4). Compound 14 was obtained
through the direct displacement of chloride of 12. Alkyne ester of CLB was obtained
through standard DCC-mediated coupling of CLB and propargyl alcohol. For the con-
struction of the final conjugate IV through click reaction, we adapted a literature method
employing an excess of CuI, sodium ascorbate, and DIEA under aqueous conditions [28].
The conjugate IV was obtained in moderate yield on the scale sufficient for further biologi-
cal evaluations.
Overall, both synthetic strategies—direct displacement and click reaction—were
proven to be effective in introducing the bioactive moiety at the last step of the synthesis.
While limited to conjugates of CLB, these strategies can prove effective in the formation of
conjugates of more chemically labile biologically active agents.

Int. J. Mol. Sci. 2021, 22, 5073
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Scheme 4. Synthesis of a click conjugate (IV).
2.2. Assessing GLUT5 Levels in Cells
Considering a strong link between GLUT5 activity and breast cancer [19], we have
selected breast cancer cells for biochemical evaluation of probes IV: GLUT5-positive
I–
breast adenocarcinoma MCF7 cell line, GLUT5-positive human breast invasive ductal
carcinoma MDA-MB-231 cell line, and GLUT5-negative normal breast 184B5 cells.
We used specific labeling of GLUT5 in the cellular membrane with the corresponding
antibody and GLUT5-specific ManCou-CH3 probe to assess the relative membrane levels
and activity of GLUT5, respectively. After immunostaining, fluorescence was recorded
using a confocal microscope and quantified using ImageJ. CTCF values were derived
for each cell (Figure 3A,B). Overall, the analysis of GLUT5 expression in the membrane
reflected a significant difference in GLUT5 levels between three cell lines, with 184B5 cells
showing the minimal presence of the transporter in the membrane. Relative to 184B5,
expression of GLUT5 in the membrane was measured ~5-fold higher for MCF7 cells and
17-fold higher for MDA-MB-231 cells.
We measured GLUT5 transport activity by monitoring the accumulation of fluores-
cence ManCou-H probe in cells. After short 10 min incubation of live cells with 25 µM
ManCou-H probe in complete culture medium, fluorescence was recorded using a confocal
microscope and quantified using ImageJ. The resulting difference in the ManCou-H uptake
reflected the relative activity of GLUT5 between cells. The uptake correlated well with the
differences in membrane GLUT5 levels, with the exception of the MDA-MB-231 cells, where
a higher accumulation of the probe (more active uptake) was observed. Overall, the three
cell lines can be categorized with respect to the expected GLUT5-assisted accumulation of
our bioactive conjugates, with the negligible uptake expected for normal 184B5 cells and
the highest uptake expected for malignant MDA-MB-231 cells.
2.3. Conjugates I–IV Show GLUT5-Dependent Uptake during Short Incubations
We have further assessed whether conjugates
Fluorescent conjugates III IV were directly incubated with GLUT5-positive MCF7 cells,
and GLUT5-negative 184B5 cells. Probe uptake was evaluated through analysis of fluo-
rescent images acquired with a confocal microscope. After incubating cells with 25
I–IV can be taken up through GLUT5.
–
µM
solutions of probes, we found both conjugates to induce significant fluorescence in MCF7
cells but not 184B5 cells (Figure 4A). The uptake of two probes appears to differ in efficiency,
with ester conjugate III inducing >3-fold higher fluorescence than IV. Considering that
both conjugates encompass the same fluorophore, a lesser GLUT5-uptake efficiency ap-
pears to be evident for conjugate IV. With respect to 184B5 cells, we have detected residual
fluorescence with conjugate III and no fluorescence for conjugate IV (image not included).
As neither conjugate was taken up by 184B5 cells, the uptake can be attributed to GLUT5
activity in MCF7 cells.

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Figure 3. Comparative analysis of ManCou uptake and membrane GLUT5 levels in MCF7, MDA-
MB-231 and 184B5 cells. (A) Confocal microscope images of cells treated with ManCou-H (blue
fluorescence) and GLUT5-specific antibody (green fluorescence). Images obtained with 60X objective.
ManCou-H imaged using DAPI filter (ex/em: 405/465 nm), GLUT5 antibody imaged using Alexa488
filter (ex/em: 488/565 nm). Images were recorded at the same laser intensity and exposure time.
(B) Corrected total cell fluorescence (CTCF) derived from fluorescence images after quantification
with ImageJ. CTCF = integrated density
readings).
−
(Area of selected cell X mean fluorescence of background
Figure 4. Uptake analysis for fluorescent conjugates III and IV. (
vs 184B5 cells shows the involvement of GLUT5 in the uptake of III and IV. (
uptake efficiency of III and IV in MCF7 cells. Corrected total cell fluorescence (CTCF) derived from
fluorescence images after quantification with ImageJ. CTCF = integrated density (Area of selected
cell X mean fluorescence of background readings). ( ) Competitive inhibition of ManCou-H (5 M)
uptake by fructose, probe , and probe II (500 M): fluorescence and bright-field/fluorescence overlay
A
) Comparative uptake in MCF7
B) Comparative
−
C
µ
I
µ
images. All fluorescence and bright fields images were obtained using confocal microscope, with 60X
objective using. Florescence images were obtained with DAPI filter (ex/em: 405/465 nm). Images
were recorded at the same laser intensity and exposure time.

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In order to assess the ability of conjugate
I
and II to pass into the cell through GLUT5,
a competitive uptake with ManCou-H probe was carried out. MCF7 cells were incubated
in parallel with ManCou-H, ManCou-H + probe , and ManCou-H + probe II. 100-fold
higher concentration of probe or probe II was used to compensate for the ~150-fold
higher binding of ManCou-H over mannitol to GLUT5 [22]. The analogous inhibition
with fructose has been carried out as a control experiment. After co-incubating 5
ManCou-H with 500 M fructose, probe or probe II, we have observed a significant
I
I
µM
µ
I
decrease in ManCou-H fluorescence, suggesting the direct competition between substrates
and ManCou-H for GLUT5-mediated uptake.
2.4. CLB Conjugates Show Structure-Activity Relationship
With a better understanding of the difference in GLUT5 expression and activity in
selected cell lines, we moved forward with assessing whether the undertaken modification
to deliver a drug to GLUT5 can improve the activity and specificity of CLB. We carried out
a continuous exposure to conjugates
compounds was given at time zero and measured cytotoxicity after 24 h and 48 h using a
96-well plate MTS cell proliferation assay. Concentrations within 0.1–500 M were selected
I–IV and CLB as control (the dose of the bioactive
µ
for the analysis. All five compounds were evaluated in one 96-well plate. Independent
experiments were carried out three times. Each plate contained a drug-free row that was
used for deriving a relative cell growth inhibition.
As summarized in Table 1 and Figure 4 (24 h data presented), all conjugates appear to
induce cytotoxicity equivalent to that of CLB in MCF7 and MDA-MB-231 cells after 24 h or
48 h incubation. Subtle differences can be observed between cytotoxic responses of two
cancer cell lines. Thus, more aggressive MDA-MB-231 cells appear to respond better to
ester conjugates I and III than to CLB, II, or IV. While this can relate to the higher presence
and activity of GLUT5 in MDA-MB-231 cells, the overall differences in cytotoxicity are not
as significant as the differences in GLUT5 levels between cell lines.
Table 1. Cytotoxicity of CLB and conjugates I–IV, IC50 (µM) *.
MCF7
MDA-MB-231
184B5
24 h incubation
CLB
I
II
III
IV
145.2 ± 28.7
179.4 ± 24.3
187.6 ± 34.3
490.9 ± 21.8
196.8 ± 11.8
155.9 ± 22.3
89.9 ± 17.4
194.2 ± 21.9
108.4 ± 29.9
>500
182.5 ± 33.7
>900
200/6 ± 18.2
203.2 ± 33.2
200.4 ± 18.4
48 h incubation
CLB
I
II
III
IV
89.7 ± 12.6
85.9 ± 27.1
103.2 ± 19.3
175.3 ± 10.8
137.6 ± 8.8
114.1 ± 14.4
83.6 ± 18.3
99.3 ± 11.1
104.1 ± 11.7
108.1 ± 15.3
270.4 ± 24.2
>900
380.5 ± 23.7
241.5 ± 13.2
350.7 ± 33.5
* IC50 measured over varied concentrations of bioactive compound in 96-well format using MTS assay. Data are
summarized for n = 3 independent experiments. The IC50 values, were calculated from dose response curves using
Quest Graph
™ IC50 Calculator (AAT Bioquest, Inc., https://www.aatbio.com/tools/ic50-calculator, accessed on
28 April 2021).
While moderately impacting the activity of CLB, sugar conjugation has contributed to
the selectivity of one of the test conjugates—Man–Ester–CLB ( ). Thus, while significant
cytotoxicity was observed for CLB and all probes in MCF7 and MDA-MB-231 cells, normal
breast 184B5 cells were not impacted by Mann–Ester–CLB ( ) conjugate. Cytotoxicity of
other probes (II IV) in the 184B5 cell line was similar to that of CLB (Figure 5). Cyto-
toxicity data for CLB and conjugates IV (1–500 M) in MCF7 (breast adenocarcinoma),
MDA-MB-231 (breast invasive ductal carcinoma), and 184B5 (normal breast) cells after 24 h.
Cytotoxicity data were measured with MTS assay using 1–500 M CLB or probe concentra-
I
I
–
I–
µ
µ

Int. J. Mol. Sci. 2021, 22, 5073
9 of 18
tions. Concentrations are presented as log10. Error bars derived from independent n = 3
repetitions of every experiment.
1.2
1.0
0.8
1.2
1.4
MCF7-24h
MDA-MB-231-24h
184B5-24h
1.0
0.8
0.6
0.4
0.2
0.0
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.6
CLB
CLB
CLB
0.4
Mann-Ester-CLB
Mann-EsterCLB
ManCou-Ester-CLB
Mann-Ester-CLB
ManCou-Ester-CLB
ManCou-Ester-CLB
0.2
0.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Concentration, log10
Concentration, log10
Concentration, log10
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.2
1
MDA-MB-231-24h
MCF7-24h
1.2
1.0
0.8
0.6
0.4
0.2
0.0
184B5-24h
0.8
0.6
0.4
0.2
0
CLB
CLB
CLB
Mann-Amide-CLB
ManCou-Click-CLB
Mann-Amide-CLB
ManCou-Click-CLB
Mann-Amide-CLB
ManCou-Click-CLB
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0
0.5
1
1.5
2
2.5
3
Concentration, log10
Concentration, log10
Concentration, log10
Figure 5. Cytotoxicity data for CLB and conjugates
(breast invasive ductal carcinoma) and 184B5 (normal breast) cells after 24 h. Cytotoxicity data measured with MTS assay
using 1–500 M CLB or probe concentrations. Untreated cells were used as a control to derive a relative % viability.
Concentrations are presented as log10 (1 M treatment concentration is presented as zero point on x-axis). Error bars
I–IV (1–500 µM) in MCF7 (breast adenocarcinoma), MDA-MB-231
µ
µ
derived from independent n = 3 repetitions of every experiment.
2.5. Analysis of CLB Conjugate Hydrophobicity
Hydrophobicity of conjugates I–IV was evaluated by assessing their relative water
solubility by n-octanol/water partition according to the reported protocol [29]. Figure 6
depicts UV spectra obtained for aqueous parts after partition with octanol. As can be seen,
there is a significant decrease in the substrate concentration in aqueous (PBS) extract upon
an increase of the compound molecular mass/carbon content. Namely, while high presence
is detected for the ester conjugate I, compound IV was completely extracted by the octanol.
Hence, conjugates can be arranged in the order of relative hydrophilicity as follows: I > II
> III > IV.
Figure 6. UV absorbance of aqueous phase from octanol/water extraction.

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3. Discussion
While GLUTs have been viewed as important therapeutic targets for several decades,
as yet the progress of specific targeting of GLUTs for drug delivery is minimal [16]. Two
factors that contribute to this limitation are the lack of approaches to target only disease-
relevant transport(s) and a little understanding of transport capacity. We report here
the first study that explores the feasibility of delivering a bioactive cargo through one
cancer-relevant transporter explicitly. We have selected fructose transporter GLUT5 as a
target due to its direct relevance to cancer, tightly regulated expression in normal cells,
and our capability to specifically deliver small fluorescent molecular probes through this
transporter into the cell [22].
Using GLUT5-directing 2,5-anhydro-D-mannitol (mannitol), we have aimed to explore
the feasibility of delivering a bioactive cargo through GLUT5 and achieving cancer speci-
ficity of the cytotoxic response. Chlorambucil (CLB) is DNA-alkylation-inducing nitrogen
mustard used under the trade name Leukeran approved as an effective antineoplastic
agent in clinical treatment against manifold malignant and nonmalignant diseases, namely
chronic lymphatic leukemia, lymphomas, and advanced ovarian and breast carcinomas [24].
Application of CLB has been restricted due to its toxicity arising from the lack of specificity
to tumors. Using chlorambucil (CLB) as a bioactive cargo, we synthesized direct ester and
amide conjugates of 2,5-anhydro-D-mannitol (I and II, Figure 1). To enhance the affinity of
conjugate-GLUT5 interaction, we have introduced an aromatic spacer (coumarin) that has
been previously shown to contribute up to 100-fold to the probe-GLUT5 interaction [22].
The synthetic approaches considered were based on achieving late-stage functionalization
with bioactive moiety and included esterification and “click” conjugation, resulting in
conjugates III and IV (Figure 2).
For biological studies, we have selected three cell lines with differential expression and
activity of GLUT5. MCF7 and MDA-MB-231 cells are breast cancer cell lines showing high
levels of GLUT5 activity [30]. Using immunofluorescence, we have established relative
levels of GLUT5 expression within the membrane for these two cell lines, reporting >3-fold
higher levels in more aggressive MBA-MB-231 cells. Using our GLUT5-specific fluorescent
ManCou-H probe, we have established that the activity of GLUT5 parallels the membrane
expression levels, suggesting that the highest uptake of GLUT5-delivered conjugate would
be expected for MDA-MB-231 cells, followed by MCF7 cells (Figure 3). With only basal
levels of GLUT5 detected in normal breast 184B5 cells, the measured membrane expression
of GLUT5 in MCF7 and MDA-MB-231 cells was, respectively, 5- and 17-fold higher. This
set of cell lines provided a good platform to assess the impact of directing CLB to GLUT5.
We have further assessed whether the synthesized conjugates can be taken up by the
cell through GLUT5. As not all conjugates exhibit fluorescence, two different approaches
were used for the analysis. Fluorescent conjugate III and IV were directly evaluated for
the uptake in GLUT5-positive MCF7 cells and GLUT5-negative 184B5 cells (Figure 4A,B).
After 10 min incubation of cells with 20 µM III or IV, significant levels of probe-induced
fluorescence were observed using a confocal microscope. The fluorescence levels were over
three-fold higher for probe III than for IV. The later could be contributed by the slower
uptake kinetics, as was observed with ManCou probes bearing hydrophobic moieties
at the C4 of a coumarin [22]. In contrast to GLUT5-positive MCF7 cells, there was no
accumulation of fluorescence in GLUT5-negative 184B5 cells. The latter provided direct
evidence for the direct involvement of GLUT5 in the uptake of III and IV over a short
incubation time.
The involvement of GLUT5 in the uptake of non-fluorescent
measuring the ability of these probes to competitively inhibit the uptake of ManCou-H
as a GLUT5-specific probe. We found that co-incubation of ManCou-H with probes and
I and II was assessed by
I
II induced almost compete inhibition of ManCou-H uptake (Figure 4C). The impact was
analogous to that of fructose, suggesting that all tested substrates compete for GLUT5.
After validating the ability of
I–IV to pass through GLUT5, their cytotoxicity was
assessed using an MTS assay. Measuring the level of cell death over a range of concentra-

Int. J. Mol. Sci. 2021, 22, 5073
11 of 18
tions reflected sufficient changes to derive the IC50 values for every conjugate (Figure 5
Table 1). We have observed that cytotoxic response was both time-dependent and cell
type-dependent. Thus, prolonged treatment of cells (48 h) induced a stronger response in
cancer cells but did not impact normal cells. Interestingly, we did not observe any correla-
tion between the relative differences in GLUT5 activity and cytotoxic response for MCF7
and MDA-MB-231 cells. We did, however, observe a remarkable selectivity for conjugate
,
I
. Thus, while being very active in cancer cells, this conjugate showed no cytotoxicity in
184B5 cells after 24 h or 48 h treatment. Conjugates II IV were observed to be cytotoxic for
normal cells, albeit with relatively higher IC50 values.
–
With the same targeting moiety employed in all four test conjugates, the observed
differences in cytotoxicity and selectivity of these conjugates directly highlight the structure-
activity relationship. Stringent requirements to substrate properties have been previously
observed for GLUTs. Earlier observations include the loss for GLUT-mediated uptake
upon functionalization of glucose or fructose hydroxyls [5], highlighting the key role of
specific H-bonding interactions of GLUT-substrate recognition. Lately, more evidence on
requirements to cargo has emerged. Namely, while GLUTs-dependent uptake is evident
for fluorescent coumarin conjugates of glucose or fructose, the lack of uptake was observed
for coumarin conjugates bearing carboxylate moieties, suggesting discrimination against
charged species by GLUTs [22]. Likewise, fluorescein conjugate of mannitol did not show
GLUT-dependent uptake [30].
Considering the fundamental role of GLUTs as transporters of hydrophilic substrates
through a hydrophobic cellular membrane, it is highly feasible that cytotoxicity of conju-
gates II
surmised that the loss in GLUT5-mediated uptake may be driven by enhanced hydropho-
bicity of conjugates II IV as compare to the conjugate . Indeed, measuring the levels of
conjugates in water after octanol/water extraction clearly reflected the lower concentration
of II IV in an aqueous phase compared to conjugate . The hydrophobicity of substrates
increased in the order II III IV, posing the conjugate as most hydrophilic and
–IV in normal 184B5 cells results from the lack of GLUT5-mediated uptake. We have
–
I
–
I
I
<
<
<
I
conjugate IV as least hydrophilic. Respectively, the loss of GLUT5-involvement in the
internalization of hydrophobic conjugates and the shift towards passive diffusion through
the cellular membrane with increased hydrophobicity would be expected to diminish
selectivity between normal and cancer cells.
4. Materials and Methods
4.1. General Methods
All reagents were used as received unless otherwise stated from Sigma-Aldrich (St
Lois, MO, USA), TCI America (New Jersey, NJ, USA), Alfa Aesar (Haverhill, MA, USA),
Ark Pharm (Arlington Heights, IL, USA), or Chem-Impex International (Wood Dale IL,
USA). Analytical TLC was carried out on commercial SiliCycle SiliaPlate® 0.2 mm F254
plates. Preparative silica chromatography was performed using SiliCycle SiliaFlash® F60
40–63
HPLC (high-pressure liquid chromatography) using reverse-phase semi-preparative col-
umn (Phenomenex® Luna® 10 10 mm, Ea). 1H, 13C, and
m C18(2) 100 Å, LC Column 100
µm (230–400 mesh). Final purification of compounds was achieved with Agilent-1200
µ
×
19F NMR spectra were recorded at room temperature with a Varian Unity Inova 400 MHz
spectrometer. CDCl3, CD3OD, and DMSO-d6 were used as solvents and referenced to the
corresponding residual solvent peaks (7.26 and 77.16 ppm for CDCl3, respectively; 3.31 and
49.0 ppm for CD3OD, respectively; 2.50 and 39.52 ppm for DMSO-d6, respectively) [31].
The following abbreviations are used to indicate the multiplicity: s—singlet; d—doublet;
t—triplet; q—quartet; m—multiplet; b—broad signal; app—approximate. The coupling
constants are expressed in Hertz (Hz). The multiplicity of carbon atoms was determined
by DEPT-135 experiment. The high-resolution (HR) MS data (ESI) were obtained using a
Thermo Fisher Orbitrap Elite
™ Hybrid Ion Trap-Orbitrap Mass Spectrometer at Chemical
Advanced Resolution Methods (ChARM) Laboratory at Michigan Technological University.
UV-vis spectra were recorded on a Cary 100 Bio-spectrophotometer from Agilent Technolo-

Int. J. Mol. Sci. 2021, 22, 5073
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gies. Fluorescence imaging was done with EVOS FLAuto inverted microscope. Confocal
images were taken with Olympus FluoViewTM FV1000 using the FluoView software.
RPMI-1640, Penicillin/Streptomycin, FBS (Fetal Bovine Serum), 25% Trypsin-EDTA (1
and PBS (phosphate buffered saline solution) were purchased from Life Technologies (Carls-
bad, CA, USA). MEM Non-Essential Amino Acids 100 were purchased from Quality
×
),
×
Biological (Gaithersburg, MD, USA). Sterile DMSO (25-950-CQC, 250 mL) was purchased
from Sigma-Aldrich (St Lois, MO, USA). MCF7, MDA-MB231, 184B5 cells were purchased
from ATCC (Manassas, VA, USA). MTS assay was performed using CellTiter 96® AQueous
One Solution Cell Proliferation Assay from Promega (Madison, WI, USA). GLUT5 primary
Mouse Monoclonal antibody (sc-271055) was purchased from Santa Cruz Biotechnology
Inc. (Dallas, TX, USA). The secondary antibody Goat anti-Mouse Alexa Fluor Plus 488
(A327230 was purchased from ThermoFisher Scientific (Waltham, MA, USA).
4.2. Organic Synthesis
2,5-Anhydro-2-carbaldehyde-D-mannitol (1) [32]: D-Glucosamine hydrochloride (4.00 g,
18.5 mmol) was dissolved in water (100 mL) and stirred at room temperature for 5 h.
Sodium nitrite (3.19 g, 45.3 mmol) was then added, followed by cautious addition of
Amberlite 120 H+ resin (90 g) by portion. The reaction mixture was maintained on ice
bath for 4 h. The completion of the reaction was confirmed by TLC. After the reaction,
the resin was removed by filtration, and the solution was then neutralized by sodium
carbonate. The remaining solution was vacuum dried, and methanol was added to the
residue to precipitate the inorganic salts. After removing the salts by filtration, the solution
was vacuum dried to get the product as a yellow sticky solid (2.49 g, 70%) that was used
directly without further purification.
((2R,3S,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)methyl 4-(4-(bis(2-
chloroethyl)amino) phenyl)butanoate (I, Man–Ester–CLB) [25]. To a solution of (2R,3S,4S,5R)-
2,5-bis(hydroxymethyl)tetrahydrofuran-3,4-diol(2,5-anhydro-2-carbaldehyde-D-mannitol)
(177 mg, 1.07 mmol) in DMF (5 mL), chlorambucil (252 mg, 0.828 mmol), HOBt (210 mg,
1.60 mmol), DIEA (0.896 mL, 5.35 mmol), and EDC HCl (300 mg, 1.60 mmol) were added
•
and stirred at room temperature for 22 h. The mixture was partitioned between EtOAc
and water (50:50 mL). Organic layer was separated, and aqueous phase was extracted
with EtOAc (2
×
20 mL). Organic phases were combined, washed with brine, dried over
Na2SO4, and concentrated under vacuum. The resulting residue was purified by silica gel
chromatography, using Methanol/DCM (1:4), to yield the target compound
I
(163 mg, 45%)
1
as a syrup: H NMR (400 MHz, CD3OD)
δ 7.46 (t, J = 8 Hz, 2H), 7.11 (t, J = 8 Hz, 2H), 4.99
(m, 1H), 4.51 (m, 2H), 3.94 (m, 1H), 3.21–3.62 (m, 8H), 2.74 (t, J = 4 Hz, 2H), 2.57 (t, J = 4 Hz,
2H), 2.32–2.40 (m, 2H). HRMS (M + H+): calc’d 389.1686, obs’d 389.1679
Tert-butyl(((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)meth-
yl)carbamate (4). To a solution of mannitolamine [22] (480 mg, 3 mmol) in MeOH (10 mL),
TEA (0.3 mL, 6 mmol) and Di-tert-butyl dicarbonate (0.96 g, 4.5 mmol) were added. Reac-
◦
tion was maintained at 47 C for 4 h and at room temperature overnight. After completion,
reaction mixture was concentrated under vacuum and the resulting residue was purified
by silica gel chromatography, using Methanol/DCM (1:9) as eluent, to yield
4 (390 mg,
1
50%) as a white solid: H NMR (400 MHz, D2O)
δ
8.31 (s, 1H), 3.8–3.9 (m, 2H), 3.76 (m, 1H),
3.5–3.65 (m, 2H), 3.04–3.21 (dd, 1H), 1.30 (s, 9H). MS (M + H+, ESI): 264.1
(2R,3R,4R,5R)-2-(Acetoxymethyl)-5-(((tert-butoxycarbonyl)amino)methyl)tetrahydrof-
uran-3,4-diyl triacetate (5) [25]: To a solution of 4 (1.745 g, 6.635 mmol) in pyridine (30 mL),
acetic anhydride (3.80 mL, 40 mmol) was added. The mixture was stirred at room tempera-
ture for 24 h. TLC in EtOAc/Hex (1:4) using vanillin and ninhydrin staining confirmed
the completion of the protection reaction. Pyridine was removed by concentration under
vacuum. The resulting syrup was extracted by EtOAc and water (2
×
15 mL). The organic
extract was washed with brine (1
vacuum to yield compound
×
15 mL), dried by Na2SO4 and concentrated under
1
5
as a dark yellow solid: H NMR (400 MHz, CDCl3),
δ
8.50

Int. J. Mol. Sci. 2021, 22, 5073
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(s, 1H), 5.02–5.10 (m, 3H), 4.96 (m, 1H), 4.00–4.21 (dd, 2H), 3.25–3.50 (dd, 2H), 2.08 (s, 3H),
2.06 (s. 6H), 1.44 (s, 9H). HRMS (M + H+): calc’d 389.1686, obs’d 389.1679.
(2R,3R,4R,5R)-2-(Acetoxymethyl)-5-(aminomethyl)tetrahydrofuran-3,4-diyldiacetate (
6).
To a solution of (0.294 g, 0.75 mmol) in DCM, TFA (0.172 g, 1.5 mmol) was added. The
5
mixture was stirred at room temperature for 30 min. After reaction completion (monitored
by TLC in EtOAc/Hex (1:4) using vanillin and ninhydrin staining) solvent was concentrated
to dryness and the resulting brown residue was used without purification.
(2R,3R,4R,5R)-2-(Acetoxymethyl)-5-((4-(4(bis(2-chloroethyl)amino)phenyl)butanamid-
o)methyl) tetrahydrofuran-3,4-diyl diacetate (7) [25]: To a solution of
in DMF (5 mL), chlorambucil (566 mg, 1.86 mmol), HOBt (0.337 g, 2.79 mmol), DIEA (1.20 g,
9.31 mmol), and EDC HCl (1.20 g, 2.79 mmol) were added and stirred at room temperature
for 22 h. The mixture was partitioned between EtOAc and water (50:50 mL). Organic layer
was separated, and aqueous phase was extracted with EtOAc (2 20 mL). Organic phases
6 (700 mg, 2.42 mmol)
•
×
were combined, washed with brine, dried over Na2SO4, and concentrated under vacuum.
The resulting residue was purified by silica gel chromatography, using Hexane/Ethyl
1
acetate (7:3) as eluent, to yield
7
(129 mg, 40%) as a syrup: H NMR (400 MHz, CD3OD)
δ
8.00 (s, 1H), 6.60 (d, J = 8 Hz, 2H), 7.05 (d, J = 8 Hz, 2H), 5.75 (m, 1H), 5.00–5.20 (m,
2H), 4.07–4.22 (m, 2H), 3.60–3.68 (m, 2H), 2.86–2.94 (dd, 8H), 2.18 (t, J = 4 Hz, 2H), 2.54 (t,
J = 4 Hz, 2H), 2.08 (s, 3H), 2.02 (s, 6H), 1.24 (p, 2H). MS (M + H+, ESI): 575.1.
4-(4-(bis(2-chloroethyl)amino)phenyl)-N-(((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxyme-
thyl)tetrahydrofuran-2-yl)methyl)butanamide (II, Man–Amide–CLB). To the solution of
7
(0.273 mg, 0.474 mmol) in methanol, sodium methoxide (3 mg, 0.074 mmol) was added and
stirred at room temperature for 4 h. The completion of deprotection was verified by TLC
analysis in hexane/ethyl acetate (7:3) as eluent and yielded compound II as a yellowish
1
solid (0.250 g, 99%). H NMR (400 MHz, CD3OD)
δ 8.00 (s, 1H), 6.60 (d, J = 8 Hz), 7.05
(d, J = 8 Hz, 2H), 5.75 (m, 1H), 5.00–5.20 (m, 2H), 4.07–4.22 (m, 2H), 3.60–3.68 (m, 2H),
2.86–2.94 (dd, 8H), 2.18 (t, J = 4 Hz, 2H), 2.54 (t, J = 4 Hz, 2H), 1.24 (p, 2H). HRMS (M + H+):
calc’d 389.1686, obs’d 389.1679
(3-Hydroxyphenyl)carbamic acid ethyl ester (9) was previously synthesized in [33]:
3-Aminophenol (10.0 g, 91.6 mmol) and ethyl acetate (40 mL) were refluxed for 1 h with
vigorous stirring. Ethyl chloroformate (4.4 mL, 45.8 mmol) was then added via addition
funnel over a 30 min. The reaction mixture was refluxed for an additional hour and then
allowed to cool to room temperature. Upon cooling, a grey/white precipitate formed
within the flask. The precipitate was removed via filtration and washed with ethyl acetate
(3
×
100 mL). The combined filtrates were concentrated to afford the target compound as a
1
grey solid (9.50 g, 58%) that was used without further purification. H NMR (400 MHz,
DMSO-d6): δ, 9.45 (s, 1H), 9.29 (bs, 1H), 7.04–7.00 (m, 2H), 6.86–6.84 (m, 1H), 6.39–6.36
(ddd, J1 = 1.2, J2 = 2.4, J3 = 8.0, 1H), 4.12–4.07 (q, J = 7.2, 2H), 1.24–1.21 (t, J = 7.2, 3H).
Ethyl (4-(chloromethyl)-2-oxo-2H-chromen-7-yl)carbamate (10) was previously syn-
thesized in [34]: (3-Hydroxyphenyl) carbamic acid ethyl ester (9) (2.00 g, 11 mmol) and
ethyl 4-chloro-3-oxobutanoate (2.17 g, 13.20 mmol) were added to a 100 mL round-bottom
flask equipped with a stir bar. 70% H2SO4 (60 mL) was added, and the mixture was stirred
at room temperature for 4 h. The reaction mixture was then poured over 100 mL of crushed
ice to give a bright yellow precipitate. The solid was filtered and washed several times
with distilled water to remove acid. It was left for several hours to dry and then yellowish
1
white precipitate was collected (2.48 g, 80%). H NMR (400 MHz, DMSO-d6):
δ 10.14 (s,
1H), 7.73–7.71 (d, J = 2.4, 1H), 7.56 (m, 1H), 7.36–7.39 (m, 1H), 6.48 (s, 1H), 4.94 (s, 2H),
4.12–4.14 (q, J = 7.2, 2H), 1.20–1.24 (t, J = 7.2, 3H).
7-Amino-4-(chloromethyl)-2H-chromen-2-one (11) was previously synthesized in [34]:
(2.48 g, 8.52 mmol) of compound (10) was added to a 50 mL round-bottom flask, followed
by H2SO4 conc. (8 mL) and glacial AcOH (8 mL). The reaction mixture was heated to
125 ◦C for 2 h under reflux condenser, after which it was cooled to room temperature and
poured over 300 mL of crushed ice. The resulting suspension was neutralized with 4M
KOH, affording a yellow precipitate. The precipitate was filtered and collected to give the

Int. J. Mol. Sci. 2021, 22, 5073
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1
target compound as a fine yellow powder (1.40 g, 80%). H NMR (400 MHz, DMSO-d6):
δ
10.14 (s, 1H), 7.43–7.45 (d, J = 2.4, 1H), 6.54–6.56 (m, 1H), 6.41 (s, 1H), 7.14–7.19 (m, 1H),
6.48 (s, 1H), 4.34 (s, 2H).
4-(Chloromethyl)-7-((((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-
2-yl)methyl)amino)-2H-chromen-2-one acetic acid salt (12) [35]: 2,5-anhydro-2-carbaldehyde-
D-mannitol (800 mg, 4.8 mmol) and compound 11 (500 mg, 2.40 mmol) were dissolved
in methanol (40 mL). AcOH (4.0 mL) was added to adjust the pH to <6, and the mixture
was stirred at room temperature for 10–15 min, followed by portion-wise addition of
NaBH3CN (4 x 120 mg, 1.92 mmol, every 30–40 min). The reaction mixture was stirred
at room temperature for overall 24 h. The mixture was concentrated to dryness under
reduced pressure, absorbed on silica gel, and purified by column chromatography on silica
gel eluting with 0%
acid salt, yellow foam in 90% yield (840 mg): H NMR (400 MHz, CD3OD):
−
10% methanol in CH2Cl2. The product was obtained as an acetic
1
δ 7.45–7.47 (d,
J = 9.2, 1H), 6.6–6.68 (dd, J1 = 2.4, J2 = 8.8, 1H), 6.48 (d, J = 2.4, 1H), 6.13–6.15 (d, J = 2.4, 1H),
5.45 (s, 1H), 4.71 (m, 2H), 3.93–3.96 (m, 2H), 3.87–3.88 (m, 1H), 3.60–3.69 (m, 2H), 3.50 (s,
1H), 3.29 (m, 2H). MS (M + H+): 356.1
(7-((((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)methyl)-am-
ino)-2-oxo-2H-chromen-4-yl)methyl 4-(4-(bis(2-chloroethyl)amino)phenyl)butanoate (III,
ManCou–Ester–CLB): to the solution of 12 (234 mg, 0.66 mmol) in dry DMF (5 mL) under
argon, chlorambucil (200 mg, 0.66 mmol) and DMAP (80 mg, 0.66 mmol) was added, and
the mixture was stirred at room temperature for 24 h. The mixture was concentrated and
purified by column chromatography, using methanol/CH2Cl2 up to 30% to yield III as a
1
light-yellow syrup. H NMR (400 MHz, CD3OD):
δ 7.37–7.39 (d, J = 9.2, 1H), 6.95–7.05 (m,
2H), 6.60–6.70 (m, 2H), 6.55 (d, J = 4, 1H), 6.05 (m, 2H), 5.22 (s, 2H), 4.90 (m, 4H), 4.61 (m,
1H), 3.93–3.96 (m, 1H), 3.69 (s, 1H), 3.61–3.63 (m, 8H), 3.54–3.56 (m, 2H), 3.29–3.32 (m, 2H),
2.40–2.44 (dd, 2H), 2.50–2.54 (dd, 2H), 1.81 (m, 2H). HRMS (ESI): m/z [M + H]+ calc’d for
C30H36Cl2N2O8: 622.18487; found: 622.18446.
Prop-2-yn-1-yl 4-(4-(bis(2-chloroethyl)amino)phenyl) butanoate (13) [36]: Prop-2-yn-
1-ol (55 mg, 0.985 mmol) was dissolved in DCM (10 mL) and cooled in an ice bath. Chlo-
rambucil (200 mg, 0.657 mmol), DMAP (10 mg, 0.065 mmol), and DCC (1 mL, 0.985 mmol)
were added to the cold solution, the mixture was purged with argon and stirred in a closed
system for 24 h at room temperature. The final mixture was concentrated and purified
by column chromatography on silica gel, using EtOAc/Hex up to 10% to extract the title
1
product in 100% yield (228 mg). H NMR (400 MHz, CDCl3):
δ 7.06–7.08 (d, J = 8, 2H),
6.63–6.65 (d, J = 8, 2H), 4.67 (s, 2H), 3.68–3.72(m, 4H), 3.61–3.64 (m, 4H), 2.55–2.59 (t, J = 8,
2H), 2.35–2.39 (t, J = 8, 2H), 2.47 (t, J = 4, 1H), 1.91–1.95 (p, J = 8, 2H). MS (M + H+): 342.1.
4-(Azidomethyl)-7-((((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-
2-yl)methyl)amino)-2H-chromen-2-one (14) [22]: To the solution of 12 (2.3 g, 11.0 mmol) in
dry DMF (30 mL) under argon, NaN3 (2.85 g, 43.8 mmol) was added, and the mixture was
stirred at room temperature for 4 h. The mixture was diluted with EtOAc (400 mL), washed
with brine (4
residue was absorbed on silica gel and purified by column chromatography on silica gel,
eluting with 0 10% methanol in CH2Cl2. The resulting solid was suspended in CH2Cl2,
×
100 mL), and dried with MgSO4. After filtration and concentration, the
−
followed by addition of hexanes and filtered, washing with hexanes, to provide the title
compound as a dark yellow solid in 78% yield (1.86 g). Prolonging the reaction time was
1
found to be detrimental for yields. H NMR (400 MHz, DMSO-d6): δ, 7.37–7.35 (d, J = 8.4,
1H), 6.58–6.55 (dd, J1 = 2.0, J2 = 8.8, 1H), 6.43 (d, J = 2.0, 1H), 6.23 (bs, 2H), 6.05 (s, 1H),
4.69 (s, 2H) ppm. HRMS (ESI): m/z [M + H]+ calc’d for C10H9N4O2: 217.07257; found:
217.07222.
(1-((7-((((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)methyl)-
amino)-2-oxo-2H-chromen-4-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl4-(4-(bis(2-chloroethyl)-
amino)phenyl)butanoate (IV, Man–Click–CLB) [28]: The salt 14 (105 mg, 0.249 mmol) was
dissolved in acetonitrile/water 1:1 mixture (v/v, up to 4 mL) and the solution was purged
with argon for few minutes. Propargyl ester (13) (114 mg, 0.274 mmol) was added, fol-

Int. J. Mol. Sci. 2021, 22, 5073
15 of 18
lowed by CuI (75 mg, 0.373 mmol), sodium L-ascorbate (750 mg, 3.735 mmol), and DIEA
(129 mg, 3.735 mmol). The headspace of the vial was purged with argon, and the resulting
suspension was stirred vigorously at room temperature for 1 h. The product mixture
was extracted by ethyl acetate (3
under vacuum. The mixtures were then concentrated to dryness under reduced pressure,
absorbed on silica, and purified by column chromatography on silica gel using 0 40%
methanol in CH2Cl2 mixtures. Compound IV was obtained in 72% yield. HRMS, 1H NMR
(400 MHz, CD3OD): 8.11 (s, 1H), 7.48–7.50 (d, J = 8, 1H), 6.96–6.98 (d, J = 8, 2H), 6.63–6.65
×
10 mL) dried over MgSO4, filtered and concentrated
−
δ
(d, J = 8, 2H), 6.50–6.51 (d, J = 8, 1H), 5.80 (s, 1H), 5.47 (s, 1H), 5.18 (m, 4H), 4.83 (m, 3H),
3.97–3.98 (m, 1H), 3.91–3.92 (m, 1H), 3.85 (m, 1H), 3.68–3.69 (m, 4H), 3.62–3.63 (m, 4H),
3.40–3.44 (m, 1H), 3.28–3.33 (m, 2H), 2.44 (m, 2H), 2.29 (m, 2H), 1.82 (m, 2H), 1.27 (m, 2H).
HRMS (ESI): m/z [M + H]+ calc’d for C10H9N4O2: 703.21757; found: 703.21744.
4.3. Fluorescence Analysis
Analysis of GLUT5 uptake with ManCou-H probe and probes III and IV: Cells were
grown in their respective media, plated (300,000/plate) in 35 mm glass-bottom confocal
dishes (MatTek) and allowed to grow in their respective growth media for 24 h. For treat-
ment, cell media was removed and 25
added. Cells were incubated with probes at 37 C for 10 min. After incubation, probe solu-
µ
M pro◦be solution in complete media (1 mL) was
tion was removed, and cells were washed with warmed PBS (3
of PBS for imaging. Cell images were taken using Olympus FluoViewTM FV1000 using the
FluoView software. 60 oil suspended lens was used to observe fluorescent activity under
DAPI (excitation 405 nm (45% intensity), emission 450/490 nm (30% intensity), 10 s/pixel.
×
1 mL) and leaving 1 mL
×
µ
Analysis of GLUT5 membrane expression through immunofluorescence: Cells were
grown in their respective media, plated (300,000/plate) in 35 mm glass-bottom confocal
dishes (MatTek), and allowed to grow in their respective growth media for 24 h. At the end
of 24 h, the cells were fixed with 4% PFA for 20 min followed by PBS washes (3 × 2 mL
for a total of 15 min. The cells were then protein blocked using bovine serum a◦lbumin
followed by incubation with primary antibody at a dilution of 1:200 overnight at 4 C. The
samples were further incubated with secondary antibody for 2 h at a dilution of 1:1000 and
)
fluorescence was imaged using Olympus FluoView FV1000 confocal microscope. 60
suspended lens was used to observe fluorescent activity with Alexa488 filter.
×
oil
Corrected total cell fluorescence (CTCF) was derived for every sample after flu-
orescence quantification with ImageJ. CTCF = Integrated Density
cell × Mean fluorescence of background readings).
−
(Area of selected
4.4. Competitive Inhibition Assay
Cells were grown in their respective media, plated (300,000/plate) in 35 mm glass-
bottom confocal dishes (MatTek) and allowed to grow in their respective growth media for
24 h. Separate dishes were incubated with the following: 5
complete growth media, a solution of 5 M ManCou-H and 500
growth media, a solution of 5 M ManCou-H and 500 M probe I in a complete growth
M probe II in a complete growth media.
µM ManCou-H solution in a
µ
µM fructose in a complete
µ
µ
media, and a solution of 5
Cells were incubated with probes at 37 C for 10 min. After incubation, treatment solution
µ
M ManCou-H and 500
µ
◦
was removed, and cells were washed with warmed PBS (3
×
1 mL) and leaving 1 mL of
PBS for imaging. Cell images (fluorescence and bright field) were taken using Olympus
FluoViewTM FV1000 using the FluoView software. 60X oil suspended lens was used
to observe fluorescent activity under DAPI (excitation 405 nm (45% intensity), emission
450/490 nm (30% intensity), 10 µs/pixel. Experiments were carried out in duplicates.
4.5. Cell Viability Assay
For biological studies, compounds I–IV were purified by reverse-phase HPLC to
achieve 99.9% analytical purity using ACN:Water. 20–70% ACN gradient for I and II and
40–90% ACN gradient was used for III and IV.

Int. J. Mol. Sci. 2021, 22, 5073
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For microplate assays, cells were seeded in 96-well flat bottom plates (10,000 cells/well)
in 150
µ
L of culture medium and allowed to grow for 24 h. Cells were then treated with
◦
probes (concentration varies) in Media and incubated at 37 C and 5% CO2 for 24, 48, 72 h.
After each incubation time, 20 µL of MTS reagent were added and then incubated for 2 and
half hours again. According to the MTS suggested protocol, the absorbance of the data was
immediately collected using automated UV 96-well plate reader at 490 nm wavelength.
Cell viability was calculated as a relative decrease in the absorbance with respect to the
untreated control: Viability, % = (ATreatment
Dose response curves are plotted using Viability, % over log10 of a concentration. The
initial zero-point on the x-axis corresponds to all 1 M concentration treatment. The IC50
IC50 Calculator,
−
AControl)
×
100 (where, A = absorbance).
µ
values, were calculated from dose response curves using Quest Graph
™
the updated AAT-Bioquest® online calculator tool (AAT Bioquest, Inc., https://www.
aatbio.com/tools/ic50-calculator, accessed on 28 April 2021).
4.6. Determination of Water Solubility via Octanol–Water Buffer Partitioning
All samples were guaranteed to be of the same weight. Octanol–water buffer partition
was performed according to an OECD Guideline for the Testing of Chemicals with a
modification using equimolar amounts of compounds [29]. A 4 mL portion of 20 mM pH
7.4 PBS and 4 mL n-octanol were introduced into a 10 mL centrifuge tube and mixed to
ensure equilibration of the PBS and octanol on a shaker for 24 h. Samples were introduced in
equimolar amounts and vortexed for 3 min. After the vortexing, the tubes were centrifuged
for 5 min with the rotation speed at 3000 rpm. The lower water layer was then separated,
and UV-vis absorbance was recorded.
5. Conclusions
Our studies represent the first proof-of-concept of specific targeting of one disease-
relevant sugar transporter with bioactive conjugates. We have shown that targeted delivery
of a bioactive cargo through one cancer-relevant transporter may prove efficient in inducing
cancer-specific cytotoxic response. Through structure-activity analysis, we have shown
that the outcomes of specific delivery depend on the ability of the conjugate to maintain
GLUT5-mediated passage long-term and that this passage is contingent upon the overall
hydrophilicity of the conjugate. The outcomes of these studies provide additional insight
into the overall requirements towards bioconjugates for specific delivery through GLUT
transporters.
Author Contributions: Investigation, N.N. and A.G.; project administration, M.T.; conceptualization,
M.T. and N.N.; methodology, N.N. and A.G.; validation, M.T., N.N. and A.G.; formal analysis, M.T.;
investigation, N.N. and A.G.; writing—original draft preparation, N.N.; writing—review and editing,
M.T.; supervision, M.T.; project administration, M.T.; funding acquisition, M.T. All authors have read
and agreed to the published version of the manuscript.
Funding: This research has been funded by the NIH-R15-AREA grant to M.T. (GRANT12736422).
Acknowledgments: All NMR and MS data were collected in the NMR and MS facility at the Michigan
Technological University. All biological data were collected at the shared CIF facility, Michigan
Technological University.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
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