Synthesis and cellular uptake of carbamoylated mannose derivatives

Article abstract of DOI:10.1016/j.carres.2019.06.008

A series of 3-carbamoyl- and 2,3-dicarbamoyl-mannose derivatives were synthesized, conjugated to a fluorescent dye (Cy5^GE, AF 647 or NBD) and their cellular uptake in A549 and THP-1 cell lines was studied by FACS. In contrast to earlier studies on carbamoyl mannosides, the observed uptake was not related to carbamoyl group on the mannose residue but rather to the cyanine dye attached, a trend previously observed for Cy5-fructose conjugates. The NBD-conjugates however, showed a temperature and concentration dependent uptake in case of mannose conjugates. These results suggest a profound impact of the dye which should be taken into consideration when studying the uptake of small molecules by dye conjugation.

Full text of DOI:10.1016/j.carres.2019.06.008

Accepted Manuscript
Synthesis and cellular uptake of carbamoylated mannose derivatives
Hidde Elferink, Kim Geurts, Stijn Jue, Somhairle MacCormick, Gerrit Veeneman,
Thomas J. Boltje
PII:
S0008-6215(19)30303-9
https://doi.org/10.1016/j.carres.2019.06.008
CAR 7729
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 14 May 2019
Revised Date: 14 June 2019
Accepted Date: 14 June 2019
Please cite this article as: H. Elferink, K. Geurts, S. Jue, S. MacCormick, G. Veeneman, T.J. Boltje,
Synthesis and cellular uptake of carbamoylated mannose derivatives, Carbohydrate Research (2019),
doi: https://doi.org/10.1016/j.carres.2019.06.008.
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Synthesis and Cellular Uptake of Carbamoylated Mannose Derivatives
Hidde Elferink[a]‡, Kim Geurts[a]‡, Stijn Jue[a], Somhairle MacCormick[b], Gerrit Veeneman[b], Thomas J.
Boltje[a]*
[a] Radboud University, Institute for Molecules and Materials, Heyendaalseweg 135, 6525 AJ, Nijmegen,
The Netherlands.
[b] PharmaCytics B.V. Noviotech Campus, Transistorweg 7-N, 6534 AT, Nijmegen, The Netherlands.
* Corresponding author, e-mail: t.boltje@science.ru.nl
‡These authors contributed equally
ABSTRACT: A series of 3-carbamoyl- and 2,3-dicarbamoyl-mannose derivatives were synthesized,
conjugated to a fluorescent dye (Cy5GE, AF 647 or NBD) and their cellular uptake in A549 and THP-1
cell lines was studied by FACS. In contrast to earlier studies on carbamoyl mannosides, the observed
uptake was not related to carbamoyl group on the mannose residue but rather to the cyanine dye attached,
a trend previously observed for Cy5-fructose conjugates. The NBD-conjugates however, showed a
temperature and concentration dependent uptake in case of mannose conjugates. These results suggest a
profound impact of the dye which should be taken into consideration when studying the uptake of small
molecules by dye conjugation.
Keywords: Glycoconjugates, Fluorescence Activated Cell Sorting, Cancer Targeting, Bleomycin

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1. INTRODUCTION:
The selective uptake of diagnostics and cytostatics in cancerous tissue is one of the main challenges in
cancer diagnosis and therapy, respectively. A number of cancers can be targeted by utilizing their aberrant
carbohydrate uptake and metabolism e.g. overexpressing of glucose transporter GLUT-1[1] and the
enzymes involved in the anaerobe glycolysis related to the Warburg effect.[2-4] For example, positron
emission tomography (PET) imaging using 2-deoxy-2-18fluoro-
D-glucose (FDG)[5] is frequently used to
diagnose cancers that display a heightened glucose metabolism. In addition, it was discovered that the
carbohydrate portion of bleomycin, a natural product used to treat various cancers, enables its selective
uptake in cancerous cells.[6] The carbohydrate portion of bleomycin is an unusual disaccharide consisting
of 3-carbamoyl-mannose (1 α-linked to ʟ-gulose (Figure 1).
→3)-
Figure 1: Studies on the selective uptake of 3-carbamoyl mannoside conjugates. Hecht and coworkers (above) showed selective
uptake of Cy5GE-coupled monocarbamoyl mono- and disaccharides in cancerous cells using a fluorescent microscopy-based
assay. This work explores the synthesis and cellular uptake of dicarbamoyl monosaccharides using fluorescence activated cell
sorting (FACS) in live cells.
Hecht and co-workers showed that microbubbles coated with bleomycin where adherent to MCF-7 human
breast carcinoma cells whilst microbubbles coated with bleomycin lacking the disaccharide were not.[7]
Critically, non-cancerous MCF-10A breast cells were not affected. Subsequent studies showed that the

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disaccharide portion itself was sufficient to enable selective uptake using disaccharides conjugated to
fluorescent dyes to assay cellular uptake.[8] Cellular uptake of the disaccharide conjugates was
temperature dependent and much higher at 37oC compared to 4oC indicating ATP-dependent uptake.
Furthermore, the induction of increased glycolysis flux in healthy cells using mitochondrial complex 1
inhibitor rotenone[9] led to enhanced uptake. Conversely, inhibition of glucose transporter GLUT1 with
cytochalasin B and phloretin led to a decrease in uptake hinting at the involvement of GLUT transporters.
A later study with six different cancerous cell lines showed that the minimal epitope for the targeting and
uptake in cancer cells is the 3-carbamoyl mannose residue.[10] When the 3-carbamoyl group was
removed, the cellular uptake was significantly lower indicating that the carbamoyl group is crucial for the
recognition and internalization.[10] Finally the position and substitution (Figure 1, highlighted in blue) of
the carbamoyl functionality in the bleomycin disaccharides was explored.[11] From these studies it
became clear that N-methyl carbamoyl group at the 2- or 3-position of mannose showed even greater
uptake than the parent 3-carbamoyl mannose.
Intrigued by these results we set out to investigate the uptake of a 2,3-dicarbamoyl mannose reasoning
that there may be a synergistic effect when installing two carbamoyl groups at once. In addition, as
determination of the uptake by confocal microscopy on fixed cells is laborious and does not allow for
high throughput screening, we opted for a more convenient method on living cells. Herein we report the
synthesis of 3-carbamoyl- and 2,3-dicarbamoyl-mannose derivatives and their conjugation to three
different fluorescent dyes. Cellular uptake was assayed by fluorescence activated cell sorting (FACS).
2. RESULTS and DISCUSSION:
2.1 Chemical Synthesis
To prepare carbamoylated mannose derivatives 9-10 in a divergent manner from known thioglycoside 1
we developed the synthetic route shown in Scheme 1. Deacetylation of 1 followed by installation of a 4,6-

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benzylidene afforded 3 in 48% yield after crystallization. Acetylation of the 2,3-diol was carried out to
ensure α-selectivity in the ensuing glycosylation. Glycosylation of 2-(2-azidoethoxy)-ethanol using 4[12]
and the NIS/TfOH promoter system afforded mannoside 5 in 64%.[13] Subsequent deacetylation afforded
2,3-diol 6 which was used to prepare carbamoylated derivatives 7-8. An unmodified mannose derivative
10[14] used for control experiments was prepared first by global deprotection of 6 using Pd/C and H2. 3-
carbamoyl-mannose derivative 7 was prepared by a regioselective reaction with benzylisocyanate.
Removal of the protecting groups was achieved using hydrogenolysis (Pd/C, H2) to afford 3-carbamoyl-
mannose derivative 9[15]. 2,3-dicarbamoylation using benzylisocyanate proved more difficult to achieve.
Fortunately, using trichloroacetyl (TCA) isocyanate dicarbomoylation was possible and after removal of
the TCA-groups using K2CO3 and MeOH 8 was obtained. Hydrogenolysis (Pd/C, H2) of 8 finally afforded
deprotected compound 11.
Scheme 1: Synthesis of mannoside conjugates 9-11: i) MeOH, K2CO3; 2, 99%; 6, quant.; ii) PhCH(OMe)2, HBF4.Et2O, DMF; 3,
48%; iii) pyridine, Ac2O; 4[12], 92%; iv) HOC2H4OC2H4N3, NIS, TfOH, DCM; 5, 64%; v) DMF, benzyl isocyanate; 7, 23% vi)
N,N-diisopropylethylamine, trichloroacetyl isocyanate, DCM vii) MeOH, K2CO3; 8, 32% over two steps; viii) H2O, tert-butanol,
EtOAc, Pd/C, H2; 9, 35%; 10, 39%; 11, 42%;.
With compounds 9-11 in hand we prepared conjugates with a fluorescent label to assay their cellular
uptake (see scheme 2). To this end, we selected three fluorescent dyes to investigate the influence of the

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dye structure on the uptake. In addition to mannose derivative 10 we also prepared glucose derivative
12[16] (see supporting information, S8-S9) as a control for the cell experiments. Although most uptake
studies involving 3-carbamoyl mannosides published used the Cy5** dye (structure presented supporting
information, S10)[17], we switched to more available dyes since Cy5** has a restricted availability.
Fluorescent dyes Cy5GE and Alexa Fluor 647 (AF 647), also used in related experiments by Bhattacharya
et al.[15], were conjugated to amines 9-12 using their corresponding N-hydroxysuccinimide esters. HPLC
purification afforded pure conjugates used for cellular uptake studies. The nitrobenzoxadiazole
(NDB)[18] dye is also frequently used to track the cellular uptake[19, 20] and hence we prepared
conjugates of this dye as well by reacting 4-chloro-7-nitrobenzofurazan with amines 9-12 to afford
conjugates 20-24.
OH
HO3S
O
HO
HO
O
NHR
O
OH
12: R = H
N
i
i
i
ii
13: R = Cy5GE
17: R = AF 647
21: R = NBD
iii
OH
OH
O
HO
HO
O
O
NHR
N
O
Cy5GE
O3S
9: R = H
ii
14: R = Cy5GE
18: R = AF 647
22: R = NBD
iii
HO3S
SO3H
OH
OH
O
HO
H2N(OC)O
N
O
NHR
O
10: R = H
ii
15: R = Cy5GE
19: R = AF 647
23: R = NBD
HO3S
iii
O
N
OH
O(CO)NH2
O
O3S
AF 647
HO
H2N(OC)O
O
O
NHR
N
N
O
11: R = H
i
NO2
ii
16: R = Cy5GE
20: R = AF 647
24: R = NBD
iii
NBD
Scheme 2: Dye conjugations to carbohydrate conjugates 9-12: i) Cy5GE-OSu, 0.2M aq. phosphate buffer; 13, 79%; 14, 62%; 15,
32%; 16, 26%; ii) AF 647-OSu, 0.2M aq. phosphate buffer; 17, 55%; 18; 69%; 19, 48%; 20, 51%; iii) NBD-Cl, 0.3M aq.
NaHCO3; 21, 15%; 22, 25%; 23, 7%; 24, 10%.

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2.2 Biological Assays
Next, we investigated the cellular uptake of 13-24 in two cancer cell lines (figure 1). We selected two
cancer cell-lines (A549 and THP-1), derived from lung cancer and acute monocytic leukemia patients,
respectively (see supporting information for specific culture conditions). The concentration of conjugates
13-24 was set at 2µM after FACS signal optimization. Cells were incubated for 1 hour at 37oC before
being rinsed with PBS twice and released from the surface by trypsin containing buffer (A549) or
mechanical stress (THP-1). Phosphate buffered albumin (PBA, 1% BSA, 0.02% NaN3 in PBS) was added
followed by propidium iodide (PI) staining (0.1% PI in PBA) to assess cell viability. Cellular uptake was
assayed using FACS based on at least 5000 live cells per measurement. To ensure cell viability during the
FACS experiments, cells were stained with trypan blue after two hours incubation in buffer. No
significant cell death was observed. In addition, the cells were isolated, resolved in medium and showed
adherence and growth to normal speed.
The uptake of the conjugates showed a striking difference depending on the fluorescent group (figure 2,
A-E). Conjugates containing Cy5GE (figure 2, A-C) and AF 647 (figure, D-E) showed a high uptake
regardless of the sugar attached. Internalization of the cyanine-dyes was confirmed by confocal
microscopy studies on conjugate 12-16 (see supporting information), which showed a non-even
distribution throughout the cells, suggesting lysosomal uptake. To assess whether the internalization is
largely mediated by an active process or passive diffusion, the uptake was studied at low temperature. The
uptake of all conjugates was lowered by incubation at 4oC, most pronounced in the AF 647 conjugates
(17-20), hinting at a specific form of uptake. In contrast, previous studies with the monosaccharide 3-
carbamoyl mannose-Cy5** conjugate showed temperature independent uptake[15], suggesting a different
mechanism of internalization. Critically, the expected difference in uptake between 3-carbamoyl mannose
vs mannose itself was not observed and both showed comparable uptake. Additionally, the glucose
control as well as the 2,3-dicarbamoyl mannoside showed equal levels of cellular uptake. Uptake of
conjugates 13-20 therefore seem to stem largely from the fluorescent label and not the sugar attached.

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These results are further emphasized by additional studies with linker-conjugates containing only the dye
and the linker but lacking a monosaccharide which also give significant signal in both Cy5GE and AF 647
(see supporting information, fig. S1). Gambhir and coworkers observed a similar effect in Cy5.5 labelled
fructose for uptake mediated by the human D-fructose transporter (GLUT5). In their case a GLUT5
independent uptake was observed with large fluorophores as Cy5.5.[19] The NBD conjugates 21-24 in
comparison show a much lower signal with most conjugates showing the same signal as the blank control.
Only mannoside conjugate 22 showed modest increased fluorescence levels, suggesting uptake.
Figure 2: Cellular uptake of fluorescent glycosyl conjugates 14-24 in A549 (A-C) and THP-1 (D-E) cancer cell lines. Cells were
incubated at warm (37oC) or cold (4oC) temperature (final concentration in all temperature experiments was set at 2µM).
Fluorescence was normalized according to the blank (H2O).
In general, the NDB dye (figure 2, C) is less bright than dyes Cy5GE and AF 647 and hence provided only
a weak signal at 2µM concentration. We therefore performed experiments at increasing concentrations of
the NDB conjugates (figure 3, A). Whilst the carbamoylated mannosides 23-24 and glucose conjugate 21
showed no increase in uptake upon increasing their concentration, mannose conjugate 22 did show a clear

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concentration dependent increase in fluorescence. Uptake of mannose conjugates 22 was temperature
dependent in A549 and THP-1 cell-lines hinting at an active uptake process. To evaluate the extent of
NDB mannose uptake, we compared the uptake of 22 to NBD-glucosamine (NBDG) a frequently used
NBD conjugated probe to measure glucose uptake.[20] In A549 cells, both mannose conjugate 22 and
glucosamine conjugate 25 performed equally well showing that the uptake of 22 is significant. In THP-1
cells, mannose conjugates 22 outperformed NBDG and was taken up to a much larger extend, which may
be related to the increased expression of the human passive glucose transporter GLUT3 in monocytes.[21,
22] Overall these results indicate that large fluorophores such as Cy5GE and AF 647 may influence the
properties of the small molecules attached, whereas small fluorophores such as NBD are more likely to
show a substrate dependent uptake.
Figure 3: Cellular uptake of fluorescent NBD-glycosides 21-25 in A549 (A-B) and THP-1 (C) cancer cell lines. Fluorescence
was normalized according to the blank (H2O). Final substrate concentrations used in B and C were set at 25 µM.

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3. Conclusion:
In conclusion, we developed a robust synthetic route towards carbamoyl functionalized mannosides for
conjugate synthesis. The mannosides were conjugated to Cy5GE, AF 647 and NBD and their temperature
dependent uptake was studied by FACS in two cancerous cell lines (A549 and THP-1). In contrast to
earlier studies, the uptake was not related to carbamoylation of the conjugates but rather to the cyanine
dye attached. The NBD-conjugates however, gave evidence of a temperature and concentration dependent
uptake in case of mannose conjugates. These results suggest a profound impact of the dye which should
be taken into consideration when studying the uptake of small molecules by dye conjugation.
Acknowledgments
The authors would like to thank Dr. M. Verdoes and F. van Dalen for kindly providing Cy5GE-OSu. Also
Dr. E. Pierson and J. M. A. Hendriks are kindly acknowledged for their help with the confocal
microscopy and the cell culture experiments respectively. The research was financed by a NWO-LIFT
grant awarded to Dr. T. J. Boltje.
Conflict of interest
The authors declare that they have no conflict of interest.

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Graphical Abstract

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•
•
(di)carbamoylated mannosides were synthesized.
The (di)carbamoylated mannose derivatives were conjugated to fluorescent dyes Cy5GE, AF 647
or NBD.
•
•
•
Cellular uptake was assed using FACS and confocal microscopy.
Cellular uptake was dependent on the fluorescent dye used.
Carbamoylation did not result in increased or selective cellular uptake.