Synthetic Fluorinated l -Fucose Analogs Inhibit Proliferation of Cancer Cells and Primary Endothelial Cells

Article abstract of DOI:10.1021/acschembio.0c00228

Fucosylation is one of the most prevalent modifications on N- and O-glycans of glycoproteins, and it plays an important role in various cellular processes and diseases. Small molecule inhibitors of fucosylation have shown promise as therapeutic agents for sickle cell disease, arthritis, and cancer. We describe here the design and synthesis of a panel of fluorinated l-fucose analogs bearing fluorine atoms at the C2 and/or C6 positions of l-fucose as metabolic fucosylation inhibitors. Preliminary study of their effects on cell proliferation revealed that the 6,6-difluoro-l-fucose (3) and 6,6,6-trifluoro-l-fucose (6) showed significant inhibitory activity against proliferation of human colon cancer cells and human umbilical vein endothelial cells. In contrast, the previously reported 2-deoxy-2-fluoro-l-fucose (1) had no apparent effects on proliferations of all the cell lines tested. To understand the mechanism of cell proliferation inhibition by the fluorinated l-fucose analogs, we performed chemoenzymatic synthesis of the corresponding GDP-fluorinated l-fucose analogs and tested their inhibitory activities against the mammalian α1,6-fucosyltransferase (FUT8). Interestingly, the corresponding GDP derivatives of 6,6-difluoro-l-fucose (3) and 6,6,6-trifluoro-l-fucose (6), which are the stronger proliferation inhibitors, showed much weaker inhibitory activity against FUT8 than that of the 2-deoxy-2-fluoro-l-fucose (1). These results suggest that FUT8 is not the major target of the 6-fluorinated fucose analogs (3 and 6). Instead, other factors, such as the key enzymes involved in the de novo GDP-fucose biosynthetic pathway and/or other fucosyltransferases involved in the biosynthesis of tumor-associated glyco-epitopes are most likely the targets of the fluorinated l-fucose analogs to achieve cell proliferation inhibition. To our knowledge, this is the first comparative study of various fluorinated l-fucose analogs for suppressing the proliferation of human cancer and primary endothelial cells required for angiogenesis.

Full text of DOI:10.1021/acschembio.0c00228

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Article
Synthetic Fluorinated L-Fucose Analogs Inhibit
Proliferation of Cancer Cells and Primary Endothelial Cells
Yuanwei Dai, Ruth Hartke, Chao Li, Qiang Yang, Jun O. Liu, and Lai-Xi Wang
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.0c00228 • Publication Date (Web): 15 Sep 2020
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Synthetic Fluorinated L-Fucose Analogs Inhibit
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Proliferation of Cancer Cells and Primary Endothelial Cells
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Yuanwei Dai1†, Ruth Hartke2†, Chao Li1, Qiang Yang1, Jun O. Liu2 and Lai-Xi Wang1,*
1. Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland
20742, USA
2. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of
Medicine, Baltimore, Maryland 21205, USA
† These authors contribute equally to this work.
*To whom correspondence should be addressed. E-mail: wang518@umd.edu
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ABSTRACT
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Fucosylation is one of the most prevalent modifications on N- and O-glycans of glycoproteins and it
plays an important role in various cellular processes and diseases. Small molecule inhibitors of
fucosylation have shown promise as therapeutic agents for sickle cell disease, arthritis and cancer. We
describe here the design and synthesis of a panel of fluorinated L-fucose analogs bearing fluorine atoms
at the C2 and/or C6 positions of L-fucose as metabolic fucosylation inhibitors. Preliminary study of their
effects on cell proliferation revealed that the 6,6-difluoro-L-fucose (3) and 6,6,6-trifluoro-L-fucose (6)
showed significant inhibitory activity against proliferation of human colon cancer cells and human
umbilical vein endothelial cells. In contrast, the previously reported 2-deoxy-2-fluoro-L-fucose (1) had
no apparent effects on proliferations of all the cell lines tested. To understand the mechanism of cell
proliferation inhibition by the fluorinated L-fucose analogs, we performed chemoenzymatic synthesis of
the corresponding GDP-fluorinated L-fucose analogs and tested their inhibitory activities against the
mammalian 1,6-fucosyltransferase (FUT8). Interestingly, the corresponding GDP derivatives of 6,6-
difluoro-L-fucose (3) and 6,6,6-trifluoro-L-fucose (6), which are the stronger proliferation inhibitors,
showed much weaker inhibitory activity against FUT8 than that of the 2-deoxy-2-fluoro-L-fucose (1).
These results suggest that FUT8 is not the major target of the 6-fluorinated fucose analogs (3 and 6).
Instead, other factors, such as the key enzymes involved in the de novo GDP-fucose biosynthetic
pathway and/or other fucosyltransferases involved in the biosynthesis of tumor-associated glyco-
epitopes are most likely the targets of the fluorinated L-fucose analogs to achieve cell proliferation
inhibition. To our knowledge, this is the first comparative study of various fluorinated L-fucose analogs
for suppressing the proliferation of human cancer and primary endothelial cells required for
angiogenesis.
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KEY WORDS: fucosylation, fluorinated L-fucose, fucosyltransferase, cancer, metabolic inhibition
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INTRODUCTION
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Fucosylated glycoconjugates play essential roles in numerous physiological and pathological
processes including inflammation, angiogenesis, immune modulation and tumor metastasis.1, 2 Aberrant
fucosylation has been reported in many cancer types,3 featuring the increased fucosylation of membrane
receptors and the overexpression of fucosylated epitopes, such as the Lewisb, sialyl Lewisa, Lewisy,
sialyl Lewisx, and Globo H carbohydrate antigens (Figure 1). Notably, these fucosylated epitopes are
typical cancer-related antigens associated with tumor progression and metastasis. It has been reported
that enhanced activity of certain fucosyltransferases such as the α1,6-fucosyltransferase (FUT8), which
is responsible for core fucosylation, i.e., α1,6-fucosylation of N-glycans, contributes to the metastasis of
melanoma4 and non-small cell lung cancer.5 A recent report has shown that the fucosylated carbohydrate
antigen, sialyl Lewisa (CA19-9), promotes pancreatitis and pancreatic cancer in mouse models.6 In
addition, fucosylated glycoproteins can serve as important biomarkers for cancer diagnosis and
prognosis.7 For example, the FDA-approved core fucosylated α-fetoprotein (AFP-L3) has been widely
used as a biomarker for early diagnosis of hepatocellular carcinoma (HCC). 8
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1-3
1-2
1-3
1-4
1-2
2-3
R
R
R
1-4
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1-3
Lewisb
sialyl Lewisa
Lewisy
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2-3
1-3
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R
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1-2
Globo H
sialyl Lewisx
Figure 1. Selected examples of fucosylated glycans as cancer-associated antigens
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The addition of L-fucose to glycans and glycoproteins is catalyzed by various fucosyltransferases
(FUTs) using GDP--L-fucose as a donor substrate. 1, 9 Two distinct pathways, the de novo pathway and
the salvage pathway, are involved in the synthesis of GDP-L-fucose in the cell. Fucosyltransferase
inhibitors can down-regulate the activity of FUTs and have shown potentials as therapeutic agents for
cancers. 10-12 Several GDP-L-fucose analogs have been reported as inhibitors of fucosyltransferases. 3, 10
However, application of those inhibitors in biological systems was hampered by the negatively charged
nature of the sugar nucleotide moieties, which have low cell permeability. On the other hand, several
small-molecule fucose analogs, including fluorinated L-fucose analogs, have been developed as
inhibitors of fucosylation.13-17 For example, 2-deoxy-2-fluoro-L-fucose has shown potential therapeutic
efficacy in animal models for treatment of sickle cell disease18 and arthritis19. Paulson and co-workers13
have reported an elegant study using peracetylated 2-deoxy-2-fluoro-L-fucose as a global metabolic
inhibitor of fucosyltransferases for remodeling cell-surface glycans. 2-Deoxy-2-fluoro-L-fucose, 6,6,6-
trifluoro-L-fucose (fucostatin I), and their per-O-acetylated derivatives were also applied as an inhibitor
for core-fucosylation to generate non-fucosylated monoclonal antibodies with enhanced antibody-
dependent cellular cytotoxicity (ADCC) against cancer.14, 17
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Despite wide applications of the 2-deoxy-2-fluoro-L-fucose as a global fucosylation inhibitor, it does
not appear to have significant effects on the proliferation of cancer cells in cell-based assays, even present
at very high concentrations. 13, 20, 21 Indeed, while 2-deoxy-2-fluoro-L-fucose was shown to retard tumor
growth in prophylactic tumor xenograft models, relatively large dosage was required.14, 21 On the other
hand, 6,6,6-trifluoro-L-fucose (fucostatin I) and its per-O-acetylated derivative have been shown to be a
potent inhibitor for core-fucosylation, clearly by primarily targeting the GDP-mannose 4,6-dehydratase
(GMD) in the de novo GDP-fucose biosynthetic pathway,17 but its effects on cancer cells have not been
described. Herein, we describe the synthesis of an array of fluorinated L-fucose analogs bearing fluorine
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atoms at C2 and/or C6 positions of L-fucose, and a study of their effects on cell proliferation in cancer
and angiogenetic cells. Our data revealed that the previously reported 2-deoxy-2-fluoro-L-fucose and 6-
fluoro-L-fucose had only marginally inhibitory activity against proliferation of several cancer cells,
while 6,6-difluoro-L-fucose and 6,6,6-trifluoro-L-fucose analogs showed significant inhibitory activity
against proliferation of human colon cancer and angiogenetic cells. To understand the putative
mechanism of cell proliferation inhibition by the fluorinated fucose analogs, we also synthesized the
corresponding fluorinated GDP-L-fucose analogs and compared their inhibitory activities against the
mammalian α1,6-fucosyltransferase (FUT8). Interestingly, the corresponding GDP derivatives of the
6,6-difluoro-L-fucose and 6,6,6-trifluoro-L-fucose analogs, which are much more efficient inhibitors of
cell proliferation than the 2-deoxy-2-fluoro-L-fucose, showed much less activity in inhibiting FUT8,
implying that the 6,6-difluoro-L-fucose and 6,6,6-trifluoro-L-fucose analogs may achieve their
inhibition of cell proliferation by targeting other components along the fucosylation pathway, concurrent
with the inhibition of FUT8.
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RESULTS AND DISCUSSION
Design. Mammalian cell fucosylation, the attachment of an -linked L-fucose to the
oligosaccharides of glycoproteins and glycolipids, is catalyzed by a respective fucosyltransferase using
GDP-L-fucose as the donor substrate. GDP-L-fucose can be generated in cells through two biosynthetic
pathways: the salvage pathway, in which free L-fucose is converted to GDP-fucose via the catalysis of
fucokinase and fucose pyrophosphorylase, and the de novo pathway where GDP-fucose is generated
from GDP-mannose via the catalysis of GDP-mannose 4,6-dehydratase (GMD) and GDP-4-keto-6-
deoxy-D-mannose-3,5-epimerase-4-reductase (FX).1 Several fluorinated L-fucose analogs, including the
2-deoxy-2-fluoro-L-fucose, the 6-fluoro-L-fucose, and the 6,6,6-trifluoro-L-fucose have been used as
global metabolic inhibitors of fucosylation in cells.13, 14, 17 A prerequisite is that the metabolic inhibitors
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must get into the cells and are converted to the corresponding fluorinated GDP-L-fucose analogs to exert
their inhibitory activities on fucosylation (Figure 2). In the present study, we sought to test a panel of
C2/C6 fluorinated L-fucose analogs (compounds 1-6, Figure 2) for their effects on proliferation of
various cancer cells. So far, no systematic comparative study of fluorinated L-fucose analogs on cancer
cell proliferation has been conducted.
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We decided to use the peracetate of the fluorinated L-fucose analogs as a “pro-drug” for the
present cellular studies, because peracetylated sugar analogs usually have better cell permeability than
the corresponding free sugars and have been frequently used for cellular metabolic incorporation
studies.20, 22-26. It should be noted that the enhancing effects of acetylation on metabolic incorporation
and inhibition of monosaccharide precursors may depend on the sugars and the cellular systems. A recent
comparative study has demonstrated that acetylation of 2-deoxy-2-fluoro-L-fucose and 5-alkynyl-L-
fucose had only moderately enhanced inhibitory effect on the core fucosylation of IgG antibodies
produced in CHO cells.26 When the peracetylated derivatives get into cells, the O-acetyl groups can be
readily removed by the cellular non-specific esterases to provide the free fluorinated L-fucose analogs,
which are then converted to the corresponding fluorinated GDP-L-fucose analogs by fucokinase and
fucose pyrophosphorylase, via the salvage biosynthetic pathway.1 The resulting fluorinated GDP-L-
fucose analogs could exert inhibition of fucosylation by a mechanism of dual action. On the one hand,
the fluorinated GDP-L-fucose could act as a competitive inhibitor of fucosyltransferases. Ample
experimental data have demonstrated that the presence of a fluorine atom proximal to the anomeric
center in the substrate provides a strong destabilizing effect on the oxocarbenium ion transition state
being developed during glycoside hydrolysis and/or glycosyl transfer.27-29 As demonstrated in the
pioneering work from Withers and co-workers, 2-deoxy-2-fluoro-glycosides can act as a class of
mechanism-based inhibitors of retaining glycosidases, which proceed to form a covalently linked
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fluorosugar-enzyme intermediate that cannot proceed further or only react slowly to form product, due
to the destabilizing effect of the proximal fluorine atom on the oxocarbonium ion transition state.27, 30-32
Mammalian -fucosyltransferases are inverting glycosyltransferases that do not form a covalently linked
enzyme-GDP-fucose intermediate, but the fluorinated GDP-L-fucose, which mimics the natural GDP-
L-fucose substrate, can be recognized by the enzyme and form a fluorinated GDP-L-fucose/enzyme
complex at the catalytic center, which might not proceed further for fucosyl transfer or might have only
a slow turnover, due to the destabilizing effect of the proximal fluorine atom(s) on the oxocarbenium ion
transition state. In fact, synthetic GDP-2-deoxy-2-fluoro-L-fucose and GDP-6-fluoro-L-fucose have
been shown to be competitive inhibitors against several fucosyltransferases including FUT3, FUT5,
FUT6, and FUT7, with a Ki of low M.33 On the other hand, the slow turnover of the fluorinated GDP-
L-fucose-enzyme complex results in the accumulation of the fluorinated GDP-L-fucose analog, which
can exert feedback inhibition of fucosylation by targeting the GDP-mannose 4,6-dehydratase (GMD), a
key enzyme in the de novo GDP-fucose biosynthetic pathway, through allosteric binding.33, 34 GDP-L-
fucose feedback inhibition is an important mechanism to regulate cellular fucosylation.34 Allen and co-
workers have solved the crystal structure of GDP-mannose 4,6-dehydratase (GMD) complexed with
synthetic GDP-6,6,6-trifluoro-L-fucose.17 The structural study has revealed that the trifluoromethyl
group is completely buried in a small pocket adjacent to the sugar moiety, and extensive Van der Waals
interactions are observed between the trifluoromethyl group and several amino acid residues in GMD.
Thus, the replacement of the methyl group in the natural substrate GDP-L-fucose with the
trifluoromethyl group leads to increased Van der Waals contacts, which may explain the increased
potency of the precursor 6,6,6-trifluoro-L-fucose in reducing core-fucosylation in cells over the 2-deoxy-
2-fluoro- or 6-fluoro-L-fucose.17 Many studies have demonstrated that, due to the strong
electronegativity and the size similarity of fluorine to hydroxyl group or hydrogen, the introduction of
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fluorine into a molecule at a selective site can influence the membrane permeability, protein and enzyme
recognition, metabolic pathways, and pharmacokinetic properties of the molecule.28, 29 In general, C–F
bond can undergo multipolar interactions with protein’s backbone amide bonds, side-chain amide
residues, and/or the positively charged guanidinium in the binding pocket, resulting in an enhancement
of protein-ligand interactions.35, 36 For example, it has been shown that introduction of a tetrafluorinated
methylene group dramatically strengthens the interactions between a fluorinated UDP-galactofuranose
analog and UDP-galactopyranose mutase.37 Thus, it will be particularly interesting to examine and
compare how the 2-deoxy-2-fluoro-, 6-fluoro-, 2-deoxy-2,2-difluoro-, 2-deoxy-2,6-difluoro-, 6,6-
difluoro- and 6,6,6-trifluoro-L-fucose analogs (compounds 1-6, Figure 2) affect the global fucosylation
and cell proliferation of cancer cells. In addition, we also sought to synthesize the corresponding
fluorinated GDP-L-fucose analogs and test their inhibitory activity against fucosyltransferases such as
FUT8, which may provide insights into the mechanism by which the fluorinated L-fucose analogs exert
their effects.
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De novo pathway
Cytosol
HO
OH
GMD, FX
OGDP
OH
O
O
Golgi
HO
HO
OH
HO
GDP--L-Fucose
OGDP
GDP-D-mannose
Feedback inhibition
Salvage pathway
Fucosyl
transferase
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R4
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R2
Competitive Inhibition
O
Esterases
OAc
3
AcO
3
R4
R5
R
Fucokinase, GFPP
R4
R5
R
OH
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O
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O
R1
OH
HO
F
R2
OH
HO
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GDP
OAc
OAc
OAc
F
OAc
OAc
F
OAc
OAc
F
OAc
OAc
O
F3C
O
O
HF2C
O
O
O
F
F
OAc
OAc
OAc
OAc
OAc
OAc
AcO
AcO
AcO
F
AcO
AcO
AcO
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Figure 2. Fluorinated L-Fucose Analogs as Metabolic Inhibitors of Fucosylation. Peracetylated
analogs of L-fucose (1-6) are up-taken by cells and converted into the corresponding GDP-fucose
analogs through the salvage pathways. The fluorinated GDP-fucose analogs may inhibit fucosylation by
two mechanisms: 1) The fluorinated GDP-fucose analogs can act as a competitive inhibitor of various
fucosyltransferases. The accumulated fluorinated GDP-fucose analogs act as a feedback inhibitor to shut
down the de novo GDP-fucose biosynthesis.
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Synthesis of the Fluorinated L-Fucose Analogs. Among the 6 fluorinated fucose analogs designed to
be examined and compared, 1, 2, 3, and 6 are known compounds, while 4 and 5 are new fluorinated L-
fucose analogs. The peracetylated 2-deoxy-2-fluoro-L-fucose (1) was synthesized by following the
previously reported procedure.38 The peracetylated 6-fluoro-L-fucose (2) was prepared starting from
1,2;3,4-di-O-isopropylidene-L-galactose, as described by Wong and co-workers.33
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Peracetylated 6,6-difluoro-L-fucose (3) has been also synthesized previously. We developed a
modified synthesis of 3, which gave an improved overall yield (Scheme 1, panel A). Oxidation of 7 with
Dess-Martin periodinane gave the aldehyde (8) in 92% yield. Deoxyfluorination was achieved by
treatment of 8 with diethylaminosulfur trifluoride (DAST) 40 to afford 9 in 77% yield. Importantly, slow
addition of DAST to the solution of 8 in dichloromethane with ice-bath cooling was essential to give a
good yield without the generation of the vinyl fluoride by-product. Notably, the long-range coupling of
fluorine and proton was observed in the proton NMR spectrum of 9 between F(6) and H(3) (5JF6, H3 = 1.3
Hz) and between F(6) and H(l) (5JF6, H1 = 1.8 Hz). The removal of isopropylidene groups was achieved
by refluxing 9 with 80% acetic acid to give the tetraol, which was acetylated with acetic anhydride in
pyridine to give the 6,6-difluorofucose derivative (3) in 91% in two steps. Thus, the synthesis of 6,6-
difluoro-L-fucose peracetate (3) was achieved in 4 steps with a 64% overall yield from the known
compound 7. Interestingly, replacement of the methyl group in fucose with the difluoromethyl group
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increased the formation of the furanose isomer. NMR analysis indicated that compound 3 was a mixture
of α,β-isomers of pyranose and furanose, which was in good agreement with the reported data. 39
7
8
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Next, we turned our attention to the synthesis of 2-deoxy-2,2-difluoro-L-fucose derivative (4) from
L-fucose as outlined in Scheme 1, panel B. Sequential electrophilic fluorination strategy was applied to
efficiently introduce the two fluorine atoms at C2 in the process. Acetylation of L-fucose, treatment with
hydrogen bromide in acetic acid, and subsequent zinc-promoted reductive elimination afforded the
diacetyl-L-fucal (10) in 78% yield over three steps. Compound 10 was treated with Selectfluor38 in dry
nitromethane, followed by adding MgBr2 as a nucleophile to furnish 2-deoxy-2-fluoro-L-fucosyl
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bromide (11). Elimination of the anomeric bromide with triethylamine in acetonitrile afforded the
desired vinyl fluoride (12) in 60% yield. Subsequent electrophilic fluorination of 12 by using Selectfluor,
followed by acetylation of anomeric position provided the peracetylated 2,2-difluorofucose (4) in 80%
yield over 2 steps. Interestingly, compound 4 was isolated as predominant α-anomer, which was
1
13
confirmed by H and C NMR analysis. Therefore, we finished the synthesis of 4 from L-fucose in 8
steps with a 28% overall yield.
A number of elegant approaches toward the synthesis of fluorinated sugars haven been reported for
mechanistic and biological studies,28, 29, 42 but fluorinated L-fucose analogs bearing fluorine atoms at
both the 2 and 6-positions of L-fucose have not been described. The synthesis of peracetylated 2-deoxy-
2,6-difluoro-L-fucose (3) was shown in Scheme 1, panel C. Deoxyfluorination and electrophilic
fluorination strategy were used to introduce the fluorine at C6 and C2 of fucose, respectively. The
preparation of the fluorinated precursor (13) was readily achieved through deoxyfluorination of 7 with
DAST in the presence of 2,4,6-collidine in refluxing dichloromethane.33 Subsequent acidolysis followed
by acetylation gave the peracetylated 6-fluoro-L-fucose (2)13, 33 in 92% yield over 2 steps. Treatment of
2 with hydrogen bromide, subsequent reductive elimination afforded 6-fluoro-L-fucal (14) in 85% yield
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in two steps. Subsequent fluorination and acetylation afforded the peracetylated 2,6-difluoro-L-fucose
(5) in 71% yield in 2 steps. Thus, the synthesis of 5 was completed in 7 steps with 42% overall yield
from 7.
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Scheme 1. Synthesis of Peracetylated Fluorinated L-Fucose Analogs 2-5.
(A)
O
O
O
O
H
HO
DAST, CH2Cl2
77%
DMP, CH2Cl2
92%
F2HC
O
O
O
O
O
O
O
O
O
O
O
O
7
8
9
OAc
OAc
OAc
1) 80% HOAc, 94%
2) Ac2O, Py, 97%
O
OAc
F2HC
O
N
F2HC
+
SF3
OAc
AcO
AcO
OAc
(P/P/F/F = 1:0.7:0.5:0.3)
DAST
3
(B)
Br
1) Ac2O, Py
2) HBr-HOAc, DCM
O
OH
OH
1) Selectfluor, MeNO2
O
F
O
3) Zn dust, EtOAc
N-methyl imidazole
2) MgBr2, MeNO2
74% for 2 steps
AcO
OAc
AcO
OH
OAc
HO
78% for 3 steps
10
11
L-Fucose
OAc
1) Selectfluor
2) Ac2O, Py
O
Cl
Et3N, CH3CN
60%
N
O
F
N
AcO
F
80% for 2 steps
(/ = 10:1)
OAc
2BF4
AcO
F
F
OAc
12
Selectfluor
4
(C)
HO
O
O
F
F
OAc
OAc
1) 80% HOAc
DAST, collidine
CH2Cl2, 75%
O
O
O
O
O
2) Ac2O, Py
O
O
O
OAc
O
AcO
92% for 2 steps
7
13
2
O
1) HBr-HOAc, DCM
2) Zn dust, EtOAc,
OAc
1) Selectfluor
2) Ac2O, Py
F
F
O
F
AcO
71% for 2 steps
(/ = 1:0.71)
N-methyl imidazole
85% for 2 steps
OAc
AcO
OAc
5
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Synthesis of Peracetylated 6,6,6-Trifluoro-L-Fucose 6. The first synthesis of 6,6,6-trifluoro-L-fucose
was reported by Toyokuni and co-workers 43 starting from a rare sugar D-lyxose. This approach required
dethioacetalization with stoichiometric amount of HgCl2 and generated a 1:1 mixture of 6,6,6-trifluoro-
L-fucose and its epimer 6,6,6-trifluoro-D-altrose, which was difficult to separate. After that, several
groups reported different approaches to the synthesis of the target molecule.44-46 However, most of the
previous syntheses suffered from low stereoselectivity and/or low yield. Inspired by the elegant synthesis
from Goddard-Borger group46, we initiated a modified synthesis from D-mannose (Scheme 2).
Isopropylidene protection of D-mannose and subsequent Swern oxidation provided mannolactone (15)47
on a 10-gram scale. Treatment of 15 with trifluoromethyl(trimethyl)silane and TBAF afforded a mixture
of diastereomers (16) in a ratio of 3.5:1, which was confirmed by NMR analysis. Next, we examined
several reagents for the stereoselective reduction of hemiketal 16, including NaBH4, LiBH4 and LiAlH4.
We found that NaBH4 in refluxing methanol was the best, which yielded the desired diastereomer 17a
as the major product (17a/17b, 7.2:1). Acetylation of the major diastereomer 17a provided 18 in 98%
yield. Remarkably, periodic acid-promoted regioselective hydrolysis of the isopropylidene group and
simultaneous oxidative cleavage of the resulting diol enabled the transformation of 18 to aldehyde 19 in
a one-pot manner. Removal of the O-acetyl and isopropylidene groups in19 afforded the 6,6,6-trifluoro-
L-fucose (20) in 90% yield in two steps. Global acetylation of free sugar 20 completed the synthesis of
6. Notably, replacement of the methyl group in fucose with the trifluoromethyl group increased the ratio
of the furanose isomer. NMR analysis indicated that compound 20 and 6 were a mixture of α,β-isomers
of pyranose and furanose, which was in consistence with the previously reported observation.45 Thus,
the synthesis of 6 was completed in 9 steps from D-mannose in a 36% overall yield. The structures of
key reaction intermediates and all fluorinated fucose analogs (1–6) were confirmed by 1H, 13C, and 19F
NMR spectroscopy and MS analysis (see the Supporting Information).
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Scheme 2. Synthesis of Peracetylated 6,6,6-Trifluoro-L-fucose 6
4
5
6
7
8
9
O
O
O
HO
HO
HO
OH
1) I2, acetone, 89%
2) Ac2O, DMSO, 92%
>10 g obtained
OH
TMSCF3, TBAF
85%
O
O
O
O
O
CF3
OH
O
O
O
O
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CF3
D-Mannose
15
16
O
O
OH
OH
OH
OH
NaBH4, MeOH, 78%
(S)
O
(R)
O
+
CF3
17a 17b
(
/
= 7.2:1)
CF3
O
O
O
O
17a
17b
O
O
O
O
H
1) NaOMe
OAc
OH
OAc
OAc
CF3
OAc
CF3
OH
H5IO6, EtOAc
85%
Ac2O, Py
98%
O
2) 80% AcOH
(R)
CF3
90% for 2 steps
O
O
O
O
O
O
(PPFF =
1:1:0.52:0.37)
17a
18
19
OAc
AcO
OAc
OAc
OH
OH
OH
O
Ac2O, Py, 98%
OAc
OAc
O
F3C
AcO
O
OH
OH
F3C
+
F3C
O
F3C
(PPFF =
0.66:0.41:0.52:1)
OAc
OH
HO
HO
6
20
Inhibition of Proliferation of Cancer Cells by the Fluorinated Fucose Analogs (1-6). We initially
screened for inhibition of cell viability by the fluorinated L-fucose analogs (1-6) in four different human
cancer cell lines of solid tumors: MDA-MB-231, PANC-1, HeLa, and HCT116, which were derived
from triple-negative breast adenocarcinoma, pancreatic epithelioid carcinoma, cervical adenocarcinoma,
and colorectal carcinoma, respectively. Previous studies investigating cellular inhibition by 2-deoxy-2-
fluoro-L-fucose analogs have used 100-500 μM12, 17 concentrations. To identify more potent hits, we
screened our compounds in each cell line at 100 μΜ and 10 μM for 72 hrs before cell viability was
determined by the Alamar blue assay. We found that inhibition (p < 0.05) in all four cancer cell lines
was demonstrated by peracetylated 6,6-difluoro-L-fucose (3) and peracylated 6,6,6-trifluoro-L-fucose
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(6) with 100 μM treatment (Figure 3). Interestingly, 3 strongly inhibited cell viability in HCT116
colorectal cancer cells by a mean of 75% (Figure 3D), whereas in each of the other cell lines, 6 exhibited
a slightly stronger effect than 3. Of note, peracetylated 6-fluoro-L-fucose (2) showed inhibition of
HCT116 cells, and to a lesser degree, HeLa cells (Figure 3C, D). These results demonstrate that the
analogs fluorinated solely at the C6 position are capable of inhibiting cell viability. In contrast, we did
not observe apparent inhibition by compounds fluorinated only at the C2 position.
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Given that HCT116 colorectal cancer cells exhibited the most sensitivity to C6 fluorinated analogs, we
investigated a dose-dependent effect of the 6-fluorinated L-fucose analogs in this cell line. We tested
compounds 2, 3, and 6 across a range of concentrations and determined the relative IC50 values of analogs
2, 3, and 6. The 6,6-difluoro-L-fucose (3) was found to be the most potent with an IC50 of 43 μM, while
the IC50 values for the 6,6,6-tifluoro-L-fucose (6) and the 6-fluoro-L-fucose (2) were 58 M and 159
M, respectively (Figure 3E).
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MDA-MB-231
PANC-1
A
B
10


M
10
M
1.5
1.0
0.5
0.0
1.5
1.0
0.5
0.0
100
M
100
M
**
*
**
**
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1
2
3
4
5
6
1
2
3
4
5
6
O
O
S
S
M
M
D
D
%
1
%
1
HeLa
HCT116
10 M
10 M
100
C1.5
D
1.5
1.0
0.5
0.0
100
M
M
1.0
0.5
0.0
*
*
**
**
**
**
1
2
3
4
5
6
1
2
3
4
5
6
O
O
S
S
M
M
D
D
%
1
%
1
Figure 3. Screen of fluorofucose Analogs 1-6 in four cancer cell lines. Cells were treated with
fluorofucose analogs for 72 hrs. at concentrations indicated. Viability was examined with Alamar blue
assay. A and B. Cells were inhibited by 100 μM compound 3 and 6. C and D. Cells were significantly
inhibited by both 2, 3 and 6. HCT116 cells experienced dramatic inhibition by both compounds 3 and 6
at 100 μM. * indicates p<0.05, ** indicates p<0.01. Panel E. Comparison of mono, di, and tri-fluorinated
fucose compounds at the C6 position. Dose response curves were generated using a titration of
compounds from 1 mM to 4 μM in HCT116 cells. All C6-fluorinated analogs had a dose-dependent
effect. The difluorinated analog resulted in the most potent IC50 of 43 μM.
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It has been well demonstrated that angiogenesis plays an important role in tumor growth and
metastasis.48, 49 We were therefore interested to know if the synthetic fluorinated L-fucose analogs could
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inhibit the viability of Human Umbilical Vein Endothelial Cells (HUVECs), an in vitro proxy of
angiogenesis. The assays of the effects on the viability of HUVECs were performed as described above
with analogs 1-6. As with HCT116 cells, the analogs fluorinated at the C6 position, including the 6-
fluoro-L-fucose (2), the 6,6-difluoro-L-fucose (3), and the 6,6,6-trifluoro-L-fucose (6), all showed strong
inhibitory activity on HUVEC viability. Once again, analogs 1, 4 and 5, fluorinated at the C2 position,
did not show apparent inhibition of HUVEC at 100 M. Interestingly, HUVEC showed increased
sensitivity to both 3 and 6, which demonstrated significant inhibitory activity at 10 μM (Figure 4).
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Figure 4. Screen of fluorinated L-fucose analogs 1-6 in HUVECs. HUVEC cells were treated with
indicated concentrations for 72 hrs before being analyzed by Alamar blue. Cells were strongly inhibited
by all compounds fluorinated at the C6 position at 100 μΜ. Cells were also significantly inhibited by 10
μM analogs di- and tri-fluorinated on C6. * indicates p<0.05, ** indicates p<0.01, *** indicates
p<0.001, **** indicates p<0.0001.
Our results demonstrated that the peracetylated 6,6-difluoro-L-fucose (3) and 6,6,6-trifluoro-L-
fucose (6) showed significant inhibitory activity against the proliferation of colon cancer cell line, while
the peracetylated 2-deoxy-2-fluoro-L-fucose (1), at 100 M, had no apparent effect on all the cancer cell
lines tested. Our results are consistent with the report described by Paulson and co-workers.13 Their
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experiments have demonstrated that 1 did not inhibit the proliferation of HL-60, Ramos, and CHO-K1
cells at up to 500 µM.13 Korytnyk and co-workers have previously studied the metabolic effects of
halogenated L-fucose and D-galactose analogs on growth, viability, and incorporation of D-
glucosamine, D-galactose, L-fucose, and L-leucine into macromolecular components (proteins and
glycoconjugates) in murine leukemia L1210, murine lymphoma P288, and human mammary tumor
SW613 cells.20 Although the fluorinated L-fucose analogs have been found to effectively inhibit the
incorporation of L-[3H]fucose into the macromolecular components, the 6-fluoro-L-fucose had only
moderate inhibition effect on cell growth of murine leukemia L1210 cells but had no inhibitory effect
on human mammary tumor SW613 cells. This study has also demonstrated that O-acetylation increased
the inhibitory activities of 2-chloro-L-fucose and 2-bromo-L-fucose analogs against murine leukemia
L1210 cells, as acetylated sugars has greater cellular permeability than the free sugars.20 Our
observations that the peracetylated 6-fluoro-L-fucose analog (2) has only marginal inhibitory effect on
the tumor cell lines so far tested are consistent with the reported results. In addition, our results further
indicated that the peracetylated 2-deoxy-2-fluoro-L-fucose (1) also had only marginal inhibitory activity
on HUVECs. In contrast, the 6-fluorinated fucose analogs (2, 3, 6) exhibited significant inhibitory effect
on HUVECs under the same testing conditions.
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Chemoenzymatic Synthesis of Fluorinated GDP-L-Fucose Analogs. To investigate the putative
mechanism of cell proliferation inhibition by the synthetic fluorinated L-fucose analogs (1-6), we
performed chemoenzymatic synthesis of the corresponding fluorinated GDP-fucose analogs and tested
their inhibitory activity on FUT8, the key enzyme responsible for core-fucosylation (1,6-fucosylation)
of N-glycans. Thus, the peracetylated fluorinated L-fucose analogs (1-6) were de-O-acetylated and the
resulting free fluorinated L-fucose analogs were converted into the corresponding fluorinated GDP-
fucose analogs (22-27) using a bifunctional enzyme, L-fucokinase/GDP-fucose pyrophosphorylase (FKP)
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as the catalyst 50 (Scheme 3). We found that most of the fluorinated L-fucose analogs (1, 2, 3, 5, and 6)
were successfully converted into the corresponding fluorinated GDP-fucose analogs (22, 23, 24, 26, and
27) in excellent yields (66-87% in 3 steps). However, the 2-deoxy-2,2-difluoro-L-fucose derivative (4),
after de-O-acetylation, was found to be a very poor substrate for the FKP enzyme, which gave only a
marginal yield for the formation of the corresponding GDP-fucose derivative (25) under the reaction
conditions. This result suggests that the 2-deoxy-2,2-difluoro-L-fucose analog might not be efficiently
incorporated in the salvage biosynthetic pathway in cells to produce the corresponding GDP-fucose
derivative, partly explaining its lack of inhibition of cell proliferation. All the fluorinated GDP-fucose
analogs (22, 23, 24, 26, and 27), except the 2-deoxy-2,2-difluoro-L-fucose analog (25) that was not
produced in sufficient amount, were purified by gel filtration chromatography and characterized by NMR
and MS analysis. The GDP-2-deoxy-2-fluoro-L-fucose (22),33 the GDP-6-fluoro-L-fucose (23) 33, 50, and
the GDP-6,6,6-trifluoro-L-fucose (27)17 have been synthesized previously by different methods.17, 33, 50
The identity of the two new compounds, GDP-6,6-difluoro-L-fucose (24) and GDP-2-deoxy-2,6-
difluoro-L-fucose (26) were characterized by 1H, 13C, 19F, and 31P-NMR analysis and MS analysis (See
Supporting Information).
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Scheme 3. Chemoenzymatic Synthesis of Fluorinated GDP-Fucose Analogs (22-27) using the
Fluorinated L-fucose Analogs as the Starting Materials
O
N
NH
OAc
R1
O
O
1. NaOMe, MeOH, rt, 2 h
2. FKP, GTP, ATP, MnCl2, MgCl2
R3
N
O
NH2
N
R3
HO
O
P
O
P
O
O
O
R1
OH
OH
Tris buffer, pH 7.5, 37 oC, 3-4 h
OAc
R2
AcO
2
OH
R
OH OH
R1 = F, R2 = H, R3 = CH3
R1 = F, R2 = H, R3 = CH3 (80%)
1
2
3
4
5
6
22
23
24
25
26
27
1
2
3
1
2
3
R = OH, R = H, R = CH2F
R1 = OH, R2 = H, R3 = CHF2
R = OH, R = H, R = CH2F (87%)
R1 = OH, R2 = H, R3 = CHF2 (72%)
1
2
3
1
2
3
R = R = F, R = CH3
R = R = F, R = CH3 (<5%)
R1 = F, R2 = H, R3 = CH2F
R1 = F, R2 = H, R3 = CH2F (66%)
1
2
3
1
2
3
R = OH, R = H, R = CF3
R = OH, R = H, R = CF3 (75%)
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Inhibition of FUT8-Catalyzed Core-Fucosylation by the Fluorinated GDP-L-Fucose Analogs. The
inhibitory effects of the fluorinated GDP-L-fucose analogs on the FUT8 catalyzed fucosylation were
assessed using GDP-fucose as the donor substrate and a synthetic N-glycan, GlcNAcMan5GlcNAc2-
AsnFmoc (Gn-Man5-AsnFmoc), as the acceptor substrate (Figure 5A). The fucosylation was measured
using the Malachite green phosphate assay through quantitative detection of the phosphate ions released
from the calf intestinal alkaline phosphatase (CIP) catalyzed hydrolysis of the GDP, which was formed
as a by-product of the FUT8 catalyzed fucosylation (Figure 5A). The Malachite green phosphate assay
is a universal method for kinetics, high-throughput screening and inhibition studies of
glycosyltransferases. 51 We optimized a previously reported method for assessing the FUT8 catalyzed
core-fucosylation.52 The results of inhibition by the fluorinated GDP-fucose analogs were shown in
Figure 5B.
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OH
O
HO
HO
HO
A
( )
OH
O
O
O
HO
OH
O
O
COOH
NHFmoc
HO
HO
HO
O
HO
HO
O
O
HO
O
O
O
O
OH
HO
HO
HO
N
HO
HO
H
NHAc
NHAc
O
O
HO
HO
HO
O
Gn-Man5-AsnFmoc
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NHAc
FUT8/GDP-Fucose
22, 23, 24
26, 27
CIP
GDP
PPi
Malachite green
OH
HO
HO
HO
O
OH
OH
OH
O
O
HO
HO
O
O
OH
O
O
COOH
NHFmoc
HO
HO
HO
O
HO
O
O
O
HO
O
O
O
O
OH
HO
HO
HO
N
HO
HO
NHAc
H
NHAc
O
O
HO
HO
HO
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NHAc
B
( )
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Control 22
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Inhibitors
Figure 5. Inhibition of FUT8 catalyzed core-fucosylation by the synthetic fluorinated GDP-fucose
analogs. (A) the reaction scheme for the FUT8-catalyzed core fucosylation and the Malachite green
phosphate assay for assessment of the inhibition. (B) the relative inhibition potency of the synthetic
fluorinated GDP-fucose analogs. The inhibition experiments were performed under the following
conditions: A mixture of GDP-Fuc (50 μM), GlcNAcMan5-AsnFmoc (50 μM), CIP (3U), MgCl2 (1
mM), and FUT8 (10 ng/μL) in a buffer (HEPES, 20 mM, pH 7.4, 80 μL) containing the respective
fluorinated GDP-L-fucose analog (5 μM) was incubated at 37 oC in a 96-well flat-bottom plate (Santa
Cruz Biotechnology) for 2 h. The GDP generated from the fucosylation reaction was measured by the
Malachite green phosphate assay (see Supporting Information for details). The inhibition experiments
were performed independently in triplicates. Standard curve was created for calibration and calculation
of inhibition potency.
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The inhibition of FUT8 activity was assessed by using a fixed concentration (5 M) of the
respective fluorinated GDP-L-fucose analog. The GDP-2-deoxy-2-fluoro-L-fucose (22) and the GDP-2-
deoxy-2,6-difluoro-L-fucose (26) showed the most potent inhibitory activity against FUT8, reaching
92% and 70% inhibition at 5 M, respectively. It was observed that an additional fluorination at the 6-
position, as in compound 26, did not lead to an enhancement of inhibitory activity, but resulted in a
moderate decrease in the inhibitory efficiency against FUT8, in comparison with 22. Interestingly, the
three 6-fluorinated L-fucose analogs, including GDP-6-fluoro-L-fucose (23), GDP-6,6-difluoro-L-
fucose (24), and GDP-6,6,6-trifluoro-L-fucose (27), showed only moderate inhibitory activities (13-28%
at 5 M) against FUT8 (Figure 5B). To the best of our knowledge, this is the first comparative study of
a series of fluorinated GDP-fucose analogs on inhibition of FUT8 for core fucosylation. Previously,
Wong and co-workers have reported that GDP-2-deoxy-2-fluoro-L-fucose (22) and GDP-6-fluoro-L-
fucose (23) are competitive inhibitors (with a Ki of 4-38 M) against several fucosyltransferases,
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including FUT3, FUT5, FUT6, and FUT7 . Senter and co-workers have reported that the GDP-2-
deoxy-2-fluoro-L-fucose (22) is an inhibitor of FUT8 and could potently inhibit core-fucosylation of
recombinant monoclonal antibodies when it is present in the mammalian expression cell culture. 14
While the observed potent inhibitory activity of the GDP-2-deoxy-2-fluoro-L-fucose analog (22)
against FUT8 is expected and consistent with previous report, 14 the finding that the three 6-fluorinated
GDP-L-fucose analogs (23, 24 and 27) showed only moderate inhibitory activity of FUT8 is somewhat
unexpected. The lack of correlation between the apparent inhibitory activity of the peracetylated 6-
fluoro-L-fucose (2), 6,6-difluoro-L-fucose (3), and 6,6,6-trifluoro-L-fucose analog (6) against cell
proliferation (Figure 3 and Figure 4) and the inhibition of FUT8 by the corresponding GDP derivatives
(23, 24 and 27) (Figure 5) suggests that other cellular factors, rather than FUT8, might be the major
targets of the 6-fluorinated L-fucose analogs (2, 3, and 6) to achieve their cell proliferation inhibitory
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activities. Given the fact that the GDP-6,6,6-trifluoro-L-fucose (27) has previously been shown to be a
potent allosteric inhibitor of GMD, a key enzyme in the de novo biosynthesis of GDP-L-fucose, which
significantly suppresses the biosynthesis of GDP-L-fucose,17 our results suggest that the significant
inhibition of proliferation of human colon cancer and angiogenetic cells by the peracetylated 6,6-
difluoro-L-fucose (3) and 6,6,6-trifluoro-L-fucose analog (6) most likely result from the targeting of
GMD by their corresponding GDP derivatives (24 and 27, respectively). Of course it should not be ruled
out that the mechanism of action of the fluorinated L-fucose analogs could also involve targeting other
key components, including the ,12- and 1,3/1,4-fucosyltransferases involved in the biosynthesis of
tumor-associated glycoepitopes such as Lewisb and sialyl Lewisa (Figure 1) and the FX enzyme (GDP-
4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase), another key enzyme in the de novo
biosynthesis of GDP-L-fucose (Figure 2). Taniguchi and co-workers have previously demonstrated that
6-alkynyl L-fucose, a potent cellular fucosylation inhibitor, achieves its inhibitory activity by selectively
binding and inhibiting FX, which leads to depletion of the GDP-L-fucose pool.16 Thus, the mechanism
by which L-fucose analogs to achieve inhibition of cellular fucosylation and the inhibition of cell
proliferation appears to be more complicated than what was previously thought. In particular, the
mechanism and correlation of fucosylation and cell growth in colon cancer remains to be characterized.
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Miyoshi and co-workers have shown that HCT116 cells have low cellular fucosylation due to an
inactive mutation in GMD, and that the deficiency of GMD actually facilitates cancer cell progression
by modulating the TRAIL signaling pathway, leading to an escape from NK cell-mediated tumor
surveillance.53 However, Fang and co-workers have demonstrated in another report that FUT8 plays a
key role in colorectal carcinoma, and that targeting FUT8 by MiR-198 dramatically represses tumor
growth and metastasis in colorectal cancer.54
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Future studies should be directed to understanding the mechanism by which the fluorinated L-
fucose analogs differentially inhibit cellular fucosylation and suppress cancer cell proliferation. These
include: 1) investigation of the potential inhibition effects of those synthetic fluorinated GDP-fucose
analogs on different types of fucosyltransferases, such as FUT1 (for α1,2-fucosylation) and FUT3 (for
α1,3/4-fucosylation); 2) analysis of the inhibitory activity of the GDP-fluorinated-L-fucose analogs on
GMD and FX, two key enzymes involved in the de novo GDP-L-fucose biosynthesis; 3) analysis of the
changes in the cellular metabolites in the presence of the fluorinated L-fucose analogs; and 4)
glycoproteomic analysis of the changes in various tumor-associated fucosylated glyco-epitopes of cancer
cells in the presence of the fluorinated L-fucose analogs.
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CONCLUSION
A facile synthesis of a panel of C2 and/or C6 fluorinated fucose analogs was achieved using L-
fucose, L-galactose, or D-mannose as the starting material. Cell-based assays revealed that the 6,6-
difluoro-L-fucose and 6,6,6-trifluoro-L-fucose analogs showed potent inhibitory activity against
proliferation of human colon cancer cells and the angiogenic HUVEC cells, while the previously
reported 2-fluoro-L-fucose did not show apparent effect on the two types of cell lines. This is the first
comparative study of an array of fluorinated L-fucose analogs for the inhibition of the proliferation of
human cancer cells and the angiogenic HUVEC cells. Notably, all the 6-fluorinated fucose analogs
exhibited significant inhibitory activity against HUVEC cells, suggesting that they may inhibit tumor
angiogenesis in vivo. The apparent no correlation between the inhibition of cell proliferation and the
inhibition of FUT8 by the fluorinated fucose analogs and their GDP derivatives, respectively, suggests
that other important factors, instead of FUT8, are most likely the targets of the fluorinated L-fucose
analogs. These may include the key enzymes (GMD and FX) essential for GDP-fucose biosynthesis
and/or other fucosyltransferases such as FUT1 and FUT3 involved in the synthesis of Lewis type tumor-
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associated glycol-epitopes. Future work should be directed to the understanding of the mechanism of
action of selected fluorinated fucose analogs. The present comparative study serves as a starting point
for designing new fluorinated L-fucose analogs as inhibitors against cancer cell proliferation. This work
also provides insights in designing inhibitors of other glycosylation processes that are potential drug
targets for cancer and other diseases.
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METHODS
Details of materials, methods, and experimental procedures are provided in the Supporting Information.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website.
Detailed experimental procedures, synthesis of the fluorinated L-fucose analogs used in the
study; chemoenzymatic synthesis of the GDP-fluorinated-L-fucose derivatives, copies of 1H, 13C,
19F and 31P NMR spectra.
AUTHOR INFORMATION
Corresponding Author
*E-mail: wang518@umd.edu
CONFLICT OF INTEREST
The authors declare that they have no conflicts of interest with the contents of this article.
ACKNOWLEDGMENT
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We thank R. Zhang for technical assistance and P. Wu for providing the FKP plasmid. This work
was supported in part by the National Institutes of Health (NIH grants R01 GM080374 to LXW
and P30 CA006973 to JOL).
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