fluorinated mannosides inhibit cellular fucosylation
TRANSCRIPT
doi.org/10.26434/chemrxiv.13082138.v1
Fluorinated Mannosides Inhibit Cellular Fucosylation.Johan Pijnenborg, Emiel Rossing, Marek Noga, Willem Titulaer, Raisa Veizaj, Dirk J. Lefeber, Thomas Boltje
Submitted date: 12/10/2020 • Posted date: 13/10/2020Licence: CC BY-NC-ND 4.0Citation information: Pijnenborg, Johan; Rossing, Emiel; Noga, Marek; Titulaer, Willem; Veizaj, Raisa;Lefeber, Dirk J.; et al. (2020): Fluorinated Mannosides Inhibit Cellular Fucosylation.. ChemRxiv. Preprint.https://doi.org/10.26434/chemrxiv.13082138.v1
Fucose sugars are expressed on mammalian cell membranes as part of glycoconjugates and mediatesessential physiological processes. The aberrant expression of fucosylated glycans has been linked topathologies such as cancer, inflammation, infection, and genetic disorders. Tools to modulate fucoseexpression on living cells are needed to elucidate the biological role of fucose sugars and the development ofpotential therapeutics. Herein, we report a novel class of fucosylation inhibitors directly targeting de novoGDP-fucose biosynthesis. We demonstrate that cell permeable fluorinated mannoside 1-phosphatederivatives (Fucotrim I & II) are metabolic prodrugs that are metabolized to their respective GDP-mannosederivatives and efficiently inhibit cellular fucosylation.
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Fluorinated mannosides inhibit cellular fucosylation.
Johan F.A. Pijnenborg[a],†, Emiel Rossing[a],†, Marek Noga[b], Willem Titulaer[a], Raisa Veizaj[c], Dirk J.
Lefeber[b,c] and Thomas J. Boltje*[a]
[a] J.F.A. Pijnenborg, E. Rossing, W. Titulaer, Dr. T.J. Boltje
Department of Synthetic Organic Chemistry
Institute for Molecules and Materials, Radboud University
Heyendaalseweg 135, 6525AJ, Nijmegen, The Netherlands
[b] Dr. M. Noga, Prof. D.J. Lefeber
Department of Laboratory Medicine, Translational Metabolic Laboratory
Radboud Institute for Molecular Life Sciences, Radboud University Medical Center
Geert Grooteplein Zuid 10, 6525GA, Nijmegen, The Netherlands
[c] R. Veizaj, Prof. D.J. Lefeber
Department of Neurology
Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center
Geert Grooteplein Zuid 10, 6525GA, Nijmegen, The Netherlands
[†] These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of the document.
Abstract: Fucose sugars are expressed on mammalian cell
membranes as part of glycoconjugates and mediates essential
physiological processes. The aberrant expression of fucosylated
glycans has been linked to pathologies such as cancer, inflammation,
infection, and genetic disorders. Tools to modulate fucose expression
on living cells are needed to elucidate the biological role of fucose
sugars and the development of potential therapeutics. Herein, we
report a novel class of fucosylation inhibitors directly targeting de novo
GDP-fucose biosynthesis. We demonstrate that cell permeable
fluorinated mannoside 1-phosphate derivatives (Fucotrim I & II) are
metabolic prodrugs that are metabolized to their respective GDP-
mannose derivatives and efficiently inhibit cellular fucosylation.
L-Fucose (Fuc) is a 6-deoxyhexose expressed at the termini of glycan
chains that decorate cell surface proteins and lipids.1 The fucose
residues on glycoconjugates are essential mediators of physiological
processes. For example, the fucose moiety in the tetrasaccharide
sialyl Lewisx (sLex) expressed on leukocytes is recognized by selectin
receptors that regulate leukocyte recruitment and extravasation. The
aberrant expression of sLex and increased fucosylation has been
linked to pathologies, most notably cancer, and has been shown to
promote tumor progression.2,3 Reducing fucosylation of glycans in
cancer using inhibitors of the fucose biosynthesis has therefore been
recognized as a promising therapeutic option. Glycans or proteins are
fucosylated by the action of thirteen fucosyltransferases (FucTs) that
differ in acceptor glycan preference but all utilize GDP-fucose as the
donor substrate.4
Scheme 1. (a) Working model of metabolic fucosylation inhibitors. (b) Previous work showing mechanism of action of different 4,6-dehydratase inhibitor acting on
CDP-D-glucose 4,6-dehydratase. Abbreviations: HK, hexokinase; PMM, phosphomannomutase; GDP, mannose-1-phosphate guanylyltransferase; GMDS, GDP-
mannose 4,6 dehydratase; FX, GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase (GMER); FUK, fucokinase; FPGT, fucose-1-phosphate
guanylyltransferase.
2
Cells generate GDP-fucose either via the recycling of
fucose released during glycan turnover in the lysosome (salvage
pathway) or via de novo biosynthesis from mannose 1-phosphate
(Scheme 1). To decrease cellular fucosylation,
fucosyltransferase (FucT) inhibitors have attracted considerable
attention.2 A common strategy has been mimicking the natural
substrate guanosine diphosphate fucose (GDP-Fuc). However,
the essential GDP moiety in these compounds is associated with
a high polarity and low stability thereby limiting their use in vivo by
poor penetration of the cell membrane. This hurdle was recently
overcome with the development of less polar, cell permeable
fucose derivatives which are metabolized to the corresponding
active GDP-fucose analogs through the salvage pathway.5,6,7,8
These compounds target FucTs by competitive inhibition and the
de novo enzymes GMDS and FX by feedback inhibition. This
strategy led to fucosylation inhibitors that are active in vitro and in
vivo, showing promising anticancer effects in liver, breast and
blood cancer models.9,10,11,12,13 Moreover, combining a
fucosylation inhibitor with immunotherapy vaccination in
immunocompetent mice completely protected against tumor
growth due to enhanced antibody-dependent cellular cytotoxicity
(ADCC) with LS174T colorectal carcinoma and A20 lymphoma
cells.11 These inhibitors enter via the fucose salvage pathway yet
it is estimated that ~90% of the GDP-Fuc pool is biosynthesized
via de novo biosynthesis from GDP-mannose.14 Thus, direct
inhibition of de novo GDP-fucose biosynthesis could result in
more potent inhibitors yet this approach remains unexplored so
far to the best of our knowledge. GDP-fucose is biosynthesized
from GDP-mannose by oxidation of the 4-carbon to a ketone and
dehydration of the 6-position by GDP-mannose-4,6-dehydratase
(GMDS, Figure 3a). Subsequent epimerization of the 3- and 5-
position followed by the reduction of the 4-ketone by FX leads to
GDP-Fucose. Hence, GMDS and FX are prime targets to develop
de novo pathway inhibitors.
Scheme 2. Synthesis of 1–10. i) Ac2O, Pyr; ii) H2NNH2·HOAc, DMF; iii) bis(S-
acetyl-2-thioethyl)N,N-diethylphosphoramidite (23), 1H-tetrazole, ACN, then
mCPBA; iv) DMP, NaHCO3, DCM; v) DAST, DCM; vi) Ac2O, H2SO4, AcOH; vii)
TMSCF3, TBAF, THF, then AcOH; viii) NBS, H2O/acetone.
Herein we report the development of highly potent inhibitors
of the de novo pathway based on fluorinated mannose 1-
phosphate derivatives (Scheme 1a).15 We were inspired by a
mechanism-based inhibitor for CDP-D-glucose 4,6-dehydratase
isolated from Yersinia pseudotuberculosis. Use of a 6-deoxy-
difluoride modified CDP-glucose formed a reactive Michael
acceptor inside the active site leading to entrapment (Scheme
1b).16 We hypothesized that GDP-D-6-deoxy-difluoro-mannose
(GDP-D-Rha6F2) could inhibit GMDS, also a 4,6-dehydratase,
through a similar mechanism. To allow passive diffusion over the
cell membrane, lipophilic metabolic precursors were designed.
Two entry points in the de novo biosynthesis were considered with
precursors based on mannose (1–5) and mannose-1-phosphate
(6–10). The lipophilic (thio)ester protecting groups are expected
to be cleaved by esterases inside the cell, allowing the derivatives
to enter the endogenous GDP-mannose biosynthetic pathway. To
investigate the degree of fluorination on the inhibitory potency we
modified the 6-position with none to three fluorides (2–5 & 7–10).
Compounds 1–10 were synthesized as described in
Scheme 2 and the supplementary information. Compounds 1 and
2 were obtained by acetylation of D-mannose and D-rhamnose,
respectively. Mono- and difluorides 3 and 4 were obtained via
DAST mediated fluorination of the 6-OH or aldehyde, respectively,
followed by acetolysis. The synthesis of protected 1-phosphate
analogs 6–9 was achieved in a two-step sequence from their
respective precursors 1–4. Selective deprotection of the anomeric
acetyl ester was achieved using hydrazinium acetate. The
resulting lactol was reacted with phosphoramidite reagent 23
bearing two S-acetyl-2-thioethyl (SATE) groups and subsequently
oxidized to corresponding phosphate triesters 6–9. Trifluorides 5
and 10 were prepared via a homologation approach by
introducing a CF3-group at the C-5 position of the pentose D-
lyxose. To this end, derivative 17 was oxidized and reacted with
a trifluoromethyl anion affording a separable mixture of the
desired 6-trifluoro D-rhamnose (19) and the byproduct L-gulose
(20) derivative. Hydrolysis of the thioacetal with NBS and water
resulted in ring closure and the resulting lactol was acetylated or
phosphorylated to afford 5 and 10, respectively. This synthetic
approach ensures pyranose formation.
Table 1. EC50 values in micromolar for fucose expression inhibition.[a]
THP-1 Jurkat EL4
Compound AAL AOL AAL AOL AAL AOL
DMSO NI NI NI NI NI NI
P-D-Man-1P (6) NI NI NI NI NI NI
P-D-Rha-1P (7) NI 137 NI 153 NI NI
P-D-Rha6F-1P (8) 267 084 174 345 NI NI
P-D-Rha6F2-1P (9) 002.0 005.3 005.9 004.1 014 038
P-D-Rha6F3-1P (10) 000.61 000.45 028 013 NI NI
P-Fuc2F 045 031 115 121 NI NI
[a] Three cell lines (THP-1, Jurkat, EL4) were cultured for 3 days with 0–512 µM
of compound 6–10, P-Fuc2F or DMSO control. The cells were stained with two
fucose specific lectins (AAL & AOL) and analyzed by flow cytometry, presented
as mean percentage lectin binding normalized to control (n=3). N.I, no inhibition
(EC50>500 µM).
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Figure 1. (a) Effect of 10 or 100 µM Fucotrim I & II and P-Fuc2F on cell surface glycosylation using different lectins, presented as percentage lectin binding compared
to DMSO control. (b) Effect of C6-modifications on potency of inhibition. (c) Potency of Fucotrim I and II compared to known salvage pathway dependent fucose
inhibitors P-Fuc2F, Fucostatin-I and Fucostatin-II using AOL lectin (n=3). (d) Onset and recovery of defucosylation. THP1 cells were incubated with 10 or 100 µM
(Figure S2) compound or DMSO control and fucosylation levels were determined with AAL lectins (Figure S2) for six days. (e) The effect on viability and cytotoxicity
on THP1 cells after incubation with 2, 10, 100 or 250 µM 6–10 or 1–5 (Figure S4) for three days was determined with an XTT and LDH respectively and presented
as percentage cell viability or cytotoxicity compared to DMSO control.
The inhibitory potency of 1–10 was assessed at different
concentrations in human THP-1 cells with fucose specific AAL
and AOL lectins (Figure S1a–d). The EC50 values were
determined, defined as the concentration where a 50% decrease
in lectin binding compared to control was observed (Table 1). The
non-phosphorylated compounds 1–5 did not show any inhibition
of fucosylation. In contrast, from the phosphorylated set 6–10, the
mono-, di-, and trifluorinated derivatives 7–10 displayed inhibition
of fucosylation. A trend was observed where increasing the
number of C-6 fluorides increased its potency (Figure 1b).
Inhibitors P-D-Rha6F2-1P (Fucotrim I, 9) and P-D-Rha6F3-1P
(Fucotrim II, 10) afforded (sub)micromolar inhibition of
fucosylation. Both Fucotrim I (9) & II (10) were more potent than
fucosylation inhibitor P-Fuc2F (1,3,4-tri-O-acetyl-2-deoxy-2-
fluoro-L-fucose), a known salvage pathway dependent inhibitor.
Also in human Jurkat cells potent inhibition for Fucotrim I and II
was observed (Table 1; Figure S1e–f). In mouse cell line EL4
however, only Fucotrim I (9) inhibited fucosylation while both
Fucotrim II (10) and P-Fuc2F did not inhibit fucosylation (Table 1;
Figure S1g–h).
Since Fucotrim I and II (9&10) are mannose-1-phosphate
derivatives, it is possible that other glycosylation pathways such
as mannosylation would be affected. Having established the
ability to inhibit cellular fucosylation with AAL and AOL lectins, the
effect on total cell surface glycosylation was evaluated using
additional lectins after 3 days incubation (Figure 1a; Figure S2).
N-glycans consist of a biantennary mannose containing backbone,
recognized by WGA, and can be branched, recognized by L-Pha.
These branches can be terminated with galactose, recognized by
PNA, or α2,6- and α2,3-linked sialic acids recognized by SNA and
MAL-II, respectively.17,18 Binding of LCA and PSA was decreased
for all fucosylation inhibitors as they are core fucose dependent.
The binding of WGA was unaffected as it recognizes core GlcNAc
residues independent of fucosylation.17 Fucotrim I and P-Fuc2F
showed a highly similar profile, not affecting the binding of L-Pha,
PNA, and MAL-II. Only the binding of SNA increased using these
inhibitors. Fucotrim II (10) on the other hand showed a less
specific profile, decreasing the binding of L-Pha, SNA and MAL-II
and increasing the binding of PNA. This would not be expected
for a selective fucosylation inhibitor as sialylation is also clearly
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affected. From these results we conclude that Fucotrim I has a
similar specificity as known fucosylation inhibitor P-Fuc2F but is
more potent.
To further evaluate the potency of Fucotrim I and II, we
made a direct comparison with known fucosylation inhibitors P-
Fuc2F, P-L-Fuc6F3 (Fucostatin I) and P-L-Fuc-1CH2P (Fucostatin
II) (Figure 1c).5,6,7 The EC50 values for known Fucostatin I and II
were determined at 1.3 µM and 0.41 µM respectively, which is in
the same range as Fucotrim I and II under these conditions (Table
1). P-Fuc2F was significantly less potent with an EC50 of 31 µM.
The onset of inhibition and duration for recovery was
evaluated on THP-1 cells with 10 and 100 µM compound using
the AAL lectin (Figure 1d–e; Figure S3). In line with the potency
data, no inhibition was observed for known inhibitor P-Fuc2F at
10 µM concentration. At the same concentration, Fucotrim I and
II decreased lectin binding by over 50% after 3 days with full
recovery to normal fucosylation levels after 4–5 days. At 100 µM
concentration Fucotrim I and II inhibit fucosylation after 1 day and
to over 50% after 3 days. Fucosylation was fully recovered to
normal levels after 6 days. Similar results were obtained using the
AOL lectin (Figure S3).
Finally, a toxicity profile of 1–10 was established by
monitoring the metabolic activity and cell death of THP-1 cells
after three days of treatment (Figure 1e; Figure S4). Importantly,
none of the compounds were cytotoxic at concentrations up to
250 μM. However, the cell viability was decreased which might be
attributed to the known relation between fucosylation and
proliferation.9,10,19
Figure 2. Nucleotide sugar analysis. THP-1 cells were incubated for indicated
time points with 10 µM Fucotrim I or II or with DMSO control. After sample
preparation, the GDP-Rha6F2 (a), GDP-Rha6F3 (b), GDP-Fuc (c) and GDP-Man
(d) levels were analyzed using reverse-phase ion pairing chromatography
coupled to a triple quadrupole mass spectrometer operating in negative ion
mode and presented as their abundance in the nucleotide sugar pool (Figure
S5) (n=3).
It has been suggested that the reduced proliferation after
incubation with known fucosylation inhibitors is caused by acting
on the de novo biosynthesis of GDP-fucose rather than acting on
fucosyltransferases which correlates with our findings.20
Figure 3. (a) Endogenous mechanism of action for human GMDS.21 (b) Natural substrate GDP-mannose (green carbons) was docked at the 4-fluoro-GDP-mannose
(G4F) binding site in human GMDS (6GPJ21, white carbons) in which also NADP+ (cyan carbons) was cocrystalized. (c–i) Unnatural substrate GDP-D-Rha6F2 (c,
green carbons) and the proposed metabolic intermediates (d–i, green carbons) were docked in the same active site. For each structure, the active site residues
Glu157 and Tyr179 were first protonated or deprotonated and either NADPH or NADP+ was present accordingly with the known mechanism of action on GDP-
mannose. Black dotted lines depict expected interactions between atoms leading to a new product, based on the natural mechanism.21
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To test our hypothesized prodrug strategy, we studied the
metabolism of Fucotrim I and II to their corresponding GDP-
analogs inside cells. To this end, intracellular nucleotide sugar
levels were analyzed. THP-1 cells were treated for different time
points with Fucotrim I and II and extracted metabolites analyzed
using reverse-phase ion pairing chromatography coupled to a
triple quadrupole mass spectrometer operating in negative ion
mode (Figure 2; Figure S5). For analysis of GDP-D-Rha6F2 and
GDP-D-Rha6F3 analogs, theoretical mass transitions were
programmed. After incubations for only 1 hour, both Fucotrim
GDP-analogs were observed with a maximum abundance after
4–8 hours (Figure 2a). Subsequently, GDP-fucose levels
decreased after 1–2 days for both inhibitors, indicating inhibition
of GDP-fucose biosynthesis by Fucotrim I and II (Figure 2b).
GDP-mannose levels were also affected by both inhibitors. For
Fucotrim I, the GDP-mannose levels recovered after 3 days and
for Fucotrim II, GDP-mannose levels didn’t recover within the 3
days of the experiment. This correlates well with the lectin panel
(Figure 1a) where after 3 days incubation Fucotrim I only shows
an effect on fucosylation and Fucotrim II shows an effect on
multiple forms of glycosylation. Importantly, for both Fucotrim I
and II no changes were observed for other nucleotide sugar levels
such as UDP-glucose and UDP-galactose (Figure S5).
Having established the metabolism towards GDP-D-Rha6F2
and GDP-D-Rha6F3 inside the cell we investigated if a mechanism
of inhibition as previously described for a bacterial CDP-glucose
4,6-dehydratase (Figure 1b) would be plausible for Fucotrim I
using in silico experiments.16 The docking experiments were
based on a recent study describing the mechanism of action for
human GMDS in detail (Figure 3a).21 The natural substrate GDP-
mannose was docked at the 4-fluoro-GDP-mannose (G4F)
binding site in a human GMDS crystal structure (6GPJ) in which
cofactor NADP+ was co-crystallized (Figure 3b).21 Docking of
GDP-D-Rha6F2 in the same binding site resulted in similar poses
as for GDP-mannose, suggesting it would still be a substrate for
GMDS. The proposed metabolic intermediates (Figure 3d–i) are
based on the endogenous mechanism of action for GMDS and a
known 4,6-dehydratase inhibitor.16,21 The catalytic residues were
positioned such that oxidation (I, Figure 3d), enolization (II,
Figure 3e), elimination (III, Figure 3f–g) and reduction (IV,
Figure 3h) do not seem to be affected sterically. In the
endogenous mechanism enolization would yield the final product.
For the difluoride derivative however, a second elimination might
occur, affording an electrophilic intermediate (Figure 3i) that
cannot be reduced due to the lack of reductant NADPH in the
active site. It is hypothesized that this intermediate either
competitively binds GMDS, or analogous to previous findings16,
reacts with a nucleophilic amino acid (e.g. Ser156) in GMDS
resulting in covalent inhibition, or with water affording a natural
intermediate for GMDS. The in silico experiments indicate that
GDP-D-Rha6F2 likely acts on GMDS but further studies are
needed to investigate the exact mechanism of action.
In conclusion, we have developed in vitro validated de novo
pathway dependent fucosylation inhibitors. Fucotrim I (P-D-
Rha6F2-1P, 9) and Fucotrim II (P-D-Rha6F3-1P, 10) are new tools
to study fucosylation function and are good candidates for further
therapeutic development. Additionally, this approach could be
considered for targeting other 4,6-dehydratases affording cell
active inhibitors.
Acknowledgements
This work was supported by an ERC-Stg awarded to T.J.B., the
Netherlands Organization for Scientific Research (VIDI Grant
91713359 to D.J.L), and the Radboud Consortium for
Glycoscience. We thank prof. Gosse Adema PhD and the
Molecular Immunology group at the Radiotherapy &
OncoImmunology lab at the Radboud University Medical Centre
for kindly supplying us with the THP-1, Jurkat and EL-4 cell lines.
We would to acknowledge John Allen from Amgen Inc. for
providing Fucostatin I and II.
Keywords: Fucosylation • Inhibition • De novo pathway • Small
molecule • GMDS • mannoside
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Entry for the Table of Contents
Fucose sugars on mammalian cell membranes play important roles in physiological and pathological processes. Inhibition of
fucosylation has been identified as a therapeutic avenue. We have developed potent cell-active fucosylation inhibitors, directly inhibiting
the de novo biosynthesis of GDP-fucose.
Institute and/or researcher Twitter usernames: @JohanPijnenborg
download fileview on ChemRxivFucotrim_Manuscript.pdf (0.97 MiB)
1
Supplementary information for:
Fluorinated mannosides inhibit cellular fucosylation.
Johan F.A. Pijnenborg[a],†, Emiel Rossing[a],†, Marek Noga[b], Willem Titulaer[a], Raisa Veizaj[c],
Dirk J. Lefeber[b,c] and Thomas J. Boltje*[a]
[a] J.F.A. Pijnenborg, E. Rossing, W. Titulaer, Dr. T.J. Boltje
Department of Synthetic Organic Chemistry
Institute for Molecules and Materials, Radboud University
Heyendaalseweg 135, 6525AJ, Nijmegen, The Netherlands
[b] Dr. M. Noga,Prof. D.J. Lefeber
Department of Laboratory Medicine, Translational Metabolic Laboratory
Radboud Institute for Molecular Life Sciences, Radboud University Medical Center
Geert Grooteplein Zuid 10, 6525GA, Nijmegen, The Netherlands
[c] R. Veizaj, Prof. D.J. Lefeber
Department of Neurology
Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center
Geert Grooteplein Zuid 10, 6525GA, Nijmegen, The Netherlands
[†] These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of the document.
Content
Supporting figures and tables ................................................................................................................ 2
Supplementary methods ........................................................................................................................ 7
Reagents ............................................................................................................................................ 7
Procedure for cell culture .................................................................................................................... 7
Procedure for lectin staining of cells ................................................................................................... 7
Procedure for lectin specificity assay ................................................................................................. 8
Procedure for determining the onset and recovery of inhibition ........................................................ 8
Viability/toxicity assays ....................................................................................................................... 9
Nucleotide sugar analysis ................................................................................................................... 9
In silico modeling ................................................................................................................................ 9
Synthetic procedures ............................................................................................................................ 11
General synthetic procedures .......................................................................................................... 11
Synthetic procedures ........................................................................................................................ 11
References ........................................................................................................................................... 23
2
Supporting figures and tables
Figure S1: Cell lines THP-1 (a-d), Jurkat (e-f) and EL4 (g-h) were cultured for 3 days with 0–512 µM
compound or DMSO control. The cells were stained with two fucose specific lectins, AAL (a, c, e, g)
& AOL (b, d, f, h) and analysed by flow cytometry, presented as mean percentage lectin binding
normalized to control (n=3).
3
Figure S2: Effect of 1, 2, 4, 6–10, P-Fuc2F and DMSO control on total cell surface glycosylation. THP-
1 cells treated for three days with 10 or 100 µM compound or DMSO control were stained with a panel
of biotinylated lectins and streptavidin-PE. Lectin binding was determined in three independent
experiments by flow cytometry and is presented as mean fluorescent intensity relative to DMSO control
± SEM. Bar diagrams show binding of AAL(a), AOL (b), LCA (c), PSA (d), WGA (e), L-Pha (f), PNA
(g), SNA (h) and MAL-II (i).
4
Figure S3: Onset and recovery of defucosylation. THP1 cells were incubated with 10 µM (b, Figure
1d) or 100 µM (a,c) compound or DMSO control and fucosylation levels were determined with AAL (a)
and AOL (b,c) lectins for six days (n=3).
5
Figure S4: The effect on viability and cytotoxicity on THP1 cells after incubation with 2, 10, 100 or
250 µM 1–5 was determined with an XTT and LDH respectively and presented as percentage cell
viability or cytotoxicity compared to DMSO control.
6
Figure S5: Full nucleotide sugar analysis. THP-1 cells were incubated for indicated time points with
10 µM Fucotrim I or II or with DMSO control. After sample preparation, the different nucleotide levels
were analyzed using reverse-phase ion pairing chromatography coupled to a triple quadrupole mass
spectrometer operating in negative ion mode and presented as their abundance in the nucleotide sugar
pool (n=3).
7
Supplementary methods
Reagents
Biotinylated AAL, SNA, MAL-II, WGA, LCA, PSA, PNA, PHA-L and GSL-1 lectins and 10x Carbo-free
Blocking Buffer were purchased from Vector laboratories Inc.. Biotinylated AOL lectin was purchased
from TCI Europe. 0.2 mg/mL Streptavidin-phycoerythrin conjugate was purchased from Fischer
Scientific (Invitrogen, eBioscience). Unnatural sugar derivatives were synthesized as described under
synthetic procedures and stored at -20°C at a concentration of 100 mM/DMSO.
Procedure for cell culture
THP-1 cells (TIB-202, ATCC) and Jurkat cells (TIB-152, ATCC) were cultured in RPMI-1640 medium
containing 2mM Glutamine and 25mM HEPES (GibcoTM, Life Technologies), supplemented with 10%
v/v heat-inactivated fetal bovine serum (FBS) (GibcoTM, Life Technologies) and 1X antibiotic-
antimycotic solution (100 units/mL of penicillin, 100 μg/mL of streptomycin, and 0.25 μg/mL Fungizone)
(GibcoTM, Life Technologies) and passaged every 3-4 day by seeding a fraction of 0.5-2 mln cells of
the culture per 10 mL medium.
EL-4 cells (TIB-39, ATCC) were cultured in DMEM medium (GibcoTM, Life Technologies),
supplemented with 10% v/v heat-inactivated fetal bovine serum (FBS) (GibcoTM, Life Technologies),
1X antibiotic-antimycotic solution (100 units/mL of penicillin, 100 μg/mL of streptomycin, and 0.25
μg/mL Fungizone) (GibcoTM, Life Technologies) and 2mM Glutamine (GibcoTM, Life Technologies) and
passaged every 2-4 days by seeding a fraction of 0.5-1 mln cells of the culture per 10 mL medium.
All cells were cultured at 37°C, 5% CO2 in a humidified incubator.
Procedure for lectin staining of cells
Protocol below was repeated until n>=3 for each compounds using cells at different passage numbers.
Cells were cultured in medium containing different concentrations of unnatural sugar derivatives on
96-wells plates (suspension cell lines; 13,000–20,000 cells per well) (Thermo Scientific). DMSO, at
same dilution as the unnatural sugar derivative stock solutions, was used as a positive control for lectin
staining; P-Fuc2F was used for all concentrations as negative control. Cells were incubated for three
days at 37°C and 5% CO2 in a humidified incubator.
Cells were harvested and washed with 100 μl 1X CF-blocking buffer (Vector Laboratories Inc.)
containing 1 mM CaCl2 and 1 mM MgCl2. The cells were then resuspended in 50 μl 0.5 μg/mL of 0.5
ng/mL biotinylated lectin in 1X carbo-free blocking buffer and incubated at 4-8°C for 45-60 min. Cells
were washed with 3x100 μl PBA (PBS containing 1% v/v FBS and 0.1% w/w sodium azide), incubated
with 40 μl 1 μg/mL Streptavidin-phycoerythrin conjugate (Invitrogen, eBioscience) in PBA for 10-15
min at 4-8°C. Cells were then washed again with 3x100 μl PBA, resuspended in PBA and fluorescence
was measured with a flow cytometer (Beckman & Dickinson FACS-Calibur). Each replicate for each
condition with >10,000 gated events. Data was processed using FlowJo (FlowJo LLC). Percentage of
lectin binding was obtained by normalizing the MFI values to the MFI values of the respective DMSO
control.
8
Procedure for lectin specificity assay
Protocol below was repeated until n>=3 for each compounds using cells at different passage numbers.
Culture medium was prepared in in 96-wells plates (Thermo Scientific). For every unnatural sugar
derivative, 11 wells containing 100 μM of unnatural sugar derivatives and 11 wells containing 10 μM
of the unnatural sugar derivatives were prepared. DMSO, at same dilution as the tested probes, was
used as a positive control for lectin staining. THP-1 Cells were cultured in the plates (20,000 cells per
well) and the cells were incubated for three days at 37°C and 5% CO2.
Cells were harvested and washed with 100 μl 1X CF-blocking buffer (Vector Laboratories Inc.)
containing 1 mM CaCl2 and 1 mM MgCl2. The cells were then resuspended in 50 μl 0.5 μg/mL of either
0.5 ng/mL biotinylated AAL, AOL, SNA, MAL-II, WGA, LCA, PSA, PNA, PHA-L or GSL-1 lectin in 1X
carbo-free blocking buffer or with 50 μl of non-supplemented 1X carbo-free blocking buffer and
incubated at 4-8°C for 45-60 min. Cells were washed with 3x100 μl PBA (PBS containing 1% v/v FBS
and 0.1% w/w sodium azide), incubated with 40 μl 1 μg/mL Streptavidin-phycoerythrin conjugate
(Invitrogen, eBioscience) in PBA for 10-15 min at 4-8°C. Cells were then washed again with 3x100 μl
PBA, resuspended in PBA and fluorescence was measured with a flow cytometer (Beckman &
Dickinson FACS-Calibur). Each replicate for each condition with >10,000 gated events. Data was
processed using FlowJo (FlowJo LLC). Percentage of lectin binding was obtained by normalizing the
MFI values to the MFI values of the respective DMSO control.
Procedure for determining the onset and recovery of inhibition
THP-1 cells were cultured in medium containing different concentrations of unnatural sugar derivatives
on 96-wells plates (suspension cell lines; 13,000-20,000 cells per well) (Thermo Scientific). DMSO, at
same dilution as the unnatural sugar derivative stock solutions, was used as a positive control for lectin
staining; P-Fuc2F was used for all concentrations as negative control. Cells were incubated for eight
days at 37°C and 5% CO2 in a humidified incubator. Medium was changed after three and six days of
incubation with cell culture medium without unnatural sugar derivatives. Cells were passaged 1:2 after
4, 6 and 8 days of incubation. Every day cells were harvested and washed with 100 μl 1X CF-blocking
buffer (Vector Laboratories Inc.) containing 1 mM CaCl2 and 1 mM MgCl2. The cells were then
resuspended in 50 μl 0.5 μg/mL of either 0.5 ng/mL biotinylated AAL, AOL, SNA, MAL-II, WGA, LCA,
PSA, PNA, PHA-L or GSL-1 lectin in 1X carbo-free blocking buffer or with 50 μl of non-supplemented
1X carbo-free blocking buffer and incubated at 4-8°C for 45-60 min. Cells were washed with 3x100 μl
PBA (PBS containing 1% v/v FBS and 0.1% w/w sodium azide), incubated with 40 μl 1 μg/mL
Streptavidin-phycoerythrin conjugate (Invitrogen, eBioscience) in PBA for 10-15 min at 4-8°C. Cells
were then washed again with 3x100 μl PBA, resuspended in PBA and fluorescence was measured
with a flow cytometer (Beckman & Dickinson FACS-Calibur). Each replicate for each condition with
>10,000 gated events. Data was processed using FlowJo (FlowJo LLC). Percentage of lectin binding
was obtained by normalizing the MFI values to the MFI values of the respective DMSO control.
9
Viability/toxicity assays
Assays were performed using an XTT Cell Viability Assay kit (Invitrogen, CyQUANT) or a LDH-toxicity
assay kit (Thermo Scientific) respectively. Assays were performed according to enclosed manuals.
THP-1 cells were treated for 3 days with unnatural sugar analogs at 37°C, 5% CO2 in a humidified
incubator before adding the XTT/LDH reagents. For the control, cells were treated with DMSO.
%Viability and %Cytotoxicity were calculated relative to the DMSO control for XTT and lysed untreated
cells for the LDH assay.
Nucleotide sugar analysis
THP-1 cells were incubated for 0, 1, 2, 4, 8, 24, 48 or 72 hrs with 10 μM Fucotrim I, Fucotrim II or
DMSO control in triplo. For the 0, 1, 2, 4, 8 hr timepoints, 3 mln cells were plated in 6-well plates, for
the 24 hour timepoint, 1.5 mln cells were plated per well, for the 48 hour timepoint, 1 million cells were
plated and for the 72 hour timepoint, 750,000 cells were plated per well to ensure a total of 3 to 3.5
million cells at time of harvesting. At each timepoint, cells were harvested, medium was removed and
the cells were washed twice with 2 mL 75 mM ammonium carbonate pH 7.4 buffer in MilliQ water. After
removing the washing buffer, cells were snap-frozen in liquid nitrogen. Samples then were stored at -
80°C until workup.
Samples were thawed at -20°C and incubated for 5 minutes in 1 mL of extraction buffer (1:2:2 MilliQ
water: acetonitrile: methanol). Samples were then spun down at 13.000 rpm in a tabletop centrifuge
and supernatant was transferred into 1.5 mL eppendorf tubes and solvent was removed with a
speedvac (Savant SC100, RVT 100 with an external oil pump). Upon redissolving in 100 μL of MilliQ
water 10 μL was injected in to a Agilent 1290 LC-MS system using a modified version of previously
published method for analysis of nucleotide sugars.1 Agilent 6490A QQQ mass spectrometer operated
in dynamic multiple-reaction monitoring MRM modes using transitions generated in silico by the use of
a script written in Python (v 3.6.7), an RDkit library (2019.09.2), and chemical structures of the target
compound as the input. All LC-MS data files were processed in Skyline version 20.1 using a target list
chosen on the basis of an in silico-generated transition list.2 Resulting peak areas were normalized to
the sum of all nucleotide sugars detected, excluding metabolites in pathways affected by treatment (all
GDP-sugars) to obtain relative abundances. Data visualization was performed in Python (v 3.8.5) using
Pandas (v 1.1.2) and Seaborn (v 0.11) libraries.
In silico modeling
Protein Preparation for Docking. The crystal structure of GMDS in complex with 4-fluoro-α-D-
mannopyrranosyl guanoside was downloaded from the PDB-redo database (https://pdb-redo.eu/ , PDB
code 6GPJ)3 and loaded into MOE. The structure was preprocessed by deleting all non- NADP+ and
G4F ligands. Next, the potential was set up to AMBER14:EHT as force field with R-field solvation
model. Addition of hydrogen atoms, removal of water molecules farther than 4.5 Å away from ligand or
receptor, correction of library errors and tethered energy minimization up to a gradient of 0.1 kcal mol-
1 Å2 was performed using MOE’s QuickPrep module. Afterwards, only the C-chain residues, ligands
and solvent molecules were kept. To mimic each intermediate step in the reaction pathway of GMDS,
changes were made to both the receptor and ligands, based on earlier finding by Pfeiffer et al. using
the MOE Builder module.3 Regarding the protein this concerned the (de)protonation of Tyr179 and
Glu157 and regarding the ligands the presence of either NADP+ or NADPH. After making the
10
adjustments, MOE’s QuickPrep module was ran again without protonation, changing the final RMS
gradient for the energy minimization to 1x10-5 kcal mol-1 Å2. Ligand preparation. Ligand structures
were drawn in ChemDraw 18.1, imported into a MOE database in sdf-file format, and energy minimized
up to a gradient of 1x10-5 using the MMFF94x force field. The saved mdb-file was used for the docking
studies. Docking. The ligands were docked into the appropriate protein model, using cocrystalized 4-
fluoro-GDP-mannose as template for placement. After 30 placements the placements were refined to
5 poses using the Induced Fit method with GBVI/WSA rescoring. From the 5 poses, the best pose was
chosen manually for each ligand by a combination of their displacement compared to the template
ligand and the calculated score.
11
Synthetic procedures
General synthetic procedures and abbreviations 1H and 13C NMR spectra were recorded on a Varian Iinova 400 MHz or Bruker Avance III 500 MHz spectrometer. Chemical shifts are reported in parts per million (ppm) relative to tetramethylsilane (TMS) as the internal standard. NMR data is presented as follows: Chemical shift, multiplicity (s = singlet, bs = broad singlet, d = doublet, t = triplet, dd = doublet of doublet, dt = doublet of triplet, m = multiplet and/or multiple resonances), integration, coupling constant in Hertz (Hz). All NMR signals were assigned on the basis of 1H, 13C, 19F NMR, COSY and HSQC experiments. Mass spectra were recorded on a JEOL JMS-T100CS AccuTOF mass spectrometer. Automatic column chromatography was performed on Biotage Isolera Spektra One, using SNAP cartridges 10-50g filled with normal silica (Biotage, 30-100 μm, 60 Å) or water resistant iatro beads. Microwave reactions were perfOCH3d on a Biotage Initiator 4.1.3. TLC analysis was conducted on TLC Silicagel, 60, F254, Merck, with detection by UV absorption (254 nm) where applicable, and by spraying with 20% sulfuric acid in methanol followed by charring at ~150 °C or by spraying with a solution of (NH-4)6Mo7O24
.H2O (25 g l-1) in 10% sulfuric acid in methanol followed by charring at ~300°C. DCM, ACN and Tol were freshly distilled. All reactions were carried out under an argon atmosphere. All synthesized samples that were used for cell tests were dissolved in water or a mixture of dioxane/water and subsequently lyophilized. Abbreviations: Ac, acetyl; ACN, Acetonitrile; AcOH Acetic acid; aq., aqueous; DCM dichloromethane; DMAP, dimethylaminopyridine; DMF, N,N-Dimethylformamide; DMP, Dess–Martin periodinane; EtOAc, ethyl acetate; eq., equivalent; Hept, heptane; hrs, hours; mCPBA, meta-chloroperoxybenzoic acid; min, minutes; NBS, N-bromosuccinimide; Pyr, pyridine; r.t., room temperature; sat., saturated; TBDMS, tert-Butyldiphenylsilyl; TEA, triethylamine; THF, tetrahydrofuran; TIPS, triisopropylsilyl; TMS, trimethylsilyl; Tol, toluene
Synthetic procedures
Scheme 1: Synthetic route towards trifluoro derivatives 5 and 10.
12
Scheme 2: Synthetic route towards phosphoramidite reagent 23.
General procedure A: Selective 1-deacetylation
Compounds 2–4 (1 eq.) were dissolved DMF (0.3 M) and hydrazine acetate (1.2 eq.) was added at
0 ̊C. After stirring for 5 minutes, the mixture was stirred till completion at r.t. which was typically after 3
hrs. The mixture was diluted with DCM, washed with sat. aq. NaHCO3 and brine. The organic layer
was dried over anhydrous Na2SO4, filtered concentrated in vacuo. The residue was purified by silicagel
flash column chromatography (EtOAc in Hept) to afford 11–13.
General procedure B: 1-Phosphorylation
Similar as described previously,4 1-hydroxy-derivatives of 6–10 (1 eq.) and di-(S-acetyl-2-thioethyl)-
N,N-diethylphosphoroamidite 23 (1.1 eq) were dissolved in in ACN (0.35 M) at 0 °C and 1H-tetrazole
(1.4 eq, 0.45 M in acetonitrile) was slowly added. After full conversion, typically after 1 hr, mCPBA (2.4
eq, 77% wt.) was added at 0 °C and stirred for 30 min. The mixture was diluted with EtOAc and 10%
aq. Na2SO3 and the organic layer was washed with sat. aq. NaHCO3 and brine. The aqueous phases
were extracted two more times with fresh EtOAc and the combined organic layers were dried over
anhydrous Na2SO4, filtered concentrated in vacuo. The residue was purified by silicagel flash column
chromatography (EtOAc in Hept) to afford 6–10.
Synthetic procedures:
1,2,3,4,6-penta-O-acetyl-D-mannopyranoside (P-D-Man, 1). Synthesis was
performed as described previously and the data is identical to the data previously reported.5. TLC:
(60:40, EtOAc:Hept v/v) Rf = 0.53. 1H NMR (500 MHz, CDCl3, major anomer) δ 6.09 (d, J = 1.9 Hz,
1H, H1), 5.36 – 5.34 (m, 2H, H3 & H4), 5.27 – 5.26 (m, 1H, H2), 4.29 (dd, J = 12.4, 4.8 Hz, 1H, H6a),
4.11 (dd, J = 12.4, 2.5 Hz, 1H, H6b), 4.06 (dtd, J = 6.2, 4.6, 2.5 Hz, 1H, H5), 2.18 (s, 3H, CH3 Ac), 2.17
(s, 3H, CH3 Ac), 2.10 (s, 3H, CH3 Ac), 2.06 (s, 3H, CH3 Ac), 2.01 (s, 3H, CH3 Ac); 13C NMR (126 MHz,
13
CDCl3, major anomer) δ 170.73 (CO Ac), 170.08 (CO Ac), 169.83 (CO Ac), 169.63 (CO Ac), 168.15
(CO Ac), 90.71 (C1), 70.72 (C5), 68.85 (C3), 68.45 (C2), 65.66 (C4), 62.21 (C6), 20.96 (CH3 Ac), 20.87
(CH3 Ac), 20.81 (CH3 Ac), 20.76 (CH3 Ac), 20.74 (CH3 Ac); HRMS (m/z): [M+Na]+ calcd for
C16H22NaO11, 413.10598; found, 413.10835.
1,2,3,4-tetra-O-acetyl-D-rhamnopyranoside (P-D-Rha, 2). D-Rhamnose (230
mg; 1.52 mmol) was dissolved in pyridine (2.2 mL; 18 eq.) and acetic anhydride (1.7 mL; 12 eq.) was
slowly added at 0 ̊C. The mixture was stirred for 16 hrs while slowly warming to r.t.. The mixture was
concentrated in vacuo and the residue was dissolved in DCM, washed with aq. HCl (1 M), sat. aq.
NaHCO3 and brine. The organic layer was dried over anhydrous MgSO4, filtered and concentrated in
vacuo. The residue was purified by silicagel flash column chromatography (0% → 20 EtOAc in Hept)
to afford 2 (466 mg; 1.40 mmol; qu.). TLC: (50:50, EtOAc:Hept v/v) Rf = 0.44. 1H NMR (500 MHz,
CDCl3, major anomer) δ 6.02 (d, J = 1.9 Hz, 1H, H1), 5.32 – 5.29 (m, 1H, H3), 5.25 (dd, J = 3.5, 2.0
Hz, 1H, H2), 5.12 (t, J = 10.0 Hz, 1H, H4), 3.94 (dq, J = 9.8, 6.2 Hz, 1H, H5), 2.17 (s, 3H, CH3 Ac),
2.16 (s, 3H, CH3 Ac), 2.07 (s, 3H, CH3 Ac), 2.01 (s, 3H, CH3 Ac), 1.24 (d, J = 6.2 Hz, 3H, 3x H6); 13C
NMR (126 MHz, CDCl3, major anomer) δ 170.17 (CO Ac), 169.92 (CO Ac), 169.90 (CO Ac), 168.47
(CO Ac), 90.76 (C1), 70.59 (C4), 68.89 (C3), 68.83 (C5), 68.76 (C2), 21.02 (CH3 Ac), 20.89 (CH3 Ac),
20.87 (CH3 Ac), 20.79 (CH3 Ac), 17.56 (C6). HRMS (m/z): [M+Na]+ calcd for C14H20NaO9, 355.10050;
found, 355.10295.
1,2,3,4-tetra-O-acetyl-6-fluoro-D-rhamnopyranoside (P-D-Rha6F, 3).
Synthesis was performed as described previously and the data is identical to the data previously
reported.6 TLC: (60:40, EtOAc:Hept v/v) Rf = 0.53. 1H NMR (500 MHz, CDCl3, major anomer) δ 6.10
(d, J = 1.9 Hz, 1H, H1), 5.39 – 5.36 (m, 2H, H3 & H4), 5.27 (td, J = 2.2, 0.6 Hz, 1H, H2), 4.54 – 4.42
(m, 2H, H6a & H6b), 4.08 – 3.99 (m, 1H, H5), 2.18 (s, 3H, CH3 Ac), 2.17 (s, 3H, CH3 Ac), 2.08 (s, 3H,
CH3 Ac), 2.02 (s, 3H, CH3 Ac); 13C NMR (126 MHz, CDCl3, major anomer) δ 170.11 (CO Ac), 169.87
(CO Ac), 169.57 (CO Ac), 168.14 (CO Ac), 90.62 (C1), 81.29 (d, J = 175.8 Hz, C6), 71.42 (d, J = 19.5
Hz, C5), 68.78 (C3), 68.36 (C2), 65.24 (d, J = 6.6 Hz, C4), 20.94 (CH3 Ac), 20.83 (CH3 Ac), 20.74 (2x
CH3 Ac); 19F NMR (470 MHz, CDCl3, major anomer) δ -232.46 (td, J = 47.1, 22.9 Hz, 6F). HRMS (m/z):
[M+Na]+ calcd for C14H19FNaO9, 373.09108; found, 373.09255.
1,2,3,4-tetra-O-acetyl-6,6-difluoro-D-rhamnopyranoside (P-D-Rha6F2, 4).
Synthesis was performed as described previously.7 TLC: (50:50, EtOAc:Hept v/v) Rf = 0.55. HRMS
(m/z): [M+Na]+ calcd for C14H18F2NaO9, 391.08166; found, 391.08289. 1H NMR (500 MHz, CDCl3) δ
6.12 (d, J = 2.0 Hz, 1H, H1), 5.83 (td, J = 54.2, 3.1 Hz, 1H, H6), 5.53 (t, J = 10.0 Hz, 1H, H4), 5.37 (dd,
J = 10.0, 3.5 Hz, 1H, H3), 5.26 (dd, J = 3.5, 2.0 Hz, 1H, H2), 4.04 (qd, J = 9.9, 3.1 Hz, 1H, H5), 2.19
14
(s, 3H, CH3 Ac), 2.18 (s, 3H, CH3 Ac), 2.06 (s, 3H, CH3 Ac), 2.02 (s, 3H, CH3 Ac). 13C NMR (126 MHz,
CDCl3) δ 170.04 (CO Ac), 169.80 (CO Ac), 169.42 (CO Ac), 167.93 (CO Ac), 113.62 (t, J = 245.6 Hz,
C6), 90.27 (C1), 70.79 (t, J = 24.3 Hz, C5), 68.28 (C3), 68.02 (C2), 63.97 (dd, J = 3.7, 2.0 Hz, C4),
20.91 (CH3 Ac), 20.82 (CH3 Ac), 20.72 (CH3 Ac), 20.67 (CH3 Ac); 19F NMR (471 MHz, CDCl3) δ -126.43
(ddd, J = 295.5, 53.8, 9.4 Hz, 6Fa), -130.59 (ddd, J = 295.6, 54.5, 11.2 Hz, 6Fb); HRMS (m/z): [M+Na]+
calcd for C14H18F2NaO9, 391.08166; found, 391.08289.
1,2,3,4-tetra-O-acetyl-6,6,6-trifluoro-D-rhamnopyranoside (P-D-Rha6F3, 5).
Compound 21 (40 mg; 0.12 mmol) was dissolved in pyridine (190 µL; 20 eq.) and acetic anhydride
(110 µL; 10 eq.) was slowly added at 0 ̊C. The mixture was then stirred for 4 hrs at r.t.. The mixture
was concentrated in vacuo and the residue was dissolved in DCM, washed with aq. HCl (1 M) and sat.
aq. NaHCO3. The organic layer was dried over anhydrous MgSO4, filtered and concentrated in vacuo.
The residue was purified by silicagel flash column chromatography (0% → 30% EtOAc in Hept) to
afford 5 (39.4 mg; 102 µmol; 88%). TLC: (50:50, EtOAc:Hept v/v) Rf = 0.46. 1H-NMR (500 MHz, CDCl3)
δ 6.17 (d, J = 2.0 Hz, 1H, H1), 5.59 (t, J = 10.0 Hz, 1H, H4), 5.37 (dd, J = 10.0, 3.4 Hz, 1H, H3), 5.26
(dd, J = 3.4, 2.1 Hz, 1H, H2), 4.22 (dt, J = 10.1, 5.6 Hz, 1H, H5), 2.20 (s, 3H, CH3 Ac), 2.19 (s, 3H, CH3
Ac), 2.06 (s, 3H, CH3 Ac), 2.03 (s, 3H, CH3 Ac). 13C-NMR (126 MHz, CDCl3) δ 169.97 (CO Ac), 169.79
(CO Ac), 169.02 (CO Ac), 167.64 (CO Ac), 122.84 (q, J = 280.8 Hz, C6), 90.16 (C1), 70.22 (q, J = 31.5
Hz, C5), 68.09 (C3), 67.80 (C2), 63.71 (C4), 20.87 (CH3 Ac), 20.81 (CH3 Ac), 20.69 (CH3 Ac), 20.58
(CH3 Ac); 19F NMR (471 MHz, CDCl3) δ -75.56 (d, J = 5.8 Hz, CF3); HRMS (m/z): [M+Na]+ calcd for
C14H17F3NaO9, 409.07224; found, 409.07328.
Di-(S-acetyl-2-thioethyl)-(2,3,4,6-tetra-O-acetyl-α-D-
mannopyranoside) phosphate (P-D-Man-1P, 6). 2,3,4,6-tetra-O-acetyl-D-mannopyranoside8 was
reacted as described in general procedure B and purified by silicagel flash column chromatography
(0% → 50% EtOAc in Hept) affording 6 (61.6 mg; 97.4 µmol; 42%). TLC: before oxidation (80:20,
EtOAc:Hept v/v) Rf = 0.76 & after oxidation (80:20, EtOAc:Hept v/v) Rf = 0.51. 1H NMR (500 MHz,
CDCl3) δ 5.65 (dd, J = 6.5, 1.7 Hz, 1H, H1), 5.38 – 5.32 (m, 3H, H2 & H3 & H4), 4.31 (dd, J = 12.4, 4.7
Hz, 1H, H6a), 4.24 – 4.16 (m, 5H, H5 & 2x CH2-O), 4.14 (dd, J = 12.4, 2.4 Hz, 1H, H6b), 3.20 (td, J =
6.4, 4.7 Hz, 4H, 2x CH2-S), 2.37 (s, 6H, 2x CH3 SAc), 2.18 (s, 3H, CH3 OAc), 2.11 (s, 3H, CH3 OAc),
2.06 (s, 3H, CH3 OAc), 2.00 (s, 3H, CH3 OAc); 13C NMR (126 MHz, CDCl3) δ 194.72 (CO SAc), 194.70
(CO SAc), 170.64 (CO OAc), 169.87 (CO OAc), 169.68 (CO OAc), 169.66 (CO OAc), 95.44 (d, J = 5.4
Hz, C1), 70.59 (C5), 68.85 (d, J = 10.9 Hz, C2), 68.34 (C3), 66.83 – 66.59 (m, 2x CH2-O), 65.41 (C4),
62.13 (C6), 30.66 (CH3 SAc), 30.64 (CH3 SAc), 29.22 (d, J = 7.3 Hz, 2x CH2-S), 20.86 (CH3 OAc),
20.83 (CH3 OAc), 20.78 (CH3 OAc), 20.72 (CH3 OAc). 31P NMR (202 MHz, CDCl3) δ -4.14 (1-OP);
HRMS (m/z): [M+Na]+ calcd for C22H33NaO15PS2, 655.08962; found, 655.08718.
15
Di-(S-acetyl-2-thioethyl)-(2,3,4-tri-O-acetyl-α-D-
rhamnopyranoside) phosphate (P-D-Rha-1P, 7). Compound 11 was reacted as described in general
procedure B and purified by silicagel flash column chromatography (0% → 50% EtOAc in Hept)
affording 7 (234 mg; 407 µmol; 43%). TLC: before oxidation (60:40, EtOAc:Hept v/v) Rf = 0.61 & after
oxidation (60:40, EtOAc:Hept v/v) Rf = 0.25. 1H NMR (500 MHz, CDCl3) δ 5.58 (dd, J = 6.3, 1.9 Hz,
1H, H1), 5.34 – 5.29 (m, 2H, H2 & H3), 5.11 (t, J = 10.0 Hz, 1H, H4), 4.23 – 4.05 (m, 5H, 2x CH2-O &
H5), 3.20 (td, J = 6.4, 4.6 Hz, 4H, 2x CH2-S), 2.37 (s, 6H, 2x CH3 SAc), 2.16 (s, 3H, CH3 OAc), 2.07
(s, 3H, CH3 OAc), 1.99 (s, 3H, CH3 OAc), 1.26 (d, J = 6.2 Hz, 3H, 3x H6); 13C NMR (126 MHz, CDCl3)
δ 194.79 (CO SAc), 194.76 (CO SAc), 169.97 (2x CO OAc), 169.78 (CO OAc), 95.56 (d, J = 5.3 Hz,
C1), 70.35 (C4), 69.14 (d, J = 11.0 Hz, C2), 68.73 (C5), 68.38 (C3), 66.72 – 66.48 (m, 2x CH2-O),
30.66 (2x CH3 SAc), 29.25 (d, J = 7.4 Hz, 2x CH2-S), 20.88 (2x CH3 OAc), 20.77 (CH3 OAc), 17.48
(C6); 31P NMR (202 MHz, CDCl3) δ -3.97 (1-OP); HRMS (m/z): [M+Na]+ calcd for C20H31NaO13PS2,
597.08414; found, 597.08291.
Di-(S-acetyl-2-thioethyl)-(2,3,4-tri-O-acetyl-6-fluoro-α-D-
rhamnopyranoside) phosphate (P-D-Rha6F-1P, 8). Compound 12 was reacted as described in
general procedure B and purified by silicagel flash column chromatography (0% → 60% EtOAc in Hept)
affording 8 (10.7 mg; 18.0 µmol; 19%). TLC: before oxidation (60:40, EtOAc:Hept v/v) Rf = 0.61 & after
oxidation (60:40, EtOAc:Hept v/v) Rf = 0.34. 1H NMR (500 MHz, CDCl3) δ 5.66 (dd, J = 6.5, 1.8 Hz,
1H, H1), 5.40 – 5.32 (m, 3H, H2 & H3 & H4), 4.58 – 4.42 (m, 2H, H6a & H6b), 4.24 – 4.15 (m, 5H, H5
& 2x CH2-O), 3.23 – 3.17 (m, 4H, 2x CH2-S), 2.37 (s, 3H, CH3 SAc), 2.36 (s, 2H, CH3 SAc), 2.17 (s,
3H, CH3 OAc), 2.08 (s, 3H, CH3 OAc), 2.01 (s, 3H, CH3 OAc); 13C NMR (126 MHz, CDCl3) δ 194.79
(CO SAc), 194.78 (CO SAc), 169.92 (CO OAc), 169.75 (CO OAc), 169.65 (CO OAc), 95.39 (d, J = 5.4
Hz, C1), 81.21 (d, J = 176.0 Hz, C6), 71.31 (d, J = 19.2 Hz, C5), 68.79 (d, J = 11.2 Hz, C3), 68.31 (C2),
66.85 – 66.62 (m, 2x CH2-O), 64.97 (d, J = 6.7 Hz, C4), 30.66 (2x CH3 SAc), 29.27 – 29.15 (m, 2x CH2-
S), 20.86 (CH3 Ac), 20.79 (CH3 Ac), 20.74 (CH3 Ac); 31P NMR (202 MHz, CDCl3) δ -4.05 (1-OP); 19F
NMR (470 MHz, CDCl3) δ -232.24 (td, J = 47.2, 23.0 Hz, 6F); HRMS (m/z): [M+Na]+ calcd for
C20H30FNaO13PS2, 615.07472; found, 615.07346.
16
Di-(S-acetyl-2-thioethyl)-(2,3,4-tri-O-acetyl-6,6-difluoro-α-D-
rhamnopyranoside) phosphate (P-D-Rha6F2-1P, 9). Compound 13 was reacted as described in
general procedure B and purified by silicagel flash column chromatography (0% → 50% EtOAc in Hept)
affording 9 (50.0 mg; 81.9 µmol; 32%). TLC: before oxidation (60:40, EtOAc:Hept v/v) Rf = 0.65 & after
oxidation (60:40, EtOAc:Hept v/v) Rf = 0.42; 1H NMR (500 MHz, CDCl3) δ 5.86 (td, J = 54.0, 3.1 Hz,
1H, H6), 5.68 (dd, J = 6.5, 2.0 Hz, 1H, H1), 5.50 (t, J = 10.0 Hz, 1H, H4), 5.38 (dd, J = 9.9, 3.4 Hz, 1H,
H3), 5.34 (dd, J = 3.5, 2.0 Hz, 1H, H2), 4.20 (dddd, J = 14.4, 9.6, 5.5, 2.5 Hz, 5H, H5 & 2x CH2-O),
3.20 (q, J = 6.3 Hz, 4H, 2x CH2-S), 2.37 (s, 3H, CH3 SAc), 2.37 (s, 3H, CH3 SAc), 2.17 (s, 3H, CH3
OAc), 2.07 (s, 3H, CH3 OAc), 2.01 (s, 3H, CH3 OAc); 13C NMR (126 MHz, CDCl3) δ 194.70 (CO SAc),
194.69 (CO SAc), 169.78 (CO OAc), 169.60 (CO OAc), 169.44 (CO OAc), 113.40 (t, J = 245.7 Hz,
C6), 94.95 (d, J = 5.3 Hz, C1), 70.42 (t, J = 23.9 Hz, C5), 68.39 (d, J = 11.0 Hz, C2), 67.74 (C3), 66.90
– 66.69 (m, 2x CH2-O), 63.87 (t, J = 2.8 Hz, C4), 30.60 (2x CH3 SAc), 29.18 – 29.08 (m, 2x CH2-S),
20.76 (CH3 OAc), 20.65 (2x CH3 OAc); 19F NMR (471 MHz, CDCl3) δ -127.31 (ddd, J = 295.3, 53.9,
10.3 Hz, 6Fa), -130.57 (ddd, J = 295.2, 54.3, 10.3 Hz, 6Fb); 31P NMR (202 MHz, CDCl3) δ -4.05 (1OP);
HRMS (m/z): [M+Na]+ calcd for C20H29F2NaO13PS2, 633.06529; found, 633.06322.
Di-(S-acetyl-2-thioethyl)-(2,3,4-tri-O-acetyl-6,6,6-trifluoro-α-D-
mannopyranoside) phosphate (P-D-Rha6F3-1P, 10). Compound 21 was reacted as described in
general procedure B and purified by silicagel flash column chromatography (0% → 40% EtOAc in Hept)
affording 10 (95.0 mg; 151 µmol; 35%). TLC: before oxidation (60:40, EtOAc:Hept v/v) Rf = 0.65 &
after oxidation (60:40, EtOAc:Hept v/v) Rf = 0.39; 1H-NMR (500 MHz, CDCl3) δ 5.71 (dd, J = 6.6, 1.9
Hz, 1H, H1), 5.58 (t, J = 9.9 Hz, 1H, H4), 5.38 (dd, J = 9.9, 3.4 Hz, 1H, H3), 5.35 (dd, J = 3.4, 2.0 Hz,
1H, H2), 4.41 (dq, J = 11.1, 5.6 Hz, 1H, H5), 4.27 – 4.14 (m, 4H, 2x CH2-O), 3.22 – 3.17 (m, 4H, 2x
CH2-S), 2.37 (s, 3H, CH3 SAc), 2.37 (s, 3H, CH3 SAc), 2.19 (s, 3H, CH3 OAc), 2.07 (s, 3H, CH3 OAc),
2.02 (s, 3H, CH3 OAc); 13C-NMR (126 MHz, CDCl3) δ 194.68 (CO SAc), 194.65 (CO SAc), 169.71 (CO
Ac), 169.60 (CO Ac), 169.03 (CO Ac), 122.84 (q, J = 280.8 Hz, C6), 94.79 (d, J = 5.3 Hz, C1), 69.81
(q, J = 31.6 Hz, C5), 68.16 (d, J = 11.0 Hz, C2), 67.54 (C3), 67.03 – 66.80 (m, 2xCH2-O), 63.48 (C4),
30.61 (CH3 SAc), 30.60 (CH3 SAc), 29.19 – 29.05 (m, 2x CH2-S), 20.75 (CH3 OAc), 20.63 (CH3 OAc),
20.55 (CH3 OAc); 19F NMR (471 MHz, CDCl3) δ -75.48 (d, J = 5.7 Hz, CF3); 31P NMR (202 MHz, CDCl3)
δ -4.05 (1-OP); HRMS (m/z): [M+Na]+ calcd for C20H28F3NaO13PS2, 651.05587; found, 651.05355.
17
2,3,4-tri-O-acetyl-D-rhamnopyranoside (11). Compound 2 was reacted as
described in general procedure A and purified by silicagel flash column chromatography (0% → 50%
EtOAc in Hept) affording 11 (323 mg; 1.11 mmol; 83%). TLC: (50:50, EtOAc:Hept v/v) Rf = 0.33. 1H
NMR (500 MHz, CDCl3, major anomer) δ 5.37 (dd, J = 10.1, 3.4 Hz, 1H, H3), 5.27 (dd, J = 3.5, 1.8 Hz,
1H, H2), 5.17 – 5.15 (m, 1H, H1), 5.08 (t, J = 10.0 Hz, 1H, H4), 4.17 – 4.10 (m, 1H, H5), 3.44 (d, J =
3.7 Hz, 1H, 1OH), 2.16 (s, 3H, CH3 Ac), 2.06 (s, 3H, CH3 Ac), 2.00 (s, 3H, CH3 Ac), 1.22 (d, J = 6.3
Hz, 3H, 3x H6). 13C NMR (126 MHz, CDCl3, major anomer) δ 170.45 (CO Ac), 170.30 (CO Ac), 170.26
(CO Ac), 92.23 (C1), 71.27 (C4), 70.43 (C2), 68.97 (C3), 66.50 (C5), 21.05 (CH3 Ac), 20.94 (CH3 Ac),
20.86 (CH3 Ac), 17.59 (C6). HRMS (m/z): [M+Na]+ calcd for C12H18NaO8, 313.08994; found,
313.09287.
2,3,4-tri-O-acetyl-6-fluoro-D-rhamnopyranoside (12). Compound 3 was reacted
as described in general procedure A and purified by silicagel flash column chromatography (0% →
35% EtOAc in Hept) affording 12 (29.0 mg; 94.1 µmol; 66%). TLC: (60:40, EtOAc:Hept v/v) Rf = 0.41. 1H NMR (500 MHz, CDCl3, major anomer) δ 5.44 (dd, J = 10.0, 3.3 Hz, 1H, H3), 5.31 – 5.27 (m, 2H,
H4 & H2), 5.25 (d, J = 1.8 Hz, 1H, H1), 4.54 – 4.42 (m, 2H, H6a & H6b), 4.24 (dddd, J = 22.9, 10.3,
4.4, 2.8 Hz, 1H, H5), 3.96 (s, 1H, 1-OH), 2.16 (s, 3H, CH3 Ac), 2.07 (s, 3H, CH3 Ac), 2.01 (s, 3H, CH3
Ac); 13C NMR (126 MHz, CDCl3, major anomer) δ 170.47 (CO Ac), 170.30 (CO Ac), 170.03 (CO Ac),
92.24 (C1), 81.85 (d, J = 174.2 Hz, C6), 70.11 (C2), 69.29 (d, J = 19.0 Hz, C5), 68.90 (C3), 65.85 (d,
J = 7.0 Hz, C4), 21.00 (CH3 Ac), 20.81 (2x CH3 Ac); 19F NMR (470 MHz, CDCl3, major anomer) δ -
231.87 (td, J = 47.2, 22.9 Hz, 6F); HRMS (m/z): [M+Na]+ calcd for C12H17FNaO8, 331.08051; found,
331.08349.
2,3,4-tri-O-acetyl-6,6-difluoro-D-rhamnopyranoside (13). Compound 4 was
reacted as described in general procedure A and purified by silicagel flash column chromatography
(0% → 40% EtOAc in Hept) affording 13 (84.0 mg; 257 µmol; 83%). TLC: (60:40, EtOAc:Hept v/v) Rf
= 0.51. 1H-NMR (500 MHz, CDCl3, major anomer) δ 5.83 (td, J = 54.3, 3.2 Hz, 1H, H6), 5.47 – 5.40
(m, 2H, H4 & H3), 5.28 – 5.24 (m, 2H, H1 & H2), 4.65 (s, 1H, 1-OH), 4.24 (ddq, J = 13.3, 6.3, 3.3 Hz,
1H, H5), 2.16 (s, 3H, CH3 Ac), 2.06 (s, 3H, CH3 Ac), 2.02 (s, 3H, CH3 Ac); 13C-NMR (126 MHz, CDCl3,
major anomer) δ 170.55 (CO Ac), 170.43 (CO Ac), 170.04 (CO Ac), 114.05 (t, J = 244.8 Hz, C6), 92.13
(C1), 69.85 (C2), 68.92 – 68.47 (m, C5 & C3), 64.84 (t, J = 3.1 Hz, C4), 20.91 (CH3 Ac), 20.74 (CH3
Ac), 20.69 (CH3 Ac); 19F NMR (471 MHz, CDCl3, major anomer) δ -127.38 (ddd, J = 293.8, 54.4, 11.4
Hz, 6Fa), -130.25 (ddd, J = 293.7, 54.3, 10.1 Hz, 6Fb). HRMS (m/z): [M+Na]+ calcd for C12H16F2NaO8,
349.07109; found, 349.07331.
18
D-lyxose diethyl dithioacetal (14). D-Lyxose (6.00 g; 40.0 mmol) was dissolved in conc.
HCl (27 mL, 1.5 M) and ethanethiol (6.3 mL; 81.9 mmol; 2.05 eq.; 97% wt.) was added dropwise at
0 ̊C. After stirring for 27 hrs at 0 ̊C the mixture was extracted with EtOAc (4x) and the combined organic
phases were neutralized by portion wise addition of solid NaHCO3, dried over Na2SO4, filtered and
concentrated in vacuo to afford 14 as white crystals (9.90 g; 38.6 mmol; 97%). TLC: (20:80,
MeOH:DCM v/v) Rf = 0.60. 1H NMR (500 MHz, (CD3)2SO)) δ 4.98 (d, J = 6.1 Hz, 1H, 2-OH), 4.46 (t, J
= 5.6 Hz, 1H, 5-OH), 4.23 (d, J = 8.2 Hz, 1H, 3-OH), 4.21 (d, J = 1.5 Hz, 1H, H1), 4.17 (d, J = 6.7 Hz,
1H, 4-OH), 3.84 (ddd, J = 9.3, 6.1, 1.6 Hz, 1H, H2), 3.71 (d, J = 6.8 Hz, 1H, H4), 3.54 (t, J = 8.5 Hz,
1H, H3), 3.44 – 3.35 (m, 2H, 2x H5), 2.68 – 2.59 (m, 4H, 2x CH2 SEt), 1.19 (dt, J = 8.7, 7.4 Hz, 6H, 2x
CH3 SEt); 13C NMR (126 MHz, (CD3)2SO)) δ 73.85 (C2), 70.34 (C3), 69.56 (C4), 62.94 (C5), 54.98
(C1), 24.84 (CH2 SEt), 24.67 (CH2 SEt), 14.74 (CH3 SEt), 14.71 (CH3 SEt); HRMS (m/z): [M+Na]+ calcd
for C9H20NaO4S2, 279.07007; found, 279.07299.
5-O-triisopropylsilyl-D-lyxose diethyl dithioacetal (15). Compound 14 (9.90 g; 38.6
mmol) and 4-dimethylaminopyridine (472 mg; 3.86 mmol; 0.1 eq.) were dissolved in pyridine (110 mL;
0.35 M) and subsequently triisopropylsilylchloride (9.3 mL; 42.5 mmol; 1.1 eq.; 97% wt.) was added
dropwise at 0 ̊C. After 48 hrs most of the starting material had converted and the mixture was
concentrated in vacuo. The residue was dissolved in EtOAc, extracted with water, dried over Na2SO4,
filtered and concentrated in vacuo. The residue was used for the next reaction without further
purification. TLC: (50:50, EtOAc:Hept v/v) Rf = 0.69; 1H NMR (500 MHz, CDCl3) δ 4.23 (d, J = 2.9 Hz,
1H, H1), 4.04 (q, J = 5.3 Hz, 1H, H4), 3.97 – 3.88 (m, 4H, H2 & H3 & H5a & H5b), 3.12 (d, J = 5.3 Hz,
1H, 3-OH), 2.90 (d, J = 5.8 Hz, 1H, 4-OH), 2.87 (d, J = 4.0 Hz, 1H, 2-OH), 2.74 (q, J = 7.4 Hz, 2H, CH2
SEt), 2.67 (q, J = 7.4 Hz, 2H, CH2 SEt), 1.29 (td, J = 7.4, 3.8 Hz, 6H, 2x CH3 SEt), 1.18 – 1.10 (m, 3H,
3x CH TIPS), 1.08 (d, J = 6.5 Hz, 18H, 3x CH3 TIPS); 13C NMR (126 MHz, CDCl3) δ 73.30 (C2), 72.20
(C3), 69.67 (C4), 66.75 (C5), 54.84 (C1), 25.94 (CH2 SEt), 25.82 (CH2 SEt), 18.06 (3x CH3 TIPS),
18.05 (3x CH3 TIPS), 14.85 (CH3 SEt), 14.69 (CH3 SEt), 11.93 (3x CH TIPS). HRMS (m/z): [M+Na]+
calcd for C18H40NaO4S2Si, 435.20350; found, 435.20401.
19
2,3,4-tri-O-acetyl-5-O-triisopropylsilyl-D-lyxose diethyl dithioacetal (16). Crude
15 was dissolved in pyridine (190 mL; 60 eq.) and acetic anhydride (109 mL; 30 eq.) was slowly added
at 0 ̊C. The mixture was stirred for 28 hrs while slowly warming to r.t.. The mixture was concentrated
in vacuo and the residue was dissolved in DCM, washed with aq. HCl (1 M), sat. aq. NaHCO3 and
brine. The organic layer was dried over anhydrous MgSO4, filtered and concentrated in vacuo. The
residue was purified by silicagel flash column chromatography (0% → 10% EtOAc in Hept) to afford
16 (12.09 g; 22.444 mmol; 58% over 2 steps). TLC: (10:90, EtOAc:Hept v/v) Rf = 0.24; 1H NMR (500
MHz, CDCl3) δ 5.76 (dd, J = 6.9, 1.8 Hz, 1H, H3), 5.39 – 5.33 (m, 2H, H2 & H4), 3.96 (d, J = 5.4 Hz,
1H, H1), 3.74 – 3.64 (m, 2H, 2x H5), 2.76 – 2.59 (m, 4H, 2x CH2 Et), 2.10 (s, 3H, CH3 Ac), 2.08 (s, 3H,
CH3 Ac), 2.07 (s, 3H, CH3 Ac), 1.25 (dt, J = 8.4, 7.4 Hz, 6H, 2x CH3 Et), 1.10 – 1.02 (m, 21H, 6x CH3
TIPS & 3x CH TIPS); 13C NMR (126 MHz, CDCl3) δ 170.36 (CO Ac), 169.87 (CO Ac), 169.74 (CO Ac),
71.65 (C2), 70.74 (C4), 70.26 (C3), 61.85 (C5), 51.76 (C1), 25.58 (CH2 Et), 25.16 (CH2 Et), 21.17 (CH3
Ac), 20.92 (2x CH3 Ac), 17.99 (6x CH3 TIPS), 14.40 (CH3 Et), 14.16 (CH3 Et), 11.99 (3x CH TIPS);
HRMS (m/z): [M+Na]+ calcd for C24H46NaO7S2Si, 561.23519; found, 561.23388.
2,3,4-tri-O-acetyl-D-lyxose diethyl dithioacetal (17). Compound 16 (12.00 g; 22.27
mmol) was dissolved in THF (150 mL; 0.15 M) in a plastic bottle and hydrogen fluoride pyridine complex
(15 mL; ~70%/30%) was added in portions over 3.5 hrs at 0 ̊C. After stirring for an additional 14 hrs at
r.t. all starting material was consumed and the mixture was diluted with EtOAc (100 mL) and carefully
quenched with sat. aq. NaHCO3. The organic layer was washed with 10% aq. CuSO4 (2x) and the
combined organic layers were dried over anhydrous MgSO4, filtered and concentrated in vacuo. The
residue was purified by silicagel flash column chromatography (0% → 40% EtOAc in Hept) to afford
17 (4.85 g; 12.7 mmol; 57%). TLC: (40:60, EtOAc:Hept v/v) Rf = 0.18. 1H NMR (400 MHz, CDCl3) δ
5.59 (dd, J = 8.8, 1.7 Hz, 1H, H3), 5.39 (dd, J = 8.8, 3.5 Hz, 1H, H2), 5.06 (ddd, J = 7.8, 6.2, 1.7 Hz,
1H, H4), 3.86 (d, J = 3.5 Hz, 1H, H1), 3.53 (dt, J = 12.0, 5.9 Hz, 1H, H5a), 3.38 (dd, J = 11.7, 7.8 Hz,
1H, H5b), 2.80 (d, J = 7.1 Hz, 1H, 5-OH), 2.71 – 2.52 (m, 4H, 2x CH2 SEt), 2.11 (s, 3H, CH3 Ac), 2.02
(s, 3H, CH3 Ac), 2.01 (s, 3H, CH3 Ac), 1.18 (td, J = 7.4, 5.9 Hz, 6H, 2x CH3 SEt); 13C-NMR (101 MHz,
CDCl3) δ 171.15 (CO Ac), 170.54 (CO Ac), 169.64 (CO Ac), 70.86 (C2), 70.62 (C4), 70.45 (C3), 60.05
(C5), 51.56 (C1), 25.81 (CH2 SEt), 25.36 (CH2 SEt), 20.93 (CH3 Ac), 20.90 (CH3 Ac), 20.70 (CH3 Ac),
14.36 (CH3 SEt), 14.15 (CH3 SEt); HRMS (m/z): [M+Na]+ calcd for C15H26NaO7S2, 405.10176; found,
405.10253.
20
2,3,4-tri-O-acetyl-5-al-D-lyxose diethyl dithioacetal (18). Compound 17 (4.85 g; 12.7
mmol) was dissolved in dry DCM (106 mL; 0.12 M) and subsequently NaHCO3 (10.7 g; 127 mmol; 10
eq.) and Dess-Martin periodinane (8.07 g; 19.0 mmol; 1.5 eq.) were added at 0 ̊C. After 10 minutes the
ice bath was removed and the mixture was stirred for an additional 3 hrs at r.t.. The mixture was diluted
with DCM (100 mL) and an aqueous solution (150 mL) of Na2SO3 (15 g) and NaHCO3 (5 g) was added
at 0 ̊C and stirred for 30 min. The organic phase was separated, dried over anhydrous MgSO4, filtered
and concentrated in vacuo. The residue was purified by silicagel flash column chromatography (0% →
60% EtOAc in Hept) to afford 18 (3.65 g; 9.59 mmol; 76%) which was immediately used for the next
step. TLC: (50:50, EtOAc:Hept v/v) Rf = 0.32. HRMS (m/z): [M+MeOH+Na]+ calcd for C16H28NaO8S2,
435.11233; found, 435.11239
2,3,4-tri-O-acetyl-6,6,6-trifluoro-D-rhamnose diethyl dithioacetal (19)
& 2,3,4-tri-O-acetyl-6,6,6-trifluoro-L-gulose diethyl dithioacetal (20). Synthetic procedure was
adapted from a previously described procedure.9 Compound 18 and trifluoromethyl(trimethyl)silane
(1.45 mL; 9.56 mmol; 1.6 eq; 97% wt.) were dissolved in dry THF (20 mL; 0.3 M) and a
tetrabutylammonium fluoride solution (598 µL; 598 µmol; 0.1 eq.; 1.0 M in THF) was added dropwise
at 0 ̊C. After stirring at 0 ̊C for 1.5 hrs the mixture was concentrated in vacuo. The residue was dissolved
in CHCl3, washed with water, dried over anhydrous Na2SO4, filtered and concentrated in vacuo. To
remove the 5-OTMS groups, the residue was dissolved in 80% AcOH (0.25 M; 24 mL) and kept for 3
hrs at 50 ̊C. The mixture was concentrated in vacuo and the residue was purified by silicagel flash
column chromatography (0% → 20% → 30% EtOAc in Hept) to afford first 19 (813 mg; 1.80 mmol;
30%) and then 20 (816; 1.81 mmol; 30%). Compound 19: TLC: (30:70, EtOAc:Hept v/v) Rf = 0.23. 1H-
NMR (500 MHz, CDCl3) δ 5.82 (dd, J = 8.4, 1.3 Hz, 1H, H3), 5.48 (dd, J = 9.5, 1.3 Hz, 1H, H4), 5.33
(dd, J = 8.4, 4.2 Hz, 1H, H2), 3.97 (d, J = 4.2 Hz, 1H, H1), 3.81 (dq, J = 9.4, 6.3 Hz, 1H, H5), 2.77 –
2.60 (m, 5H, OH & 2x CH2 SEt), 2.22 (s, 3H, CH3 Ac), 2.10 (s, 3H, CH3 Ac), 2.07 (s, 3H, CH3 Ac), 1.26
(q, J = 7.3 Hz, 7H, 2x CH3 SEt); 13C-NMR (126 MHz, CDCl3) δ 172.28 (CO), 169.85 (CO), 169.47 (CO),
124.34 (q, J = 282.2 Hz, C6), 71.14 (C3), 71.06 (C2), 68.05 – 67.27 (m, C5&C4), 51.67 (C1), 26.06
(CH2 SEt), 25.68 (CH2 SEt), 21.22 (CH3 Ac), 20.95 (CH3 Ac), 20.68 (CH3 Ac), 14.40 (CH3 SEt), 14.26
(CH3 SEt). 19F NMR (471 MHz, CDCl3) δ -75.75 (d, J = 6.4 Hz, CF3); HRMS (m/z): [M+Na]+ calcd for
C16H25F3NaO7S2, 473.08915; found, 473.08916. Compound 20: TLC: (30:70, EtOAc:Hept v/v) Rf =
0.18. 1H-NMR (500 MHz, CDCl3) δ 5.79 (dd, J = 8.0, 2.5 Hz, 1H, H3), 5.60 (dd, J = 2.5, 1.4 Hz, 1H,
21
H4), 5.33 (dd, J = 8.0, 4.2 Hz, 1H, H2), 4.29 – 4.21 (m, 1H, H5), 3.93 (d, J = 4.2 Hz, 1H, H1), 2.79 (d,
J = 9.8 Hz, 1H, OH), 2.74 – 2.61 (m, 4H, 2x CH2 SEt), 2.13 (s, 3H, CH3 Ac), 2.13 (s, 3H, CH3 Ac), 2.10
(s, 3H, CH3 Ac), 1.25 (q, J = 7.5 Hz, 7H, 2x CH3 SEt); 13C-NMR (126 MHz, CDCl3) δ 170.13 (CO),
169.94 (CO), 169.14 (CO), 123.68 (q, J = 283.1 Hz, C6), 72.06 (C3), 71.20 (C2), 70.38 (q, J = 31.4
Hz, C5), 65.43 (d, J = 1.9 Hz, C4), 51.40 (C1), 25.86 (CH2 SEt), 25.68 (CH2 SEt), 21.07 (CH3 Ac),
20.93 (CH3 Ac), 20.68 (CH3 Ac), 14.41 (CH3 SEt), 14.21 (CH3 SEt); 19F NMR (471 MHz, CDCl3) δ -
78.18 (d, J = 6.8 Hz, CF3); HRMS (m/z): [M+Na]+ calcd for C16H25F3NaO7S2, 473.08915; found,
473.08860.
2,3,4-tri-O-acetyl-6,6,6-trifluoro-D-rhamnopyranoside (21). Compound 19 (763
mg; 1.69 mmol) was dissolved in a 5:1 acetone/water mixture (34 mL; 0.05 M) and NBS (1.51 g; 8.47
mmol; 5 eq.) was added at 0 ̊C. After stirring for 45 min at 0 ̊C, subsequently sat. aq. NaHCO3 (15 mL)
and sat. aq. Na2S2O3 (15 mL) were added and the mixture was stirred for 15 min. The organic solvent
was evaporated in vacuo and the remaining mixture was extracted with Et2O (2x), dried over anhydrous
MgSO4, filtered and concentrated in vacuo. The residue was purified by silicagel flash column
chromatography (0% → 30% EtOAc in Hept) to afford 21 (225 mg; 654 µmol; 39%). TLC: (50:50,
EtOAc:Hept v/v) Rf = 0.40; 1H-NMR (500 MHz, CDCl3) δ 5.54 (t, J = 9.9 Hz, 1H, H4), 5.44 (dd, J = 10.0,
3.3 Hz, 1H, H3), 5.32 (s, 1H, H1), 5.26 (dd, J = 3.3, 1.9 Hz, 1H, H2), 4.42 (dq, J = 9.9, 5.9 Hz, 1H, H5),
4.20 (s, 1H, OH), 2.17 (s, 3H, CH3 Ac), 2.06 (s, 3H, CH3 Ac), 2.02 (s, 3H, CH3 Ac); 13C-NMR (126 MHz,
CDCl3) δ 170.50 (CO Ac), 170.31 (CO Ac), 169.54 (CO Ac), 123.47 (q, J = 280.4 Hz, C6), 92.26 (C1),
69.56 (C2), 68.71 – 67.90 (m, C5 & C3), 64.32 (C4), 20.93 (CH3 Ac), 20.74 (CH3 Ac), 20.62 (CH3 Ac); 19F NMR (471 MHz, CDCl3) δ -75.44 (d, J = 6.0 Hz, 3x 6F); HRMS (m/z): [M+Na]+ calcd for
C12H15F3NaO8, 367.06167; found, 367.06424.
S-acetyl-2-thioethyl alcohol (22). Synthesis was performed as described previously
and the data was identical.10 TLC: (5:95, EtOAc:DCM v/v) Rf = 0.15. 1H NMR (500 MHz, CDCl3) δ 3.67
(t, J = 6.3 Hz, 2H, CH2-O), 3.01 (t, J = 6.3 Hz, 2H, CH2-S), 2.30 (s, 3H, CH3 Ac); 13C NMR (126 MHz,
CDCl3) δ 196.42 (CO), 61.48 (CH2OH), 31.89 (CH2-S), 30.60 (CH3 Ac)
Di-(S-acetyl-2-thioethyl)-N,N-diethylphosphoroamidite (23). Compound 22
(5.88 g; 48.9 mmol; 2 eq.) was dissolved in anhydrous THF (31 mL; 1.6 M) and subsequently molecular
sieves (4Å) and anhydrous TEA (6.1 mL; 44.0 mmol; 1.8 eq.) were added. The mixture was cooled to
0° C, diethylphosphoramidous dichloride (3.56 mL; 24.5 mmol; 1 eq.). After stirring for 2 hrs the mixture
was diluted with heptane, filtered and the filtrate was concentrated and directly purified by silicagel
flash column chromatography (3% TEA in Hept) to afford 23 (3.90 g; 11.4 mmol; 47%). The dry
phosphoroamidite reagent was stored at -80 ̊C and after 11 months no changes in 1H NMR were
22
observed. TLC: (30:67:3, EtOAc:Hept:TEA v/v) Rf = 0.53. 1H NMR (500 MHz, CDCl3) δ 3.73 (dddt, J
= 32.3, 10.7, 8.4, 6.5 Hz, 4H), 3.11 (t, J = 6.6 Hz, 4H), 3.06 (dq, J = 9.5, 7.1 Hz, 4H), 2.34 (s, 6H), 1.06
(t, J = 7.1 Hz, 6H); 13C NMR (126 MHz, CDCl3) δ 195.08, 61.76 (d, J = 16.0 Hz), 37.31 (d, J = 20.7
Hz), 30.49, 30.37 (d, J = 6.5 Hz), 14.98 (d, J = 3.2 Hz); 31P NMR (202 MHz, CDCl3) δ 147.22
(PNEt2(OCH2)2); HRMS (m/z): [M+Na]+ calcd for C12H24NNaO5PS2, 380.07312; found, 380.07340.
23
References
1. van Scherpenzeel, M.; Conte, F.; Büll, C.; Ashikov, A.; Hermans, E.; Willems, A.; van Tol, W.; Kragt, E.; Moret, E. E.; Heise, T.; Langereis, J. D.; Rossing, E.; Zimmermann, M.; Rubio-Gozalbo, M. E.; de Jonge, M. I.; Adema, G. J.; Zamboni, N.; Boltje, T.; Lefeber, D. J., Dynamic analysis of sugar metabolism reveals the mechanisms of action of synthetic sugar analogs. bioRxiv 2020, 2020.09.15.288712. 2. MacLean, B.; Tomazela, D. M.; Shulman, N.; Chambers, M.; Finney, G. L.; Frewen, B.; Kern, R.; Tabb, D. L.; Liebler, D. C.; MacCoss, M. J., Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 2010, 26 (7), 966-968. 3. Pfeiffer, M.; Johansson, C.; Krojer, T.; Kavanagh, K. L.; Oppermann, U.; Nidetzky, B., A Parsimonious Mechanism of Sugar Dehydration by Human GDP-Mannose-4,6-dehydratase. ACS Catalysis 2019, 9 (4), 2962-2968. 4. Yu, S.-H.; Boyce, M.; Wands, A. M.; Bond, M. R.; Bertozzi, C. R.; Kohler, J. J., Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners. Proceedings of the National Academy of Sciences 2012, 109 (13), 4834-4839. 5. Timmer, B. J. J.; Flos, M. A.; Jørgensen, L. M.; Proverbio, D.; Altun, S.; Ramström, O.; Aastrup, T.; Vincent, S. P., Spatially well-defined carbohydrate nanoplatforms: synthesis, characterization and lectin interaction study. Chemical Communications 2016, 52 (83), 12326-12329. 6. Khan, S. H.; Jain, R. K.; Abbas, S. A.; Matta, K. L., Synthesis of some monodeoxyfluorinated methyl and 4-nitrophenyl α-d-mannobiosides and a related 4-nitrophenyl α-d-mannotrioside. Carbohydrate Research 1990, 198 (2), 259-273. 7. Crich, D.; Vinogradova, O., Synthesis and Glycosylation of a Series of 6-Mono-, Di-, and Trifluoro S-Phenyl 2,3,4-Tri-O-benzyl-thiorhamnopyranosides. Effect of the Fluorine Substituents on Glycosylation Stereoselectivity. Journal of the American Chemical Society 2007, 129 (38), 11756-11765. 8. Tosin, M.; Murphy, P. V., Synthesis of Structurally Defined Scaffolds for Bivalent Ligand Display Based on Glucuronic Acid Anilides. The Degree of Tertiary Amide Isomerism and Folding Depends on the Configuration of a Glycosyl Azide. The Journal of Organic Chemistry 2005, 70 (10), 4107-4117. 9. Nakai, K.; Takagi, Y.; Tsuchiya, T., Synthesis and antitumor activity of 7-O-[2,6-dideoxy-2-fluoro-5-C-(trifluoromethyl)-α-l-talopyranosyl]-daunomycinone and -adriamycinone. Carbohydrate Research 1999, 316 (1), 47-57. 10. Liras, M.; García, O.; Guarrotxena, N.; Palacios-Cuesta, M.; Quijada-Garrido, I., Versatile thiolated thermosensitive polymers synthesized by ATRP of MEO2MA and AcSEMA, a new methacrylic monomer with a protected thiol group. Polymer Chemistry 2013, 4 (24), 5751-5759.
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