Fluorous-tag assisted synthesis of a glycosaminoglycan mimetic tetrasaccharide as a high-affinity FGF-2 and midkine ligand

Article abstract of DOI:10.1016/j.bmc.2018.01.022

Here, we present the preparation of a sulfated, fully protected tetrasaccharide derivative following the glycosaminoglycan (GAG)-related sequence GlcNAc-β(1 → 4)-Glc-β(1 → 3). The tetramer was efficiently assembled via an iterative glycosylation strategy using monosaccharide building blocks. A fluorous tag was attached at position 6 of the reducing end unit enabling the purification of reaction intermediates by simple fluorous solid phase extraction. Fluorescence polarization competition experiments revealed that the synthesized tetrasaccharide strongly interacts with two heparin-binding growth factors, midkine and FGF-2 (IC₅₀ of 270 nM and 2.4 μM, respectively). Our data indicate that this type of oligosaccharide derivatives, displaying sulfates, hydrophobic protecting groups and a fluorinated tail can be considered as interesting GAG mimetics for the regulation of relevant carbohydrate-protein interactions.

Full text of DOI:10.1016/j.bmc.2018.01.022

Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Fluorous-tag assisted synthesis of a glycosaminoglycan mimetic
tetrasaccharide as a high-affinity FGF-2 and midkine ligand
⇑
⇑
Susana Maza, Noel Gandia-Aguado, José L. de Paz , Pedro M. Nieto
Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), cicCartuja, CSIC and Universidad de Sevilla, Americo Vespucio, 49, 41092 Sevilla, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Here, we present the preparation of a sulfated, fully protected tetrasaccharide derivative following the
glycosaminoglycan (GAG)-related sequence GlcNAc-b(1 ? 4)-Glc-b(1 ? 3). The tetramer was efficiently
assembled via an iterative glycosylation strategy using monosaccharide building blocks. A fluorous tag
was attached at position 6 of the reducing end unit enabling the purification of reaction intermediates
by simple fluorous solid phase extraction. Fluorescence polarization competition experiments revealed
that the synthesized tetrasaccharide strongly interacts with two heparin-binding growth factors, midkine
and FGF-2 (IC50 of 270 nM and 2.4 mM, respectively). Our data indicate that this type of oligosaccharide
derivatives, displaying sulfates, hydrophobic protecting groups and a fluorinated tail can be considered as
interesting GAG mimetics for the regulation of relevant carbohydrate-protein interactions.
Ó 2018 Elsevier Ltd. All rights reserved.
Received 19 December 2017
Revised 22 January 2018
Accepted 24 January 2018
Available online xxxx
Keywords:
Fluorescence polarization
Oligosaccharide synthesis
Glycosaminoglycans
Fluorous tag
Carbohydrate-protein interactions
1. Introduction
There is a great interest in the chemical synthesis of GAG
oligosaccharides13 that specifically interact with protein receptors,
Glycosaminoglycans (GAGs) constitute
a
family of linear,
paving the way for the regulation of relevant biological processes.
However, the preparation of well-defined, long GAG oligosaccha-
rides is a formidable challenge. Thus, the synthesis of more easily
accessible GAG mimetics, which retain the biological properties
of the natural polysaccharides, is an attractive alternative.14 For
instance, different types of multivalent scaffolds displaying short
and easily prepared GAG oligomers have been proposed as promis-
ing GAG mimetics.15–21 These multivalent systems include den-
drimers and polymers functionalized with CS and heparin
oligosaccharide sequences. Sulfated non-GAG oligosaccharides,
such as mannose PI-88 and glucose PG545 derivatives, have also
been reported as GAG mimetics with potent anticancer activity
and high binding affinities to angiogenic growth factors.22–24 Addi-
tionally, non-sugar, sulfated compounds bearing an aromatic scaf-
fold are also potent modulators of GAG-protein binding.25–27 The
interaction of these highly hydrophobic analogues usually involves
an important non-ionic contribution to binding energy. In fact,
although GAG-protein binding is mainly driven by electrostatic
forces between positively charged amino acid residues of the pro-
tein and anionic sulfate and carboxylate moieties of the sugar,
hydrophobic interactions can also play an important role in these
recognition events.28
heterogeneous polysaccharides that include hyaluronic acid, chon-
droitin sulfate (CS) and heparin, among others. GAGs interact with
a plethora of proteins and these molecular recognition processes
regulate a broad range of biological phenomena including cell
growth and differentiation, blood coagulation and inflamma-
tion.1–5 Generally, GAGs are formed by disaccharide repeating
units that are decorated with sulfate groups at different positions,
giving rise to polysaccharidic chains with enormous structural
diversity. It is well-known that defined GAG oligosaccharide
sequences are responsible for specific protein recognition and sub-
sequent activity.6–9 For example, the heparin-antithrombin III
binding, responsible for the anticoagulant activity of this polysac-
charide, is mediated by a specific pentasaccharide structure with
a well-defined sequence and sulfate group distribution.10 On the
other hand, a particular class of CS (CS-E), characterized by the
disulfated sequence GalNAc(4,6-di-OSO3)-b(1 ? 4)-GlcA-b(1 ? 3),
has an important role in the central nervous system development
and it has been demonstrated that CS-E tetrasaccharides specifi-
cally interact with several neurotrophins, controlling the survival,
development and growth of neurons.11,12
In this context, several research groups have shown that the
incorporation of hydrophobic scaffolds or groups improves the bio-
logical activities and the protein binding affinities of sulfated com-
pounds.22,23,29 In addition, we have previously discovered that
⇑
Corresponding authors.
E-mail addresses: jlpaz@iiq.csic.es (J.L. de Paz), pedro.nieto@iiq.csic.es
(P.M. Nieto).
https://doi.org/10.1016/j.bmc.2018.01.022
0968-0896/Ó 2018 Elsevier Ltd. All rights reserved.
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2
S. Maza et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
sulfated, fully protected oligosaccharides, typical intermediates in
the chemical synthesis of CS oligomers, strongly bound to certain
heparin-binding proteins and could be considered as promising
CS mimetics.30 Importantly, the binding affinities of these com-
pounds were much higher than those corresponding to the depro-
tected natural sequences. Therefore, our results also indicated that
the presence of hydrophobic protecting groups significantly
enhances the protein binding.
The preparation of these CS intermediates involved the use of
expensive galactosamine (GalN) units and poorly reactive glu-
curonic acid (GlcA) building blocks. In order to speed up the access
to these CS analogues and explore new structure-activity relation-
ships, we decided to replace the GalN and GlcA moieties by glu-
cosamine (GlcN) and glucose (Glc) units, respectively. Thus, we
present here the synthesis of tetrasaccharide 1, displaying the
sequence GlcN(4,6-di-OSO3)-b(1 ? 4)-Glc-b(1 ? 3), closely related
to the CS-E structure (Scheme 1). For this purpose, we envisioned
the use of a fluorous-tag assisted strategy. The interaction between
1 and two proteins, midkine and FGF-2, was then evaluated.
First, reducing end glycosyl acceptor 2 was prepared from
known diol 542 by selective acylation with heptadecafluorounde-
canoyl chloride at 0 °C (Scheme 2). Similar reaction conditions
(acyl chloride, triethylamine, DMAP in CH2Cl2 at 0 °C) were used
for the preparation of the 6-O-pivaloylated derivative 6. Levulinoy-
lation at position 4, followed by oxidative removal of the 4-meth-
oxyphenyl group with cerium (IV) ammonium nitrate (CAN)
afforded 1-hydroxy sugar 8 in good yield. 1-O-benzoylated 2-
hydroxy sugar, derived from the migration of the benzoyl group
to the anomeric position, was detected as a minor side product.
Treatment with trichloroacetonitrile and potassium carbonate in
CH2Cl2 gave trichloroacetimidate donor 3. Compound 9 was
converted into derivative 10 in excellent yield by treatment with
levulinic anhydride and DMAP, followed by removal of the
4-methoxyphenyl group at 0 °C. Treatment with CAN at room tem-
perature resulted in lower yields due to partial benzylidene
hydrolysis. Purification of 10 by silica gel column chromatography
was avoided, due to its instability and the formation of H-2/H-3
levulinate elimination side products, as previously reported.43 To
obtain the glycosyl donor 4, crude hemiacetal 10 was treated with
trichloroacetonitrile and catalytic 1,8-diazabicycloundec-7-ene
(DBU).
2. Results and discussion
With the appropriate building blocks in hand, fluorous-assisted
synthesis of tetrasaccharide 1 was accomplished (Scheme 3). Fluo-
rous acceptor 2 was coupled with 2 equiv of glycosyl donor 4 using
trimethylsilyl trifluoromethanesulfonate (TMSOTf) as promoter
and CH2Cl2 as solvent at 0 °C to give disaccharide 11 in high yield.
This product was isolated by F-SPE. Non-fluorinated side products
were removed by elution with MeOH/H2O 80:20 and the desired
fluorinated compound was recovered using acetone as the eluent.
NMR spectroscopy and mass spectrometry confirmed the structure
of disaccharide 11. Only the b anomer was formed due to neighbor-
ing-group participation of the N-phthalimido group. In previous
reports,44 we observed a loss of fluorous-tagged material during
F-SPE purification steps due to sample breakthrough, and fluori-
nated compounds were detected in the fluorophobic elution. To
For the synthesis of tetramer 1, we followed a 1 + 1 modular
approach, using the monosaccharide building blocks shown in
Scheme 1. A fluorous tag was introduced at position 6 of reducing
end unit 2. Fluorous-tag assisted approaches have been success-
fully applied to the synthesis of complex oligosaccharides,31–37
including GAG oligomers.38–40 The use of a fluorous tag reduces
the number of silica gel chromatographic purifications required
for the preparation of oligosaccharides since fluorinated intermedi-
ates can be easily separated from nonfluorinated side products by a
simple fluorous solid-phase extraction (F-SPE).41 Thus, the attach-
ment of a C8F17 tail to the 6 position of reducing end sugar 2
enabled the purification of growing chains by F-SPE. Moreover,
the fluorous-tag assisted reactions are run in solution and can be
monitored by standard TLC, NMR and mass spectrometry. Due to
the homogeneous solution-phase reaction conditions, lower
amounts of glycosyl donors are typically needed for the glycosyla-
tions compared to solid phase strategies.
Benzylidene acetals were chosen as temporary and orthogonal
protecting groups for further installation of sulfates and benzoyl
(Bz) and N-phthalimido (N-Phth) functionalities ensured the selec-
tive formation of the desired b glycosidic linkages (Scheme 1).
Levulinoyl (Lev) esters were employed to protect position 4 of glu-
cose 3 and position 3 of glucosamine 4 for subsequent chain elon-
gation. The trichloroacetimidate method was selected for the
construction of the glycosidic bonds.
overcome this problem, we employed here
a 9:1 DMF/H2O
mixture, instead of 100% DMF, as loading solvent.
Deprotection of the temporary levulinoyl group with hydrazine
monohydrate in pyridine/acetic acid buffer smoothly afforded
derivative 12 (Scheme 3). Synthesis of trisaccharide 13 proved to
be a challenging task. Initial glycosylation trial between fluorous
acceptor 12 and trichloroacetimidate 3, under TMSOTf activation
(5 mol% with respect to the donor) resulted in the formation of
trisaccharide 13 and the corresponding orthoester side product.
Trimethylsilyl etherification of 12 was also detected in the reaction
mixture and, for this reason, TMSOTf was replaced by
Scheme 2. Reagents and conditions: (a) C8F17(CH2)2C(@O)Cl, Et3N, DMAP, CH2Cl2,
0 °C, 81%; (b) PivCl, Et3N, DMAP, CH2Cl2, 0 °C, 82%; (c) Lev2O, DMAP, CH2Cl2, 99%; (d)
CAN, CH2Cl2/CH3CN/H2O, 81%; (e) Cl3CCN, K2CO3, CH2Cl2, 88%; (f) Lev2O, DMAP,
CH2Cl2, 90%; CAN, CH2Cl2/CH3CN/H2O, 0 °C, 96%; (g) Cl3CCN, DBU, CH2Cl2, 76%.
DMAP = 4-(N,N-dimethylamino)pyridine.
Scheme 1. Building blocks required for the synthesis of target tetrasaccharide 1.
Bn = benzyl; MP = 4-methoxyphenyl; Piv = pivaloyl; NPhth = N-phthalimido; Bz =
benzoyl; Lev = levulinoyl.
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S. Maza et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
3
Scheme 3. Reagents and conditions: (a) TMSOTf, CH2Cl2, 0 °C, 92%; (b) NH2NH2ꢀH2O, Py/AcOH, CH2Cl2, 91% (12); (c) 3, TBSOTf, CH2Cl2, 0 °C; (d) 4, TBSOTf, CH2Cl2, 0 °C, 52%
(three steps, from 12); (e) TFA, H2O, CH2Cl2, 95%; (f) SO3ꢀMe3N, DMF, 100 °C, MW heating, 72%.
tert-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf).45
When the glycosylation was run with higher amounts of Lewis acid
(20 mol% of TBSOTf with respect to the donor), orthoester forma-
tion was prevented but byproducts derived from hydrolysis of
the benzylidene group were observed. Therefore, careful adjust-
ment of the amount of promoter was required to avoid both the
acid-mediated cleavage of benzylidene functionalities and the for-
mation of orthoesters. Gratifyingly, glycosylation of 12 with 2.6
equiv. of 3 using TBSOTf (10 mol% with respect to the donor) as
activator at 0 °C cleanly gave trimer 13, as confirmed by NMR
and mass spectrometry analysis. After isolation by F-SPE, 13 was
selectively delevulinoylated to provide glycosyl acceptor 14. The
latter compound was condensated with donor 4 (3 equiv.) using
TBSOTf as the promoter at 0 °C. Pure tetramer 15 was obtained
in excellent yield after F-SPE and standard silica gel column chro-
matography. The overall yield for the assembly of the tetrasaccha-
ride, starting from monosaccharide 2, was 44%, which corresponds
to an 85% yield per reaction step. It is important to note that
selected building blocks displayed high reactivity in all the glyco-
sylations and just one cycle with a small excess of sugar donors
(2–3 equiv.) was required to complete these reactions.
Scheme 4. Reagents and conditions: (a) TBSOTf, CH2Cl2, 0 °C; (b) TFA, H2O, CH2Cl2,
78% (two steps, from 2); (c) SO3ꢀMe3N, DMF, 100 °C, MW heating, 93%; (d) ethylene
diamine, n-BuOH, 120 °C, MW heating; NaOH, MeOH; Ac2O, Et3N, MeOH, 76%; (e)
H2, Pd(OH)2/C, H2O/MeOH, 75%.
desired deacylated amine derivative and a benzoylated byproduct.
To completely remove the 2-O-benzoate group, further treatment
with aqueous NaOH in MeOH was needed. Selective N-acetylation
with acetic anhydride and triethylamine in MeOH proceeded
smoothly to afford disaccharide 19 in good yield. Finally,
hydrogenolysis of 19 gave disaccharide 20, demonstrating that
our protecting group design can deliver fully deprotected
derivatives.
Next, we studied the interaction between midkine and the syn-
thesized fully protected derivative 1. Midkine is a heparin-binding
growth factor involved in development, survival and migration of
target cells.48,49 This cytokine is overexpressed in most of human
malignant tumors. Notably, midkine plays important roles in sev-
eral diseases of the central nervous system, such as glioblastoma
and multiple sclerosis. Furthermore, midkine is implicated in cell
development during embryogenesis.50 There is a great interest in
the discovery of high-affinity ligands that could modulate the
activity of this protein.15,51,52 For the analysis of the tetramer-mid-
kine interaction, we employed a fluorescence polarization compe-
tition experiment previously developed in our group.52–54 In this
experiment, we measured the ability of compound 1 to block the
formation of a complex between midkine and a fluorescein labelled
heparin hexasaccharide,54 which is characterized by a high polar-
ization value. Thus, the fluorescence polarization (FP) of samples
Finally, 15 was transformed into the target molecule
1
(Scheme 3). The benzylidene groups were removed using trifluo-
roacetic acid (TFA) in wet CH2Cl2. The resulting tetraol 16 was
extensively sulfated, under microwave irradiation,46,47 to provide
1 in good yield. In this case, standard silica gel chromatography
was required to separate tetrasulfated sugar from partially sulfated
byproducts. The structure of 1 was confirmed by NMR and mass
spectroscopic analysis. NMR spectra showed the characteristic
downfield shifts of the proton and carbon signals at positions bear-
ing a sulfate group (H-4/H-6 GlcN: d = 3.75–3.26 ppm in 16; d =
4.74–3.96 ppm in 1; C-4 GlcN: d = 70.7–70.5 ppm in 16; d = 77.8–
75.9 ppm in 1; C-6 GlcN: d = 62.7–62.0 ppm in 16; d = 68.8–68.1
ppm in 1).
After successful assembly of sulfated tetrasaccharide 1, global
deprotection reactions were tested at the disaccharide stage
(Scheme 4). Disaccharide 17 was obtained by glycosylation
between 2 and 4 under TBSOTf catalysis followed by TFA-mediated
benzylidene hydrolysis. Microwave-assisted sulfation of com-
pound 17 gave derivative 18 in excellent 93% yield. 18 was then
treated with ethylene diamine in n-butanol at 120 °C under micro-
wave heating. Interestingly, mass spectrometry and TLC analysis
indicated the formation of a mixture of two compounds: the
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S. Maza et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
containing fixed amounts of protein and fluorescent probe in the
presence of increasing concentrations of 1 were recorded (Fig. 1).
We observed a decrease in the fluorescence polarization indicating
that 1 bound to midkine and displaced the fluorescent probe from
the complex. The measurements were carried out in PBS buffer
containing bovine serum albumin (BSA, 0.5% w/v) to prevent
non-specific binding. Compound 1 was soluble in PBS buffer at
100 mM concentration in the presence of 1% of DMSO. Mathemati-
cal curve fitting provided an IC50 value (Table 1, IC50 = 270 70
nM), defined as 1 concentration required for 50% inhibition. The
IC50 value and the error represent the average and the standard
deviation from three independent experiments (see Supporting
Information, Fig. S1). Interestingly, this relative binding affinity
was significantly higher than that reported for a fully protected
CS-like tetrasaccharide following the sequence GalNAc(4,6-di-
OSO3)-GlcA (IC50 = 1.3 mM).30 The binding affinity of 1 was also
much higher than that displayed by a deprotected, natural CS-E
tetramer (IC50 = 254 mM).30
The interaction between midkine and disulfated dimer 18 was
also evaluated (Fig. 1 and Supporting Information, Fig. S2). The
FP competition assay showed that 18 strongly bound to midkine,
in the high nanomolar range (Table 1, IC50 = 700 220 nM from
four independent experiments). Once again, this relative affinity
was remarkably higher than those previously reported for disul-
fated, fully protected disaccharides lacking the perfluorinated tag
(IC50 ranging from 15 to 20 mM).30 Our results suggest that the
presence of a fluorous tail enhances the binding of oligosaccharide
precursors to midkine. On the other hand, it is well known that
fully deprotected CS/GAG disaccharides exhibit weak binding affin-
ity for proteins. In fact, compounds 19 and 20 were tested using
our FP assay and no significant inhibition was detected at 250
mM concentration (Table 1, IC50 > 250 mM). These observations
demonstrate that the display of hydrophobic protecting groups,
150
100
50
-3
0
3
log [inhibitor concentration ( M)]
Fig. 2. Representative competition curve showing the ability of compound 1 to
inhibit the interaction between FGF-2 (97 nM) and fluorescent probe (10 nM). All
the FP values are the average of three replicate wells, with error bars showing the
standard deviations for these measurements. The reported IC50 value and the error
(2.4 1.3 mM) represent the average and the standard deviation from three
independent experiments.
in particular fluorinated tags, on oligosaccharide derivatives
strongly increases their interactions with midkine.
In addition, we studied the interaction between tetrasaccharide
1 and a different GAG-binding protein, FGF-2 (basic fibroblast
growth factor), with a key role in angiogenesis and tumor cell
growth.22,24,55 Compounds that selectively recognize one GAG-
binding protein are highly demanded. The analysis of the FP com-
petition curves afforded an IC50 value of 2.4 1.3 mM (see Fig. 2 and
Supporting Information, Fig. S3) that was again lower than the IC50
displayed by a CS-E persubstituted tetrasaccharide derivative
without the fluorous tag (42 mM).30 It is known that the intensity
of binding of FGF-2 toward CS-E is lower than that for heparin.56
Interestingly, our CS-E mimetic 1 displayed an FGF-2 binding affin-
ity comparable with those shown by heparin oligosaccharides, in
the low micromolar range (for example, KD = 0.4–6 mM for heparin
tetrasaccharides).8,57 However, the FGF-2 binding affinity of 1 was
clearly higher than that previously reported for a CS-E tetrasaccha-
ride (IC50 = 2.4 mM against 271 mM).30 On the other hand, although
1 strongly bound to FGF-2, our results suggest that this compound
presents a slight degree of selectivity for midkine over FGF-2
(IC50 = 0.27 mM versus 2.4 mM).
180
18
1
160
140
120
100
80
60
3. Conclusions
40
-3
-2
-1
0
1
2
3
4
Compounds that strongly interact with GAG-binding proteins,
such as midkine and FGF-2, are highly demanded because these
molecules can potentially regulate biological processes like angio-
genesis and tumor cell growth. We previously found that sulfated
CS-E oligosaccharides, displaying the hydroxyl, carboxylate and
amine functionalities masked by different protecting groups, are
excellent ligands for these proteins.30 Based on these results, we
envisaged the preparation of tetrasaccharide 1 as a CS-E mimetic.
In this molecule, GalN and GlcA units, typical of CS structure, were
replaced by more accessible and reactive GlcN and Glc building
blocks, while maintaining the cluster of sulfate groups at positions
4 and 6 of the hexosamine moiety. For the synthesis of this CS-E
log [inhibitor concentration ( M)]
Fig. 1. Representative competition curves showing the ability of compounds 1 and
18 to inhibit the interaction between midkine and the fluorescent probe. The
fluorescence polarization of samples containing midkine (63 nM), fluorescent probe
(10 nM) and increasing concentrations of inhibitors were measured and the
resulting curves were fitted to the equation for a one-site competitive interaction
model in order to calculate the IC50 values. All the polarization values are the
average of three replicate wells. At least three independent experiments were
carried out for each IC50 calculation.
Table 1
IC50 values for compounds 1, 18–20 obtained from FP midkine competition assays.
analogue, we successfully employed
a
fluorous-supported
approach. The fluorous tag at position 6 of the reducing end
allowed the purification of reaction intermediates by simple and
quick F-SPE, facilitating the access to these molecules. Importantly,
Compound
1
18
19
20
IC50 (mM)
0.27 0.07
0.70 0.22
>250
>250
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S. Maza et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
5
the designed monosaccharide building blocks showed high reactiv-
ity in all glycosylation couplings.
organic layer was dried (MgSO4), filtered and concentrated in
vacuo. The residue was purified by column chromatography
(CH2Cl2-MeOH 100:0 ? 99.5:0.5) to afford 2 (894 mg, 81%) as a
Our FP experiment revealed that tetramer 1, and also disaccha-
ride 18, interacted with midkine in the nanomolar range (IC50 of
270 and 700 nM, respectively). On the other hand, tetrasaccharide
1 bound to FGF-2 in the low micromolar range (IC50 = 2.4 mM). All
these relative binding affinities were significantly higher than
those obtained for other fully protected oligosaccharide precursors
with the same length, but lacking the fluorinated tag.30 We
hypothesized that the presence of the fluorous protecting group
strongly enhanced the protein binding, although the influence of
other structural differences cannot be ruled out. Importantly, nat-
ural deprotected CS-E di- and tetrasaccharides present very low
protein affinities (in the high micromolar range) compared to fully
protected oligosaccharides. Overall, our results provide valuable
information for the design and synthesis of sulfated oligosaccha-
ride derivatives as potent GAG mimetics.
white amorphous solid. TLC (toluene-EtOAc 3:1) Rf 0.58;
20
[a
]
ꢁ11° (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3/CD3OD 3:1): d
D
7.97 (m, 2H, Ar), 7.59 (m, 1H, Ar), 7.44 (m, 2H, Ar), 7.18–7.09 (m,
5H, Ar), 6.85 (m, 2H, Ar), 6.73 (m, 2H, Ar), 5.38 (t, 1H, H-2), 5.00
(d, 1H, J1,2 = 8.0 Hz, H-1), 4.82 (d, 1H, CH2(Bn)), 4.70 (d, 1H, CH2(Bn)),
4.53 (dd, 1H, J5,6 = 1.6 Hz, J6,6 = 11.8 Hz, H-6a), 4.39 (dd, 1H, J5,6 = 5.8
Hz, H-6b), 3.75–3.68 (m, 6H, H-3, H-4, H-5, Me (OMP)), 2.72–2.44
(m, 4H, –CH2–CH2–); 13C NMR (100 MHz, CDCl3/CD3OD 3:1): d
171.3, 165.7 (2 ꢂ CO), 155.5–114.4 (Ar), 100.6 (C-1), 82.0 (C-3),
74.8 (CH2(Bn)), 74.0 (C-4 or C-5), 73.2 (C-2), 70.3 (C-4 or C-5), 64.0
(C-6), 55.3 (Me (OMP)), 26.3 (–CH2–), 25.3 (–CH2–); 19F NMR (376
MHz, CDCl3/CD3OD 3:1):
d
ꢁ81.19 (t, 3F), ꢁ114.91 (m, 2F),
ꢁ122.05 (m, 6F), ꢁ122.98 (m, 2F), ꢁ123.66 (m, 2F), ꢁ126.43 (m,
2F); HR MS: m/z: calcd for
977.1577 [M+Na]+.
C38H31F17O9Na: 977.1589; found:
4. Experimental
4.4. 4-Methoxyphenyl 2-O-benzoyl-3-O-benzyl-6-O-pivaloyl-b-
D-glu-
4.1. General synthetic procedures
copyranoside (6)
Thin layer chromatography (TLC) analyses were performed on
silica gel 60 F254 precoated on aluminium plates (Merck) and the
compounds were detected by staining with sulfuric acid/ethanol
(1:9), with cerium (IV) sulfate (10 g)/phosphomolybdic acid (13
g)/sulfuric acid (60 mL) solution in water (1 L), or with anisalde-
hyde solution [anisaldehyde (25 mL) with sulfuric acid (25 mL),
ethanol (450 mL) and acetic acid (1 mL)], followed by heating at
over 200 °C. Column chromatography was carried out on silica
gel 60 (0.2–0.5 mm, 0.2–0.063 mm or 0.040–0.015 mm; Merck).
A solution of DMAP (99 mg, 0.81 mmol), triethylamine (0.3 mL,
2.15 mmol) and diol 5 (516 mg, 1.07 mmol) in CH2Cl2 (15 mL) was
cooled to 0 °C, and treated with pivaloyl chloride (172 mL, 1.39
mmol). After stirring for 2 h at 0 °C, the reaction mixture was
diluted with CH2Cl2, and washed with saturated NaHCO3 aqueous
solution and brine. The organic layer was dried (MgSO4), filtered
and concentrated in vacuo. The residue was purified by column
chromatography (toluene-EtOAc 4:1) to afford 6 (498 mg, 82%) as
a
[a]
white amorphous solid. TLC (toluene-EtOAc 4:1) Rf 0.49;
20
ꢁ1° (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): d 8.09 (m, 2H,
Optical rotations were determined with
a Perkin-Elmer 341
D
polarimeter. 1H, 19F and 13C NMR spectra were acquired on Bruker
DPX-300, Avance III-400 and DRX-500 spectrometers. Unit A refers
to the reducing end monosaccharide in the NMR data. Electrospray
mass spectra (ESI MS) were carried out with an Esquire 6000 ESI-
Ion Trap from Bruker Daltonics. High resolution mass spectra (HR
MS) were carried out by CITIUS (Universidad de Sevilla). Micro-
wave-based sulfation reactions were performed using a Biotage
Initiator Eight synthesizer in sealed reaction vessels. Compound 9
was purchased from Carbosynth. FluoroFlash silica gel was pur-
chased from Sigma-Aldrich.
Ar), 7.61 (m, 1H, Ar), 7.49 (m, 2H, Ar), 7.24 (m, 5H, Ar), 6.93 (m,
2H, Ar), 6.76 (m, 2H, Ar), 5.50 (dd, 1H, J1,2 = 8.1 Hz, J2,3 = 9.1 Hz,
H-2), 5.01 (d, 1H, H-1), 4.76 (2d, 2H, CH2(Bn)), 4.42 (m, 2H, H-6),
3.78–3.69 (m, 6H, H-3, H-4, H-5, Me (OMP)), 1.27 (s, 9H, C(CH3)3);
13C NMR (100 MHz, CDCl3): d 179.1, 165.2 (2 ꢂ CO), 155.6–114.4
(Ar), 101.0 (C-1), 82.0 (C-3), 74.7 (CH2(Bn)), 74.2 (C-4 or C-5), 73.4
(C-2), 70.3 (C-4 or C-5), 63.4 (C-6), 55.6 (Me (OMP)), 39.0 (C
(CH3)3), 27.2 (C(CH3)3); HR MS: m/z: calcd for C32H36O9Na:
587.2252; found: 587.2258 [M+Na]+.
4.5. 2-O-Benzoyl-3-O-benzyl-4-O-levulinoyl-6-O-pivaloyl-a,b-D-glu-
copyranose (8)
4.2. General procedure for F-SPE
FluoroFlash silica gel (5 g, Fluorous Technologies, Inc) was
placed in a glass chromatography column (1.7 cm diameter). The
F-SPE column was washed with DMF (2 mL) and then precondi-
tioned with MeOH/H2O 80:20 (15 mL). Next, the crude sample
(100–300 mg) was dissolved in DMF/H2O 9:1 (0.8 mL) and loaded
on the column. The fluorophobic elution was carried out with 15
mL of MeOH/H2O 80:20. The fluorous compounds were then eluted
using 100% acetone (20 mL). To regenerate the F-SPE column, we
washed with additional acetone (20 mL). DMF washing step can
be omitted when reusing the F-SPE column.
LevOH (1.5 mL, 14.3 mmol) was added at 0 °C to a solution of
1,3-dicyclohexylcarbodiimide (1.5 g, 7.2 mmol) in CH2Cl2 (20 mL).
After stirring for 5 min at room temperature, the mixture was
cooled (0 °C) and filtered, and the urea precipitate was washed
with additional CH2Cl2 (4 mL). The resulting Lev2O solution (7.2
mmol) was added at room temperature to a mixture of 6 (1.35 g,
2.39 mmol) and DMAP (44 mg, 0.36 mmol). The mixture was stir-
red for 1.5 h, diluted with CH2Cl2, and washed with saturated
aqueous NaHCO3, and H2O. The organic phase was dried (MgSO4),
filtered and concentrated to dryness. The residue was suspended in
hexane/EtOAc (1:1, 19 mL) and the mixture was filtered. The solid
was washed with additional hexane/EtOAc (1:1) to give 7 as a
white amorphous solid (1.58 g, 99%) that was used without further
purification. TLC (toluene-EtOAc 5:1) Rf 0.42; 1H NMR (300 MHz,
CDCl3): d 8.02 (m, 2H, Ar), 7.60 (m, 1H, Ar), 7.46 (m, 2H, Ar), 7.16
4.3. 4-Methoxyphenyl 2-O-benzoyl-3-O-benzyl-6-O-4,4,5,5,6,6,7,7,8,8,
9,9,10,10,11,11,11-heptadecafluoroundecanoyl-b-d-glucopyranoside (2)
A solution of DMAP (35 mg, 0.29 mmol), triethylamine (320 mL,
2.3 mmol) and diol 5 (556 mg, 1.16 mmol) in CH2Cl2 (8 mL) was
cooled to 0 °C, and treated with heptadecafluoroundecanoyl chlo-
ride (4 mL of a 0.32 M solution in dry CH2Cl2). After stirring for 2
h at 0 °C, the reaction mixture was diluted with CH2Cl2, and
washed with saturated NaHCO3 aqueous solution and brine. The
(m, 5H, Ar), 6.91 (m, 2H, Ar), 6.73 (m, 2H, Ar), 5.54 (dd, 1H, J2,3
9.1 Hz, H-2), 5.22 (t, 1H, J3,4 = J4,5 = 9.5 Hz, H-4), 5.01 (d, 1H, J1,2
=
=
7.7 Hz, H-1), 4.63 (2d, 2H, CH2(Bn)), 4.32 (dd, 1H, H-6), 4.13 (dd,
1H, H-6), 3.94 (t, 1H, H-3), 3.81 (m, 1H, H-5), 3.74 (s, 3H, Me
(OMP)), 2.76–2.49 (m, 4H, CH2(Lev)), 2.17 (s, 3H, CH3(Lev)), 1.23
Please cite this article in press as: Maza S., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.01.022

6
S. Maza et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
(s, 9H, C(CH3)3); ESI MS: m/z: calcd for C37H42O11Na: 685.3; found:
685.3 [M+Na]+.
CH2Cl2/MeCN (1:2; 54 mL). After stirring for 1.5 h at 0 °C, the reac-
tion mixture was diluted with EtOAc, washed with H2O, saturated
aqueous NaHCO3, and H2O. The organic phase was dried (MgSO4),
filtered and concentrated to dryness. EtOAc (2.5 mL) and then hex-
ane (7.5 mL) were added to the residue at 0 °C. After stirring, the
resulting suspension was filtered and the solid was washed with
cold hexane to afford 10 as a yellow amorphous solid (904 mg,
96%). Analytical data were in good agreement with those described
in literature previously.43
CAN (9.0 mL of a 1.06 M solution in H2O) was added to a solu-
tion of 7 (1.58 g, 2.38 mmol) in CH2Cl2/MeCN (1:2; 81 mL). After
stirring for 1.5 h at room temperature, the reaction mixture was
diluted with EtOAc, washed with H2O, saturated aqueous NaHCO3,
and H2O. The organic phase was dried (MgSO4), filtered and con-
centrated to dryness. The residue was purified by column chro-
matography (toluene-EtOAc 4:1) to afford 8 as a yellow foam
(1.08 g, 81%). TLC (toluene-EtOAc 2:1) Rf 0.40 and 0.37 (
a/b anom-
ers); 1H NMR (400 MHz, CDCl3) (data for
a
anomer): d 8.04 (m, 2H,
4.8. O-(4,6-O-Benzylidene-2-deoxy-3-O-levulinoyl-2-phthalimido-
b- -glucopyranosyl) trichloroacetimidate (4)
a,
Ar), 7.58 (m, 1H, Ar), 7.44 (m, 2H, Ar), 7.18 (m, 5H, Ar), 5.56 (t, 1H,
J1,2 = J1,OH = 3.0 Hz, H-1), 5.20 (t, 1H, J3,4 = J4,5 = 9.7 Hz, H-4), 5.08
(dd, 1H, J2,3 = 9.9 Hz, H-2), 4.74, 4.67 (2d, 2H, CH2(Bn)), 4.26–4.18
(m, 3H, H-3, H-5, H-6a), 4.12 (dd, 1H, J5,6b = 3.7 Hz, J6a,6b = 12.1
Hz, H-6b), 3.75 (d, 1H, OH), 2.75–2.38 (m, 4H, CH2(Lev)), 2.14 (s,
3H, CH3(Lev)), 1.21 (s, 9H, C(CH3)3); 13C NMR (100 MHz, CDCl3)
D
Trichloroacetonitrile (1.2 mL, 12 mmol) and catalytic DBU (18
mL, 0.12 mmol) were added to a solution of 10 (596 mg, 1.2 mmol)
in dry CH2Cl2 (5 mL). After stirring for 3 h at room temperature, the
reaction mixture was concentrated to dryness. The residue was
purified by a short silica gel column (toluene-EtOAc 4:1 + 1%
Et3N) to afford 4 as a yellow foam (584 mg, 76%). Analytical data
were in good agreement with those described in literature
previously.43
(data for
a
anomer): d 206.5, 178.6, 171.4, 165.9 (4 ꢂ CO), 138.1–
125.4 (Ar), 90.4 (C-1), 77.1 (C-3), 75.0 (CH2(Bn)), 74.1 (C-2), 70.0
(C-4), 67.9 (C-5), 61.8 (C-6), 39.0 (C(CH3)3), 38.0 (CH2(Lev)), 29.9
(CH3(Lev)), 28.0 (CH2(Lev)), 27.2 (C(CH3)3); HR MS: m/z: calcd for
C
30H36O10Na: 579.2201; found: 579.2192 [M+Na]+.
4.9. 4-Methoxyphenyl O-(4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-
4.6. O-(2-O-Benzoyl-3-O-benzyl-4-O-levulinoyl-6-O-pivaloyl-
a
,b-
D
-
2-phthalimido-b-D-glucopyranosyl)-(1 ? 4)-O-(2-O-benzoyl-3-O-benzyl-
glucopyranosyl) trichloroacetimidate (3)
6-O-pivaloyl-b-
D-glucopyranosyl)-(1 ? 3)-O-(4,6-O-benzylidene-2-
deoxy-2-phthalimido-b-
O-benzyl-6-O-4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluo-
roundecanoyl-b- -glucopyranoside (15)
D-glucopyranosyl)-(1 ? 4)-2-O-benzoyl-3-
Trichloroacetonitrile (2.4 mL, 26 mmol) and K2CO3 (214 mg,
1.55 mmol) were added to 8 (717 mg, 1.29 mmol) in dry CH2Cl2
(8 mL) under an argon atmosphere. After stirring at room temper-
ature for 4 h, the mixture was filtered and concentrated in vacuo.
The residue was purified by using a short silica gel column
(toluene-EtOAc 3:1 + 1% Et3N) to give 3 as a white amporphous
D
Donor 4 (130 mg, 0.20 mmol) and aceptor 2 (97 mg, 0.10 mmol)
were dissolved in dry CH2Cl2 (4 mL) in the presence of activated 4
Å molecular sieves (MS, 300 mg). The reaction mixture was stirred,
under an argon atmosphere, for 10 min at 0 °C and TMSOTf (200 mL
of a 0.14 M solution in dry CH2Cl2) was added. After stirring for 1.5
h at 0 °C, the reaction mixture was quenched with triethylamine,
filtered, and then concentrated under reduced pressure. The crude
product was purified using a fluorous solid-phase extraction (F-
SPE) column. Nonfluorous compounds were eluted with 80:20
MeOH/water and the fluorous product was eluted by 100% acetone.
This acetone fraction was concentrated to give disaccharide 11 as a
white amporphous solid (134 mg, 92%). TLC (hexane-EtOAc 3:2) Rf
0.42; 1H NMR (400 MHz, CDCl3): d 8.01 (m, 2H, Ar), 7.89–7.76 (m,
4H, Ar), 7.61 (m, 1H, Ar), 7.49–7.23 (m, 12H, Ar), 6.82 (m, 2H, Ar),
6.70 (m, 2H, Ar), 5.90 (t, 1H, J2,3 = J3,4 = 9.7 Hz, H-30), 5.64 (d, 1H,
solid (795 mg, 88%, mixture of
a/b anomers). TLC (CH2Cl2-MeOH
60:1) Rf 0.44 and 0.38; 1H NMR (400 MHz, CDCl3) (data for b
anomer): d 8.61 (s, 1H, NH), 7.97 (m, 2H, Ar), 7.57 (m, 1H, Ar),
7.43 (m, 2H, Ar), 7.18 (m, 5H, Ar), 5.95 (d, 1H, J1,2 = 7.8 Hz, H-1),
5.58 (dd, 1H, J2,3 = 8.8 Hz, H-2), 5.26 (t, 1H, J3,4 = J4,5 = 9.2 Hz, H-
4), 4.64 (br s, 2H, CH2(Bn)), 4.30–4.11 (m, 2H, H-6a, H-6b), 3.95
(t, 1H, H-3), 3.90 (ddd, 1H, J5,6a = 2.7 Hz, J5,6b = 5.5 Hz, H-5), 2.76–
2.39 (m, 4H, CH2(Lev)), 2.16 (s, 3H, CH3(Lev)), 1.21 (s, 9H, C
(CH3)3); 13C NMR (100 MHz, CDCl3) (data for b anomer): d 206.2,
178.3, 171.4, 164.8 (4 ꢂ CO), 161.4 (C@NH), 137.6–125.4 (Ar),
96.0 (C-1), 90.5 (CCl3), 79.2 (C-3), 74.0 (CH2(Bn)), 73.3 (C-5), 71.8
(C-2), 69.7 (C-4), 61.7 (C-6), 39.0 (C(CH3)3), 38.0 (CH2(Lev)), 29.9
(CH3(Lev)), 28.0 (CH2(Lev)), 27.2 (C(CH3)3); ESI MS: m/z: calcd for
J1,2 = 8.2 Hz, H-10), 5.45 (m, 2H, PhCHO, H-2), 5.03 (d, 1H, J1,2
=
C
32H36Cl3NO10Na: 722.1; found: 722.2 [M+Na]+.
6.5 Hz, H-1), 4.83 (s, 2H, CH2(Bn)), 4.46 (br d, 1H, H-6), 4.33 (t,
1H, H-20), 4.11 (m, 2H, H-60, H-4), 3.90 (t, 1H, J2,3 = J3,4 = 7.3 Hz,
H-3), 3.78–3.66 (m, 6H, H-40, H-5, H-6, Me (OMP)), 3.59–3.46 (m,
2H, H-50, H-60), 2.59–2.21 (m, 8H, –CH2–CH2–, CH2(Lev)), 1.88 (s,
3H, CH3(Lev)); 13C NMR (100 MHz, CDCl3, selected data from HSQC
experiment): d 101.5 (PhCHO), 99.4 (C-1), 98.9 (C-10), 80.7 (C-3),
78.7 (C-40 or C-5), 77.1 (C-4), 73.6 (CH2(Bn)), 72.4 (C-2), 72.2 (C-
40 or C-5), 69.4 (C-30), 68.2 (C-60), 65.9 (C-50), 62.3 (C-6), 55.4 (C-
20), 55.2 (Me (OMP)); 19F NMR (376 MHz, CDCl3): d ꢁ80.70 (t,
3F), ꢁ114.52 (m, 2F), ꢁ121.74 (m, 6F), ꢁ122.65 (m, 2F), ꢁ123.36
(m, 2F), ꢁ126.06 (m, 2F); ESI MS: m/z: calcd for C64H54F17NO17Na:
1454.3; found: 1454.2 [M+Na]+.
Compound 11 (146 mg, 0.10 mmol) was dissolved in CH2Cl2
(1.5 mL) and hydrazine monohydrate (0.41 mL of a 0.5 M solution
in Py/AcOH 3:2) was added. After stirring at room temperature for
1 h, the reaction mixture was quenched with acetone (0.2 mL). The
mixture was diluted with CH2Cl2 and washed with 1 M HCl aque-
ous solution, saturated NaHCO3 aqueous solution and H2O. The
organic layer was dried (MgSO4), filtered and concentrated in
vacuo. The crude product was purified using a fluorous solid-phase
extraction (F-SPE) column. Nonfluorous compounds were eluted
4.7. 4,6-O-Benzylidene-2-deoxy-3-O-levulinoyl-2-phthalimido-a,b-D-
glucopyranose (10)
LevOH (4.2 mL, 41 mmol) was added at 0 °C to a solution of 1,3-
dicyclohexylcarbodiimide (4.2 g, 20.5 mmol) in CH2Cl2 (40 mL).
After stirring for 5 min at room temperature, the mixture was
cooled and filtered, and the urea precipitate was washed with
additional CH2Cl2 (5 mL). The resulting Lev2O solution (20.5 mmol)
was added at room temperature to a mixture of 9 (3.44 g, 6.83
mmol) and DMAP (125 mg, 1.0 mmol). The mixture was stirred
for 1.5 h, diluted with CH2Cl2, and washed with saturated aqueous
NaHCO3, and H2O. The organic phase was dried (MgSO4), filtered
and concentrated to dryness. The residue was suspended in hex-
ane/EtOAc (1:1, 30 mL) and the mixture was filtered. The solid
was washed with additional hexane/EtOAc (1:1, 10 mL) to give
the 3-O-levulinated monosaccharide as a white amorphous solid
(3.70 g, 90%) that was used without further purification.
CAN (6.0 mL of a 1.27 M solution in H2O) was added at 0 °C to a
solution of the levulinated compound (1.15 g, 1.91 mmol) in
Please cite this article in press as: Maza S., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.01.022

S. Maza et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
7
with 80:20 MeOH/water and the fluorous product was eluted by
100% acetone. This acetone fraction was concentrated to give dis-
accharide 12 as a white amporphous solid (124 mg, 91%). TLC (hex-
ane-EtOAc 1:1) Rf 0.61; 1H NMR (400 MHz, CDCl3): d 7.99 (m, 2H,
Ar), 7.87 (m, 2H, Ar), 7.74 (m, 2H, Ar), 7.58 (m, 1H, Ar), 7.46–7.21
(77 mg). TLC (hexane-EtOAc 3:2) Rf 0.5; 1H NMR (400 MHz, CDCl3):
d 7.96 (m, 2H, Ar), 7.57 (m, 1H, Ar), 7.51–7.38 (m, 12H, Ar), 7.27–
7.18 (m, 9H, Ar), 7.05 (m, 3H, Ar), 6.95 (m, 2H, Ar), 6.74 (m, 2H,
Ar), 6.65 (m, 2H, Ar), 5.53 (s, 1H, PhCHO), 5.37 (t, 1H, J1,2 = J2,3
7.2 Hz, H-2A), 5.30 (d, 1H, J1,2 = 8.5 Hz, H-1B), 4.93 (d, 1H, J1,2
=
=
(m, 12H, Ar), 6.79 (m, 2H, Ar), 6.68 (m, 2H, Ar), 5.49 (d, 1H, J1,2
=
6.7 Hz, H-1A), 4.92 (br t, 1H, H-2C), 4.75 (m, 2H, CH2(Bn)), 4.68
(br dd, 1H, H-3B), 4.63 (d, 1H, J1,2 = 8.1 Hz, H-1C), 4.48, 4.42 (2d,
2H, CH2(Bn), 4.27 (m, 2H, H-2B, H-6aA), 4.09 (m, 2H, H-6aC, H-
6aB), 3.98 (m, 2H, H-6bC, H-4A), 3.80 (t, 1H, J3,4 = 7.7 Hz, H-3A),
3.77 (t, 1H, J3,4 = J4,5 = 9.0 Hz, H-4B), 3.67 (s, 3H, Me (OMP)), 3.60
(dd, 1H, J5,6b = 4.5 Hz, J6a,6b = 12.0 Hz, H-6bA), 3.56–3.50 (m, 2H,
8.4 Hz, H-10), 5.44 (s, 1H, PhCHO), 5.42 (br t, 1H, H-2), 5.00 (d,
1H, J1,2 = 6.8 Hz, H-1), 4.80 (s, 2H, CH2(Bn)), 4.60 (br t, 1H, H-30),
4.44 (br d, 1H, H-6a), 4.25 (dd, 1H, J2,3 = 10.3 Hz, H-20), 4.07 (m,
2H, H-60a, H-4), 3.87 (t, 1H, J2,3 = J3,4 = 7.6 Hz, H-3), 3.79 (dd, 1H,
J5,6b = 4.4 Hz, J6a,6b = 12.0 Hz, H-6b), 3.71–3.64 (m, 4H, H-5, Me
(OMP)), 3.52–3.43 (m, 3H, H-40, H-50, H-60b), 2.41 (br d, 1H, OH),
2.24 (m, 4H, –CH2–CH2–); 13C NMR (100 MHz, CDCl3, selected data
from HSQC experiment): d 101.5 (PhCHO), 99.5 (C-1), 98.8 (C-10),
81.7 (C-40), 80.3 (C-3), 76.8 (C-4), 73.4 (CH2(Bn)), 72.3 (C-2), 72.2
(C-5), 68.3 (C-60), 68.0 (C-30), 66.0 (C-50), 62.4 (C-6), 56.7 (C-20),
55.1 (Me (OMP)); 19F NMR (376 MHz, CDCl3): d ꢁ80.70 (t, 3F),
ꢁ114.54 (m, 2F), ꢁ121.73 (m, 6F), ꢁ122.64 (m, 2F), ꢁ123.35 (m,
2F), ꢁ126.06 (m, 2F); ESI MS: m/z: calcd for C59H48F17NO15Na:
1356.3; found: 1356.0 [M+Na]+.
H-6bB, H-5A), 3.48–3.41 (m, 2H, H-4C, H-5B), 3.36 (t, 1H, J2,3
=
J3,4 = 9.2 Hz, H-3C), 2.91 (m, 1H, H-5C), 2.67 (d, 1H, J4,OH = 3.3 Hz,
OH), 2.27–2.10 (m, 4H, –CH2–CH2–), 1.22 (s, 9H, C(CH3)3); 13C
NMR (100 MHz, CDCl3, selected data from HSQC experiment): d
101.8 (PhCHO), 100.3 (C-1C), 99.4 (C-1A), 98.8 (C-1B), 82.1 (C-
3C), 80.8 (C-3A, C-4B), 77.0 (C-4A), 75.7 (C-3B), 74.4 (CH2(Bn)),
74.0 (CH2(Bn)), 73.7 (C-2C, C-5C), 72.6 (C-2A), 72.5 (C-5A), 69.7
(C-4C), 68.7 (C-6B), 66.3 (C–5B), 62.6 (C-6A), 62.3 (C-6C), 55.7 (C-
2B), 55.5 (Me (OMP)); ESI MS: m/z: calcd for C84H76F17NO22Na:
1796.4; found: 1795.9 [M+Na]+.
Donor 4 (82 mg, 0.128 mmol) and aceptor 14 (76 mg, 0.043
mmol) were dissolved in dry CH2Cl2 (1.6 mL) in the presence
of activated 4 Å MS (120 mg). The reaction mixture was stirred,
under an argon atmosphere, for 10 min at 0 °C and TBSOTf
(140 mL of a 0.064 M solution in dry CH2Cl2) was added. After
stirring for 35 min at 0 °C, the reaction mixture was quenched
with triethylamine, filtered, and then concentrated under
reduced pressure. The crude product was first purified using a
fluorous solid-phase extraction (F-SPE) column. Nonfluorous
compounds were eluted with 80:20 MeOH/water and the fluo-
rous product was eluted by 100% acetone. This acetone fraction
was concentrated in vacuo and then purified by a silica gel col-
umn (toluene-EtOAc 7:1) to afford 15 (54 mg, 52% from 12; 3
Donor 3 (83 mg, 0.118 mmol) and aceptor 12 (62 mg, 0.046
mmol) were dissolved in dry CH2Cl2 (1.4 mL) in the presence
of activated 4 Å MS (105 mg). The reaction mixture was stirred,
under an argon atmosphere, for 10 min at 0 °C and TBSOTf
(91 mL of a 0.13 M solution in dry CH2Cl2) was added. After stir-
ring for 50 min at 0 °C, the reaction mixture was quenched with
triethylamine, filtered, and then concentrated under reduced
pressure. The crude product was purified using
a fluorous
solid-phase extraction (F-SPE) column. Nonfluorous compounds
were eluted with 80:20 MeOH/water and the fluorous product
was eluted by 100% acetone. This acetone fraction was concen-
trated to give trisaccharide 13 as a white amporphous solid
(85 mg). TLC (hexane-EtOAc 3:2) Rf 0.36; 1H NMR (400 MHz,
CDCl3): d 7.96 (m, 2H, Ar), 7.57 (m, 1H, Ar), 7.52–7.39 (m,
12H, Ar), 7.27–7.16 (m, 9H, Ar), 7.02 (m, 3H, Ar), 6.90 (m, 2H,
Ar), 6.74 (m, 2H, Ar), 6.65 (m, 2H, Ar), 5.53 (s, 1H, PhCHO),
5.37 (t, 1H, J1,2 = J2,3 = 7.2 Hz, H-2A), 5.29 (d, 1H, J1,2 = 8.4 Hz,
H-1B), 5.09 (t, 1H, J3,4 = J4,5 = 9.5 Hz, H-4C), 5.02 (br t, 1H,
H-2C), 4.93 (d, 1H, J1,2 = 6.7 Hz, H-1A), 4.75 (m, 2H, CH2(Bn)),
4.69 (t, 1H, J2,3 = J3,4 = 9.4 Hz, H-3B), 4.63 (d, 1H, J1,2 = 8.1 Hz,
H-1C), 4.36–4.24 (m, 4H, CH2(Bn), H-6aA, H-2B), 4.06 (m, 2H,
H-6aB, H-6aC), 3.96 (dd, 1H, J3,4 = 7.7 Hz, J4,5 = 9.4 Hz, H-4A),
3.80 (t, 1H, H-3A), 3.77–3.70 (m, 2H, H-4B, H-6bC), 3.66 (s, 3H,
Me (OMP)), 3.60 (dd, 1H, J5,6b = 4.4 Hz, J6a,6b = 12.0 Hz, H-6bA),
3.58–3.49 (m, 3H, H-3C, H-6bB, H-5A), 3.43 (td, 1H, J5,6 = 4.5
Hz, J5,6 = J4,5 = 9.5 Hz, H-5B), 3.09 (m, 1H, H-5C), 2.65–2.12 (m,
8H, –CH2–CH2–, CH2(Lev)), 2.09 (s, 3H, CH3(Lev)), 1.22 (s, 9H, C
(CH3)3); 13C NMR (100 MHz, CDCl3, selected data from HSQC
experiment): d 101.8 (PhCHO), 100.3 (C-1C), 99.6 (C-1A), 98.8
(C-1B), 80.7 (C-3A, C-4B), 80.4 (C-3C), 77.0 (C-4A), 75.7 (C-3B),
74.1 (CH2(Bn)), 73.5 (C-2C, CH2(Bn)), 72.8 (C-2A), 72.5 (C-5A),
71.9 (C-5C), 69.1 (C-4C), 68.7 (C-6B), 66.4 (C-5B), 62.6 (C-6A),
61.1 (C-6C), 55.8 (C-2B), 55.5 (Me (OMP)); ESI MS: m/z: calcd
for C89H82F17NO24Na: 1894.5; found: 1894.1 [M+Na]+.
Compound 13 (85 mg, 0.045 mmol) was dissolved in CH2Cl2
(1.7 mL) and hydrazine monohydrate (182 mL of a 0.5 M solution
in Py/AcOH 3:2) was added. After stirring at room temperature
for 1.5 h, the reaction mixture was quenched with acetone (0.26
mL). The mixture was diluted with CH2Cl2 and washed with 1 M
HCl aqueous solution, saturated NaHCO3 aqueous solution and
H2O. The organic layer was dried (MgSO4), filtered and concen-
trated in vacuo. The crude product was purified using a fluorous
solid-phase extraction (F-SPE) column. Nonfluorous compounds
were eluted with 80:20 MeOH/water and the fluorous product
was eluted by 100% acetone. This acetone fraction was concen-
trated to give trisaccharide 14 as a white amporphous solid
steps) as a white amorphous solid. TLC (toluene-EtOAc 5:1) Rf
20
0.28; [
a]
+4° (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
d
D
7.96–7.81 (m, 6H, Ar), 7.56 (m, 1H, Ar), 7.50–7.16 (m, 23H, Ar),
7.07–6.90 (m, 8H, Ar), 6.74 (m, 2H, Ar), 6.64 (m, 2H, Ar), 5.80
(t, 1H, J2,3 = J3,4 = 9.8 Hz, H-3D), 5.40 (s, 1H, PhCHO), 5.39 (d, 1H,
J1,2 = 7.8 Hz, H-1D), 5.36 (t, 1H, J1,2 = J2,3 = 7.2 Hz, H-2A), 5.35 (s,
1H, PhCHO), 5.26 (d, 1H, J1,2 = 8.4 Hz, H-1B), 4.92 (d, 1H, J1,2
=
6.7 Hz, H-1A), 4.85 (t, 1H, J1,2 = J2,3 = 8.6 Hz, H-2C), 4.72 (m, 2H,
CH2(Bn)), 4.65 (d, 1H, CH2(Bn)), 4.55 (dd, 1H, J2,3 = 10.2 Hz,
J3,4 = 8.7 Hz, H-3B), 4.44 (d, 1H, J1,2 = 8.1 Hz, H-1C), 4.35 (d, 1H,
CH2(Bn)), 4.25 (br dd, 1H, H-6aA), 4.22–4.13 (m, 3H, H-2B, H-
6aC, H-2D), 4.07 (dd, 1H, J5,6a = 4.8 Hz, J6a,6b = 10.7 Hz, H-6aD),
3.99 (dd, 1H, J5,6a = 4.6 Hz, J6a,6b = 10.4 Hz, H-6aB), 3.94 (dd, 1H,
J3,4 = 8.2 Hz, J4,5 = 9.3 Hz, H–4A), 3.88 (t, 1H, J3,4 = J4,5 = 9.2 Hz,
H-4C), 3.78 (t, 1H, H-3A), 3.66 (s, 3H, Me (OMP)), 3.63 (t, 1H,
J4,5 = 9.0 Hz, H-4B), 3.58 (dd, 1H, J5,6b = 4.8 Hz, J6a,6b = 12.1 Hz,
H-6bA), 3.57–3.43 (m, 4H, H-4D, H-5A, H-6bB, H-5D), 3.40 (t,
1H, H-3C), 3.36 (td, 1H, J5,6b = 9.5 Hz, H-5B), 3.26 (t, 1H, J5,6b
=
10.4 Hz, H-6bD), 3.00 (dd, 1H, J5,6b = 2.6 Hz, J6a,6b = 12.1 Hz,
H-6bC), 2.69 (m, 1H, H-5C), 2.56–2.06 (m, 8H, –CH2–CH2–,
CH2(Lev)), 1.86 (s, 3H, CH3(Lev)), 1.24 (s, 9H, C(CH3)3); 13C NMR
(100 MHz, CDCl3):
d 205.7, 177.8, 172.0, 170.2, 167.7, 165.1,
164.4 (10 ꢂ CO), 155.5–114.4 (Ar), 101.8, 101.5 (2 ꢂ PhCHO),
100.2 (C-1C), 99.7 (C-1A), 98.8 (C-1B), 97.7 (C-1D), 81.0 (C-4B),
80.7 (C-3A), 80.6 (C-3C), 79.1 (C-4D), 77.1 (C-4A), 75.7 (C-3B),
75.4 (C-4C), 74.3, 74.0 (2 ꢂ CH2(Bn)), 73.6 (C-2C), 72.7 (C-2A),
72.5 (C-5C), 72.4 (C-5A), 69.7 (C-3D), 68.5, 68.4 (C-6B, C-6D),
66.2 (C-5B), 66.0 (C-5D), 62.5 (C-6A), 60.9 (C-6C), 55.7, 55.6 (C-
2B, C-2D), 55.5 (Me (OMP)), 39.0 (C(CH3)3), 37.7 (CH2(Lev)), 29.5
(CH3(Lev)), 27.8 (CH2(Lev)), 27.4 (C(CH3)3), 26.2 (t, JC,F = 22.0 Hz,
–CH2–CF2–),
C
24.7
(–CH2–);
HR
MS:
m/z:
calcd
for
110H99F17N2O30Na: 2273.5903; found: 2273.5871 [M+Na]+.
Please cite this article in press as: Maza S., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.01.022

8
S. Maza et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
4.10. 4-Methoxyphenyl O-(2-deoxy-3-O-levulinoyl-2-phthalimido-b-
(dd, 1H, J1,2 = 7.6 Hz, J2,3 = 8.5 Hz, H-2A), 5.17 (d, 1H, H-1A), 4.90
(dd, 1H, J1,2 = 7.6 Hz, J2,3 = 8.9 Hz, H-2C), 4.81, 4.78 (2d, 2H,
CH2(Bn)), 4.74 (m, 2H, H-6aB, H-3B), 4.62 (d, 1H, H-1C), 4.56 (dd,
1H, J5,6a = 1.8 Hz, J6a,6b = 11.4 Hz, H-6aD), 4.52, 4.49 (2d, 2H,
CH2(Bn)), 4.37 (br dd, 1H, H-6aA), 4.34 (br dd, 1H, H-6aC), 4.31
(br t, 1H, H-4B), 4.29 (br dd, 1H, H-4D), 4.21–4.08 (m, 5H, H-3A,
H-2D, H-2B, H-6bB, H-6bA), 4.05–3.96 (m, 4H, H-6bD, H-4C,
H-5A, H-5B), 3.95 (t, 1H, J3,4 = J4,5 = 9.0 Hz, H-4A), 3.81 (m, 1H,
H-5D), 3.77 (dd, 1H, J5,6b = 5.8 Hz, J6a,6b = 11.8 Hz, H-6bC), 3.65 (s,
3H, Me (OMP)), 3.56 (t, 1H, J3,4 = 8.6 Hz, H-3C), 3.44 (m, 1H,
H-5C), 2.62–2.32 (m, 8H, –CH2–CH2–, CH2(Lev)), 1.87 (s, 3H,
CH3(Lev)), 1.21 (s, 9H, C(CH3)3); 13C NMR (100 MHz, CD3OD): d
208.8, 179.8, 173.9, 172.2, 169.9, 169.1, 167.2, 166.9 (10 ꢂ CO),
156.7–115.4 (Ar), 100.6 (C-1C), 100.4 (C-1A), 98.3 (C-1D), 96.7
(C-1B), 81.2 (C-3C), 78.9 (C-3A), 77.8 (C-4B), 77.1 (C-4C), 76.5,
76.4 (C-3B, C-4A), 75.9 (C-4D), 75.1, 75.0 (CH2(Bn), C-2C, C-5B,
C-5D), 74.6 (C-2A), 74.4 (C-5C), 73.9 (C-5A), 73.2 (CH2(Bn)), 71.9
(C-3D), 68.8 (C-6B), 68.1 (C-6D), 64.6 (C-6A), 64.0 (C-6C), 57.4 (C-
2B), 56.4 (C-2D), 55.9 (Me (OMP)), 39.9 (C(CH3)3), 38.5 (CH2(Lev)),
29.2 (CH3(Lev)), 29.1 (CH2(Lev)), 27.8 (C(CH3)3), 27.1 (t, JC,F = 21.5
Hz, –CH2–CF2–), 25.9 (–CH2–); 19F NMR (376 MHz, CD3OD): d
ꢁ82.30 (t, 3F), ꢁ115.56 (m, 2F), ꢁ122.71 (m, 6F), ꢁ123.66 (m,
2F), ꢁ124.33 (m, 2F), ꢁ127.19 (m, 2F); ESI MS: m/z: calcd for
D
-glucopyranosyl)-(1 ? 4)-O-(2-O-benzoyl-3-O-benzyl-6-O-pivaloyl-b-
-glucopyranosyl)-(1 ? 3)-O-(2-deoxy-2-phthalimido-b- -glucopy-
D
D
ranosyl)-(1 ? 4)-2-O-benzoyl-3-O-benzyl-6-O-4,4,5,5,6,6,7,7,8,8,9,9,
10,10,11,11,11-heptadecafluoroundecanoyl-b- -glucopyranoside (16)
D
TFA (150
(55 mg, 24
l
L) and H2O (10
lL) were added to a solution of 15
l
mol) in CH2Cl2 (1.5 mL) at 0 °C. The solution was stir-
red for 2 h at room temperature, then diluted with CH2Cl2, and
washed with saturated NaHCO3 aqueous solution and brine. The
organic layer was dried (MgSO4), filtered, and concentrated in
vacuo. The residue was purified by column chromatography
(toluene/acetone 4:1) to give 16 (48 mg, 95%) as a white amor-
20
phous solid. TLC (toluene/acetone 2:1) Rf 0.37; [
a]
+2° (c 1.0,
D
CHCl3); 1H NMR (400 MHz, CDCl3): d 7.93–7.70 (m, 6H, Ar), 7.56–
7.37 (m, 9H, Ar), 7.19–7.06 (m, 11H, Ar), 6.97 (m, 2H, Ar), 6.72 (m,
2H, Ar), 6.63 (m, 2H, Ar), 5.67 (dd, 1H, J2,3 = 10.7 Hz, J3,4 = 8.8 Hz,
H-3D), 5.38 (d, 1H, J1,2 = 8.3 Hz, H-1D), 5.34 (t, 1H, J1,2 = J2,3 = 7.7
Hz, H-2A), 5.05 (t, 1H, J1,2 = J2,3 = 8.4 Hz, H-2C), 5.02 (d, 1H, J1,2
8.3 Hz, H-1B), 4.84 (d, 1H, J1,2 = 7.2 Hz, H-1A), 4.76, 4.70, 4.62, 4.40
(4d, 4H, CH2(Bn)), 4.37 (m, 2H, H-1C, H-6aC), 4.31 (dd, 1H, J2,3
=
=
10.7 Hz, J3,4 = 8.2 Hz, H-3B), 4.19–4.12 (m, 3H, H-2D, H-6aA, H-2B),
3.98 (br s, 1H, OH), 3.88 (t, 1H, J3,4 = J4,5 = 8.8 Hz, H-4A), 3.82 (t,
1H, J3,4 = J4,5 = 8.8 Hz, H-4C), 3.75–3.53 (m, 10H, H-3A, H-6bC, H-
6aB, H-6aD, Me (OMP), H-4D, H-3C, H-6bA), 3.51–3.26 (m, 7H, H-
5C, H-5D, H-6bB, H-6bD, H-4B, H-5A, H-5B), 3.18 (br s, 1H, OH),
2.63–2.18 (m, 8H, –CH2–CH2–, CH2(Lev)), 1.98 (s, 3H, CH3(Lev)),
1.12 (s, 9H, C(CH3)3); 13C NMR (100 MHz, CDCl3): d 207.3, 177.8,
173.1, 170.4, 168.2, 165.2, 164.6 (10 ꢂ CO), 155.6–114.4 (Ar), 101.1
(C-1C), 100.1 (C-1A), 98.3 (C-1B), 97.5 (C-1D), 81.9 (C-3B), 80.1 (C-
3A, C-3C), 76.7 (C-4A), 75.9 (C-4C), 75.7 (C-5B), 75.6 (C-5D), 74.8,
74.6 (2 ꢂ CH2(Bn)), 73.9 (C-5C, C-3D), 73.3 (C-2C), 72.7 (C-5A),
72.6 (C-2A), 70.7 (C-4B), 70.5 (C-4D), 62.9 (C-6A), 62.7 (C-6B or C-
6D), 62.3 (C-6C), 62.0 (C-6B or C-6D), 55.5 (Me (OMP)), 55.2, 54.9
(C-2B, C-2D), 38.8 (C(CH3)3), 38.3 (CH2(Lev)), 29.6 (CH3(Lev)), 28.1
(CH2(Lev)), 27.0 (C(CH3)3), 26.3 (t, JC,F = 21.7 Hz, –CH2–CF2–), 24.9
(–CH2–); HR MS: m/z: calcd for C96H91F17N2O30Na: 2097.5277;
found: 2097.5282 [M+Na]+.
C
96H87F17N2O42S4Na22ꢁ: 1218.2; found: 1217.6 [M+2Na]2ꢁ
.
4.12. 4-Methoxyphenyl O-(2-deoxy-3-O-levulinoyl-2-phthalimido-b-
-glucopyranosyl)-(1 ? 4)-2-O-benzoyl-3-O-benzyl-6-O-4,4,5,5,6,6,
D
7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecanoyl-b-
ranoside (17)
D-glucopy-
Donor 4 (161 mg, 0.251 mmol) and acceptor 2 (120 mg, 0.126
mmol) were dissolved in dry CH2Cl2 (4 mL) in the presence of acti-
vated 4 Å MS (300 mg). The reaction mixture was stirred, under an
argon atmosphere, for 10 min at 0 °C and TBSOTf (295 mL of a
0.085 M solution in dry CH2Cl2) was added. After stirring for 40
min at 0 °C, the reaction mixture was quenched with triethy-
lamine, filtered, and then concentrated under reduced pressure.
The crude product was purified using a fluorous solid-phase
extraction (F-SPE) column. Nonfluorous compounds were eluted
with 80:20 MeOH/water and the fluorous product was eluted by
100% acetone. This acetone fraction was concentrated to give dis-
accharide 11 as a white amporphous solid (175 mg).
4.11. 4-Methoxyphenyl O-(2-deoxy-3-O-levulinoyl-2-phthalimido-
4,6-di-O-sulfo-b-
D
-glucopyranosyl)-(1 ? 4)-O-(2-O-benzoyl-3-O-benzyl-
-glucopyranosyl)-(1 ? 3)-O-(2-deoxy-2-phthalimido-
-glucopyranosyl)-(1 ? 4)-2-O-benzoyl-3-O-benzyl-
6-O-pivaloyl-b-
D
4,6-di-O-sulfo-b-
D
TFA (189 lL) and H2O (30 lL) were added to a solution of 11
6-O-4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecanoyl-
(175 mg, 0.122 mmol) in CH2Cl2 (1.9 mL) at 0 °C. The solution
was stirred for 90 min at room temperature. Then, it was diluted
with CH2Cl2, and washed with saturated NaHCO3 aqueous solution
and brine. The organic layer was dried (MgSO4), filtered, and con-
centrated in vacuo. The residue was purified by column chro-
matography (toluene/EtOAc 1:1) to give 17 (132 mg, 78% from 2;
b-D-glucopyranoside (1)
Compound 16 (22 mg, 11 mmol) and sulfur trioxide–trimethy-
lamine complex (59 mg, 0.42 mmol) were dissolved in dry DMF
(2.0 mL) and heated at 100 °C for 30 min using microwave radia-
tion (18 W average power). The reaction vessel was cooled and
Et3N (300 mL), MeOH (1.5 mL) and CH2Cl2 (1.5 mL) were added.
The solution was purified by Sephadex LH 20 chromatography
(CH2Cl2-MeOH 1:1). The residue was submitted to a second sulfa-
tion cycle with new sulfating reagent (sulfur trioxide–trimethy-
lamine complex, 59 mg, 0.42 mmol) in dry DMF (2 mL). Then, the
reaction vessel was again cooled and Et3N (300 mL), MeOH (1.5
mL) and CH2Cl2 (1.5 mL) were added. The solution was purified
by Sephadex LH 20 chromatography (CH2Cl2-MeOH 1:1) and silica
gel column chromatography (EtOAc-MeOH-H2O 32:5:3 ? EtOAc-
MeOH-H2O 24:5:3). The residue was finally eluted from a Dowex
50WX2-Na+ column (MeOH) to obtain 1 as sodium salt (19 mg,
72%, white amorphous solid). TLC (EtOAc-MeOH-H2O 24:5:3) Rf
0.27; 1H NMR (400 MHz, CD3OD): d 7.96–7.79 (m, 6H, Ar), 7.61–
7.35 (m, 12H, Ar), 7.06–6.94 (m, 10H, Ar), 6.77 (m, 2H, Ar), 6.68
(m, 2H, Ar), 5.83 (dd, 1H, J2,3 = 10.8 Hz, J3,4 = 8.9 Hz, H-3D), 5.45
(d, 1H, J1,2 = 8.4 Hz, H-1D), 5.37 (d, 1H, J1,2 = 8.4 Hz, H-1B), 5.26
2 steps) as a white amorphous solid. TLC (toluene/EtOAc 1:1) Rf
20
0.29; [
a]
+2° (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): d 7.97
D
(m, 2H, Ar), 7.84 (m, 2H, Ar), 7.73 (m, 2H, Ar), 7.56 (m, 1H, Ar),
7.42 (m, 2H, Ar), 7.27–7.17 (m, 5H, Ar), 6.78 (m, 2H, Ar), 6.67 (m,
2H, Ar), 5.67 (dd, 1H, J2,3 = 10.7 Hz, J3,4 = 8.8 Hz, H-30), 5.52 (d, 1H,
J1,2 = 8.4 Hz, H-10), 5.42 (dd, 1H, J1,2 = 7.1 Hz, J2,3 = 8.2 Hz, H-2), 4.94
(d, 1H, H-1), 4.85, 4.73 (2d, 2H, CH2(Bn)), 4.40 (dd, 1H, J5,6a = 2.1
Hz, J6a,6b = 12.0 Hz, H-6a), 4.24 (dd, 1H, H-20), 4.05 (dd, 1H, J3,4
=
8.2 Hz, J4,5 = 9.4 Hz, H-4), 3.86 (t, 1H, H-3), 3.80 (dd, 1H, J5,6b = 5.2
Hz, H-6b), 3.75 (m, 1H, H-60a), 3.71–3.68 (m, 4H, H-40, Me (OMP)),
3.62 (ddd, 1H, H-5), 3.51–3.47 (m, 2H, H-50, H-60b), 3.27 (br s, 1H,
OH), 2.63–2.27 (m, 8H, –CH2–CH2–, CH2(Lev)), 1.99 (s, 3H, CH3(Lev));
13C NMR (125 MHz, CDCl3): d 207.3, 173.1, 170.5, 168.4, 167.8, 165.3
(6 ꢂ CO), 155.7–114.5 (Ar), 100.1 (C-1), 98.1 (C-10), 80.3 (C-3), 76.8
(C-4), 75.7 (C-50), 74.4 (CH2(Bn)), 73.9 (C-30), 72.9, 72.8 (C-2, C-5),
70.3 (C-40), 63.2 (C-6), 62.1 (C-60), 55.5 (Me (OMP)), 55.1 (C-20),
38.2 (CH2(Lev)), 29.5 (CH3(Lev)), 28.1 (CH2(Lev)), 26.3 (t, JC,F = 21.7
Please cite this article in press as: Maza S., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.01.022

S. Maza et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
9
Hz, –CH2–CF2–), 25.0 (–CH2–); HR MS: m/z: calcd for
57H50F17NO17Na: 1366.2699; found: 1366.2683 [M+Na]+.
(dd, 1H, J2,3 = 10.5 Hz, H-30), 3.75–3.66 (m, 4H, H-2, H-6b, H-5, H-
50), 2.05 (s, 3H, NHAc); 13C NMR (100 MHz, D2O; selected data from
HSQC experiment)): d 100.8 (C-1), 99.8 (C-10), 81.4 (C-3), 76.8
(C-40), 75.1 (C-50), 74.9 (C-4), 73.5 (CH2(Bn)), 72.2 (C-2, C-5), 71.9
(C-30), 67.1 (C-60), 59.9 (C-6), 55.8 (Me (OMP)), 55.5 (C-20), 21.8
(NHAc); ESI MS: m/z: calcd for C28H35NO18S2Naꢁ: 760.1; found:
760.0 [M+Na]ꢁ.
C
4.13. 4-Methoxyphenyl O-(2-deoxy-3-O-levulinoyl-2-phthalimido-4,6-
di-O-sulfo-b- -glucopyranosyl)-(1 ? 4)-2-O-benzoyl-3-O-benzyl-6-O-
D
4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecanoyl-b-
D-
glucopyranoside (18)
Compound 17 (132 mg, 0.098 mmol) and sulfur trioxide–
trimethylamine complex (273 mg, 1.96 mmol) were dissolved in
dry DMF (6.5 mL) and heated at 100 °C for 30 min using microwave
radiation (26 W average power). The reaction vessel was cooled
and Et3N (600 mL), MeOH (2 mL) and CH2Cl2 (2 mL) were added.
The solution was purified by Sephadex LH 20 chromatography
(CH2Cl2-MeOH 1:1) and the residue was eluted from a Dowex
50WX2-Na+ column (MeOH) to obtain 18 as sodium salt (141
mg, 93%, white amorphous solid). TLC (EtOAc-MeOH-H2O 24:5:3)
Rf 0.34; 1H NMR (400 MHz, CD3OD): d 8.00 (m, 2H, Ar), 7.80–
7.45 (m, 7H, Ar), 7.11–7.04 (m, 5H, Ar), 6.83 (m, 2H, Ar), 6.72 (m,
2H, Ar), 5.88 (dd, 1H, J2,3 = 10.8 Hz, J3,4 = 8.8 Hz, H-30), 5.71 (d, 1H,
J1,2 = 8.4 Hz, H-10), 5.35 (br t, 1H, H-2), 5.24 (d, 1H, J1,2 = 7.7 Hz,
H-1), 4.87 (d, 1H, CH2(Bn)), 4.65–4.60 (m, 2H, H-60a, CH2(Bn)),
4.53 (br dd, 1H, H-6a), 4.33 (br dd, 1H, H-40), 4.29–4.20 (m, 3H,
H-3, H-20, H-6b), 4.16–4.08 (m, 3H, H-5, H-4, H-60b), 3.98 (m, 1H,
H-50), 3.67 (Me (OMP)), 2.65–2.33 (m, 8H, –CH2–CH2–, CH2(Lev)),
1.87 (s, 3H, CH3(Lev)); 13C NMR (100 MHz, CD3OD): d 208.9,
174.0, 172.3, 169.8, 169.1, 167.0 (6 x CO), 156.7–115.4 (Ar),
100.5 (C-1), 96.9 (C-10), 79.2 (C-3), 76.6 (C-4), 76.1 (C-40), 74.7
(C-50), 74.4 (C-2), 73.9 (C-5), 73.2 (CH2(Bn)), 71.8 (C-30), 68.3 (C-
60), 64.6 (C-6), 56.4 (C-20), 55.8 (Me (OMP)), 38.4 (CH2(Lev)), 29.2,
29.1 (CH3(Lev), CH2(Lev)), 27.1 (t, JC,F = 21.4 Hz, –CH2–CF2–), 25.9
(–CH2–); 19F NMR (376 MHz, CD3OD): d ꢁ82.30 (t, 3F), ꢁ115.54
(m, 2F), ꢁ122.70 (m, 6F), ꢁ123.67 (m, 2F), ꢁ124.34 (m, 2F),
ꢁ127.21 (m, 2F); ESI MS: m/z: calcd for C57H48F17NO23S2Naꢁ:
1524.2; found: 1524.2 [M+Na]ꢁ.
4.15. 4-Methoxyphenyl O-(2-acetamido-2-deoxy-4,6-di-O-sulfo-b-
glucopyranosyl)-(1?4)-b- -glucopyranoside (20)
D-
D
A solution of 19 (7.6 mg, 9.7 mmol, sodium salt) in H2O/MeOH
(4.5 mL/0.5 mL) was hydrogenated in the presence of 20% Pd
(OH)2/C (15 mg). After 24 h, the suspension was filtered over Celite
and concentrated. The residue was purified by Sephadex LH 20
chromatography column which was eluted with H2O-MeOH (9:1)
to obtain 20. This compound was then dissolved in H2O (2 mL)
and Amberlite IR-120H+ resin was added (pH 3.0). The mixture
was immediately filtered and treated with 0.04 M NaOH (pH 7.1)
to obtain 20 as a white amorphous solid (5.1 mg, sodium salt,
75%) after lyophilization. 1H NMR (400 MHz, D2O): d 7.12 (m, 2H,
Ar), 6.99 (m, 2H, Ar), 5.06 (d, 1H, J1,2 = 8.0 Hz, H-1), 4.67 (d, 1H,
J1,2 = 8.3 Hz, H-10), 4.55 (dd, 1H, J5,6a = 2.0 Hz, J6a,6b = 11.4 Hz, H-
60a), 4.23 (dd, 1H, J3,4 = 8.5 Hz, J4,5 = 9.7 Hz, H-40), 4.18 (dd, 1H,
J5,6b = 7.9 Hz, H-60b), 3.94 (m, 1H, H-50), 3.92–3.81 (m, 6H, H-20,
H-6a, H-30, Me (OMP)), 3.78 (br dd, 1H, H-3), 3.72–3.63 (m, 3H,
H-5, H-6b, H-4), 3.59 (dd, 1H, J2,3 = 9.3 Hz, H-2), 2.08 (s, 3H, NHAc);
13C NMR (100 MHz, D2O; selected data from HSQC experiment)): d
101.4 (C-10), 100.5 (C-1), 79.6 (C-4), 76.6 (C-40), 74.5 (C-3, C-5),
72.3 (C-50), 72.2 (C-2), 71.7 (C-30), 67.2 (C-60), 59.5 (C-6), 55.6
(Me (OMP)), 55.1 (C-20), 21.8 (NHAc); ESI MS: m/z: calcd for
C
21H29NO18S2Naꢁ: 670.1; found: 669.9 [M+Na]ꢁ.
4.16. Fluorescence polarization assays
Fluorescence polarization measurements were performed in
384-well microplates (black polystyrene, non-treated, Corning).
The fluorescence polarization was recorded using a TRIAD multi-
mode microplate reader (from Dynex), with excitation and emis-
sion wavelengths of 485 and 535 nm, respectively. The
fluorescent probe (a fluorescein labelled heparin-like hexasaccha-
ride previously prepared in our lab)54 was dissolved in PBS buffer
(10 mM, pH 7.4). Recombinant human midkine and FGF-2 (Pepro-
tech) were dissolved in PBS buffer (10 mM, pH 7.4) containing 1%
BSA (bovine serum albumin). Compounds 19 and 20 were dis-
solved in PBS buffer (10 mM, pH 7.4). 1 mM stock solutions of com-
pounds 1 and 18 were prepared in PBS/DMSO 9:1 (v/v) and serial
dilutions were then performed in PBS buffer (10 mM, pH 7.4).
For the determination of the IC50 values, we recorded the fluo-
rescence polarization from wells containing 20 mL of protein solu-
tion (125 nM midkine solution or 194 nM FGF-2 solution) and 10
mL of a 40 nM probe solution in the presence of 10 mL of inhibitor
solution, with concentrations ranging from 100 mM to 50 nM. The
microplate was shaked in the dark for 5 min, before reading. The
total sample volume in each well was 40 mL and the final buffer
composition was PBS + 0.5% BSA. The final concentrations of fluo-
rescent probe and midkine/FGF-2 in each well were 10 nM and
63/97 nM, respectively, while the final inhibitor concentration ran-
ged from 25 mM to 12.5 nM. The average polarization values of
three replicates were plotted against the logarithm of inhibitor
concentration. Two control samples were included in the competi-
tion experiment. The first one only contained fluorescent probe
and afforded the expected minimum polarization value for 100%
inhibition; the second one contained midkine/FGF-2 and probe,
in the absence of inhibitor, and gave the maximum polarization
value corresponding to 0% inhibition. Blank wells contained 20 mL
4.14. 4-Methoxyphenyl O-(2-acetamido-2-deoxy-4,6-di-O-sulfo-b-
glucopyranosyl)-(1 ? 4)-3-O-benzyl-b- -glucopyranoside (19)
D-
D
Ethylene diamine (130
lL, 1.94 mmol) was added to a solution
of 18 (20 mg, 13 mol) in n-BuOH (1.5 mL) under an argon atmo-
l
sphere, and the reaction mixture was subjected to microwave irra-
diation (30 W average power) for 90 min at 120 °C (3 cycles, 30
min each). The reaction vessel was cooled, and the mixture was
concentrated to dryness. The residue was dissolved in MeOH
(2.5 mL) and an aqueous solution of NaOH (4 M, 645 mL) was
added. After stirring for 19 h at room temperature, the reaction
mixture was neutralized with Amberlite IR-120 (H+) resin, filtered,
and concentrated to give the desired amine intermediate. Triethy-
lamine (24 mL, 0.17 mmol) and acetic anhydride (24 mL, 0.26 mmol)
were added to a cooled (0 °C) solution of this amine derivative in
MeOH (2.5 mL). After stirring for 2 h at room temperature, addi-
tional triethylamine (24 mL, 0.17 mmol) and acetic anhydride (24
mL, 0.26 mmol) were added at 0 °C to complete the reaction. After
further stirring for 3 h at room temperature, Et3N (300 mL) was
added and the mixture was concentrated to dryness. The residue
was then purified by silica gel column choromatography (EtOAc-
MeOH-H2O 40:5:3 ? EtOAc-MeOH-H2O 24:5:3) and finally eluted
from a Dowex 50WX2-Na+ column (H2O) to obtain 19 as sodium
salt (7.7 mg, 76%, white amorphous solid). TLC (EtOAc-MeOH-
H2O 24:5:3) Rf 0.26; 1H NMR (400 MHz, D2O): d 7.56–7.39 (m,
5H, Ar), 7.12 (m, 2H, Ar), 7.00 (m, 2H, Ar), 5.03 (d, 1H, J1,2 = 7.8
Hz, H-1), 4.99, 4.94 (2d, 2H, CH2(Bn)), 4.66 (d, 1H, J1,2 = 8.1 Hz, H-
10), 4.41 (dd, 1H, J5,6a = 1.9 Hz, J6a,6b = 11.4 Hz, H-60a), 4.13 (dd,
1H, J3,4 = 8.6 Hz, J4,5 = 9.8 Hz, H-40), 3.94 (t, 1H, J3,4 = J4,5 = 9.1 Hz,
H-4), 3.92–3.80 (m, 7H, H-6a, H-60b, H-20, H-3, Me (OMP)), 3.78
Please cite this article in press as: Maza S., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.01.022

10
S. Maza et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
18. Oh YI, Sheng GJ, Chang S-K, Hsieh-Wilson LC. Angew Chem Int Ed.
of protein solution and 20 mL of a 50 mM inhibitor solution and
their measurements were subtracted from all values. The curve
was fitted to the equation for a one-site competition: y = A2 + (A1
ꢁ A2)/[1 + 10^(x ꢁ logIC50)] where A1 and A2 are the maximal
and minimal values of polarization, respectively, and IC50 is the
inhibitor concentration that results in 50% inhibition. At least three
independent experiments were carried out for each IC50
calculation.
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