Chemical synthesis of a sulfated D-glucosamine library and evaluation of cell proliferation capabilities

Full text of DOI:10.1016/j.carres.2020.108085

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Chemical synthesis of a sulfated d-glucosamine library and evaluation of cell
proliferation capabilities
Aisling Ní Cheallaigh, Scott E. Guimond, Stefan Oscarson, Gavin J. Miller
PII:
S0008-6215(20)30280-9
DOI:
https://doi.org/10.1016/j.carres.2020.108085
CAR 108085
Reference:
To appear in: Carbohydrate Research
Received Date: 6 May 2020
Revised Date: 16 June 2020
Accepted Date: 17 June 2020
Please cite this article as: A. Ní Cheallaigh, S.E. Guimond, S. Oscarson, G.J. Miller, Chemical synthesis
of a sulfated d-glucosamine library and evaluation of cell proliferation capabilities, Carbohydrate
Research (2020), doi: https://doi.org/10.1016/j.carres.2020.108085.
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Chemical synthesis of a sulfated -glucosamine library
D
and evaluation of cell proliferation capabilities
Aisling Ní Cheallaigh,1,2,4 Scott E. Guimond,3 Stefan Oscarson2 and Gavin J.
Miller1*
1Lennard-Jones Laboratory, School of Chemical and Physical Sciences, Keele University,
Keele, Staffordshire, ST5 5BG, U.K.
2 Centre for Synthesis and Chemical Biology, University College Dublin, Belfield, Dublin 4,
Ireland
3School of Medicine, Keele University, Keele, Staffordshire, ST5 5BG, UK.
4Current address: Manchester Institute of Biotechnology, University of Manchester,
Manchester, M1 7DN, U.K.
*Corresponding author. Email: g.j.miller@keele.ac.uk
Keywords: glycosaminoglycan,
assay, FGF-2
D-glucosamine, sulfation, heparan sulfate, proliferative
1. Introduction
Amongst the multitude of sugar components in nature,
D-glucosamine (D-GlcN) and
N-substituted derivatives thereof are found in many biologically important molecules,
including cell surface glycoproteins, cell wall components and carbohydrate effector ligands.
Within this broad classification, sulphated and acetylated forms of
D-GlcN are commonly
associated with the glycosaminoglycan (GAG) polysaccharide family due to their presence in
the repeating disaccharide unit in heparin and heparan sulfate (H/HS). These highly
functionalised GAGs are involved in a myriad of important biological recognition
processes.1–3 and consequently there has been an intense focus on using chemical4–10 and
enzymatic11–16 methods to provide structurally defined, homogenous H/HS materials for
biological evaluations and applications.
More specifically, the role of sulfation and/or acetylation microhetereogeneity
(Figure 1) and its contribution to H/HS biological ubiquity has resulted in the need for access
to libraries of sulfation-site programmed oligosaccharide sequences.17 Whilst many groups
have successfully synthesised defined H/HS di- and oligosaccharide sequences,18–28 there is
still a need to access the simplest monosaccharide components of H/HS (D-GlcN, D-GlcA, L-
IdoA) with defined sulfation and acetylation patterns. This will enable their application, not
only in chemical biology and biomedical contexts,29,30 but within the field of carbohydrate
metrology, which is rapidly expanding as new analytical techniques for glycan analysis are
developed.31,32
Figure 1. Representative illustration of
N-acetylation are shown in blue.
D-GlcN within a HS chain. Possibilities for O/N-sulfation and
1

Herein, a convenient synthetic route to a small matrix of N- and 6-O-substituted D-
GlcN monosaccharides, capped as the O-methyl glycoside with α- and β-anomeric linkages
has been developed (Figure 2). This will enable their use as comparative tools in analytical
and biological contexts. To initiate such applications, their ability to affect cell proliferation
and the FGF-2 signaling system regulated by HS has been examined here.
Common intermediate to
OH
introduce 6-O-sulfate
OH
O
O
HO
HO
HO
HO
NH
P
OMe
H2N
OH
Access to α + β
linked systems
Commerical
D
-GlcN
Temporary
N-protecting group
Figure 2. Strategy to an N/O-substituted D-GlcN monosaccharide library.
2. Results and Discussion
As we required access to α- and β-linked O-methyl glycoside derivatives of D-GlcN,
parallel synthetic routes from N-protected monosaccharides 1 and 8 towards the six target
structures 5-7 and 12-14 were developed.
2.1. Synthesis of α-OMe-
Our synthesis of α-OMe linked targets were accessed from N-CBz-protected
glucosamine derivative 1, obtained from commercial
-GlcN in two steps.33,34 The route was
D-GlcN derivatives
D
designed to generate a key 6-O-sulfated intermediate 4, from which all targets could then be
acquired in a divergent manner (Scheme 1). Accordingly, the primary hydroxyl group of 1
was selectively protected as a TBDPS ether and the remaining secondary hydroxyl positions
O-benzoylated in the same reaction vessel, delivering 2 in very good yield (88%). Attempts
to remove the primary TBDPS group, to enable O-sulfation, found that treatment of 2 with
TBAF at room temperature resulted in ester migration, with a 3,6-di-O-benzoylated
regioisomer observed as the major migration product. This unwanted migration persisted
even when trialling lower reaction temperatures (-20 °C and 0 °C). However, switching to
.
Lewis acidic deprotection conditions using BF3 Et2O successfully removed the group with no
observed migration, delivering 3 successfully, albeit in a lower than expected yield of 42%.
The primary hydroxyl group of 3 was then O-sulphated using sulfur trioxide-pyridine
complex in a microwave reactor at 100 °C. This delivered the key 6-O-sulfated intermediate 4
in essentially quantitative yield, isolated as the ammonium salt.
Scheme 1. Synthesis of differentially sulphated
D-glucosamines 5-7. a) TBDPSCl, imidazole, DMF then BzCl,
2

.
.
DMAP, pyridine, 88% b) BF3 Et2O, CH2Cl2, 42% c) SO3 pyridine, pyridine, 100°C, microwave, 96% d) Pd/C,
H2, MeOH, then NaOMe, MeOH, 63% e) MeOH, Ac2O, 78% f) SO3.pyridine, NaHCO3, H2O, 38%.
Hydrogenolysis of 4 using Pd/C as catalyst was then employed to remove the N-Cbz
group, followed by treatment with NaOMe to remove the OBz groups and deliver the first 6-
OS-
D
-GlcNH2 target 5 in 63% yield over the two steps. From this free amine, independent N-
-GlcNHAc 6 and 6-OS- -GlcNS 7 in 78%
acetylation and N-sulfation steps delivered 6-OS-
D
D
and 38% yields respectively. The lower than expected final yield for 7 was attributed to loss
of material during extensive desalting, required for obtaining pure quantities of material and
was also observed for β-linked target 14. Overall, this synthetic route delivered three
homogenous, differentially sulphated or acetylated monosaccharides from commercial
GlcNH2 in only six steps to the key intermediate 5; this compares favourably to a recently
reported synthesis accessing 5 in nine steps from
-GlcN.29
D-
D
2.2. Synthesis of β-OMe-
D-GlcN derivatives
The comparative series of β-OMe-linked derivatives, was accessed through a second
synthetic route from N-phthalimido glucosamine 8, accessed from D-GlcN in 4 steps (Scheme
2).35,36 Similar one-pot regioselective 6-OH protection and 3,4-di-O-benzolyation proceeded
smoothly in excellent yield (96%), delivering 9, and was followed by Lewis acid mediated
deprotection to furnish 10. Sulfation of the D-GlcN 6-OH was again accomplished in high
yield (96%) to give 11 which, in contrast to the method established for 5, could then be
simultaneously N- and O-3,4 deprotected using ethylene diamine in hot ethanol to furnish 12
in an acceptable yield of 56%. 6-O-sulfated free amine 12 was then N-acetylated and N-
sulfated to access novel targets 13 and 14 in 83% and 51% yields respectively.
Scheme 2. Synthesis of differentially sulphated D-glucosamines 12-14 a) i) TBDPSCl, imidazole, DMF then
.
.
BzCl, DMAP, pyridine, 96% b) BF3 Et2O, CH2Cl2, 46% c) SO3 pyridine, pyridine, 100 °C, microwave, 95% d)
.
ethylene diamine, EtOH, 70 °C, 56% e) MeOH, Ac2O, 83% f) SO3 pyridine, NaHCO3, H2O, 51%.
Completing a parallel synthesis of α- and β-OMe derivatives enabled a comparative
examination of their NMR spectra. Illustrated in Figure 3 are the 1H NMR spectra for 6S-
D-
GlcNS derivatives 7 and 14, highlighting the observed differences for H1 and H2 between
these two anomeric forms. Chemical shifts for these protons were downfield for 7 (δH = 4.94
ppm [d, 3JH1-H2 = 3.6 Hz, H1]) compared to 14 (δH = 4.41 ppm [d, 3JH1-H2 = 8.4 Hz, H1]) and
13
this enabled their easy distinction. In addition, C NMR for 12 and 14 showed distinctive
chemical shift differences at C2 for the free amine (55.8 ppm) vs N-sulfated (59.9 ppm)
3

forms. These observations were mirrored in the α-series (C2 53.8 ppm for 5 and 57.2 ppm for
7). With multi-milligram amounts of site-specifically sulfated glucosamine derivatives 5-7
and 12-14 in hand, their ability to support HS-mediated proliferative pathways was evaluated.
Figure 3. Comparison of key 1H NMR chemical shifts for 7 and 14, highlighting differences for H1 and H2 in
the α- and β-anomers. See SI for comparative overlay of 12 and 14 highlighting chemical shift differences for
H2 in the free amine vs N-sulfated forms.
2.3. Biological evaluations
The saccharide constituents of the glycosaminoglycan HS are generally considered
non-toxic to cells, but it is unknown if the monosaccharide components thereof (including 5-
7 and 12-14) have any adverse effect on cells. To examine this possibility, the ability of
compounds 5-7 and 12-14 to affect cells was examined using toxicity tests in lymphocytic
(BaF3), fibroblastic (NIH 3T3) and epithelial cell lines (Vero). None of the compounds tested
exhibited any toxicity towards these cell types. Figure 4 demonstrates that monosaccharide
concentrations of 100 µg/ml (100-1000 times a typical biologically relevant concentration37)
did not inhibit cell proliferation in response to 10% FBS in 3T3 cells. Similarly, compounds
5-7 and 12-14 were not able to counter the ability of Il-3 to suppress apoptosis in BaF3 cells
and did not inhibit cell proliferation in response to 10% FBS in Vero cells (see
Supplementary Information).
Figure 4. Ability of compounds 5-7 and 12-14 to affect a proliferative response in response to 10% FBS using
fibroblastic (NIH 3T3) cell line. Monosaccharide concentrations were 100 ug/ml with 10% FBS. Triton X-100
4

was 0.1% with 10% FBS. Cells were incubated with saccharides for 72 hours and results are representative of
three independent experiments with triplicate wells in each experiment.
HS oligosaccharides shorter than a hexasaccharide do not have the ability to
positively regulate protein activity, but those smaller than hexasaccharide can act as
competitive inhibitors of H/HS to suppress protein activity.38 In light of this, compounds 5-7
and 12-14 were also tested for their ability to suppress FGF-2 signalling in BaF3 cells
expressing FGFR1c in the presence of heparin (Figure 5).
Figure 5. Compounds 5-7 and 12-14 do not affect the ability of heparin to support FGF-2 signalling.
Monosaccharide concentrations were 100 ug/ml with 0.3 ng/ml FGF-2 + 1 ug/ml heparin. Il-3 = 1 ng/ml.
Results are representative of three independent experiments with triplicate wells in each experiment.
In these cells, FGF-2 signalling can substitute for Il-3 signalling when Il-3 is
withdrawn, allowing the cells to proliferate normally. This only happens in the presence of
heparin or an HS saccharide that supports FGF-2 activity. Saccharides that do not bind to
FGF-2, FGFR1c or would otherwise inhibit FGF-2 signalling would not support BaF3 cell
proliferation. None of compounds 5-7 and 12-14 had a statistically significant effect on the
ability of heparin to support FGF-2 signalling, indicating that they do not interfere with FGF-
2 activity.
3. Conclusion
A six-step chemical synthesis of α- and β-OMe D-GlcN intermediates enables simple
acylation or N-sulfation to afford a small matrix of six differentially sulphated or acetylated
derivatives. These constituent monosaccharides of heparin/heparan sulfate were shown not to
support proliferative pathways in lymphocytic (BaF3), fibroblastic (NIH 3T3) and epithelial
(Vero) cell lines or an ability to interfere with FGF-2 signalling. They will however provide
important, structurally defined standards for alternative biological and analytical applications.
4. Experimental section
4.1. General Methods and Materials
5

1H NMR spectra were recorded on a Bruker Avance 400 (400 MHz) instrument using
deuterochloroform (or other indicated solvent) as reference. The chemical shift data for each
signal are given as δ in units of parts per million (ppm) relative to tetramethylsilane (TMS)
where δ (TMS) = 0.00 ppm. The multiplicity of each signal is indicated by: s (singlet); brs
(broad singlet); d (doublet); t (triplet); dd (doublet of doublets); ddd (doublet of doublet of
doublets); dddd (doublet of doublet of doublet of doublets); ddt (doublet of doublet of
triplets); sp (septet) or m (multiplet). The number of protons (n) for a given resonance is
indicated by nH. Coupling constants (J) are quoted in Hz and are recorded to the nearest 0.1
13
Hz. C NMR spectra were recorded on a Bruker Avance 400 (100 MHz) instrument using
the PENDANT sequence and internal deuterium lock. The chemical shift data for each signal
are given as δ in units of ppm relative to TMS where δ (TMS) = 0.00 ppm. Analytical thin
layer chromatography (TLC) was carried out on pre-coated 0.25 mm ICN Biomedicals
GmbH 60 F254 silica gel plates. Visualisation was by absorption of UV light or thermal
development after dipping in 5% H2SO4 in MeOH. Optical activities were recorded on
automatic Rudolph Autopol I or Bellingham and Stanley ADP430 polarimeters
(concentration in g/100mL).HRMS (ESI) were obtained on Agilent 6530 Q-TOF, LQT
Orbitrap XL1 or Waters (Xevo, G2-XS TOF or G2-S ASAP) Micromass LCT spectrometers
using a methanol mobile phase in positive/negative ionisation modes as appropriate. Manual
column chromatography was carried out on silica gel (Sigma Aldrich 40-63 micron) under a
positive pressure of compressed air. Automatic Column Flash chromatography was carried
out on silica gel (Reveleris® X2 system) under a positive pressure of compressed N2. Dry
CH2Cl2 and DMF was acquired from an Inovative Technology solvent purification system.
Anhydrous MeOH was dried over 4 Å molecular sieves. Chemicals were purchased from
Acros UK, Aldrich UK, Avocado UK, Fisher UK or Fluka UK. All solvents and reagents
were purified and dried where necessary, by standard techniques. Where appropriate and if
not stated otherwise, all non-aqueous reactions were performed under an inert atmosphere of
nitrogen, using a vacuum manifold with nitrogen passed through 4 Å molecular sieves and
self-indicating silica gel. In vacuo refers to the use of a rotary evaporator attached to a
diaphragm pump. Brine refers to a saturated aqueous solution of sodium chloride. Hexane
refers to n-hexane and petroleum ether to the fraction boiling between 40 and 60 °C.
4.2. General method for TBDPS and benzoate introduction
Compound 1 or 8 (1.0 mmol) was dissolved in DMF (3.3 mL) at room temperature. The
solution was cooled to 0 °C and imidazole (350 mg, 5.10 mmol) was added followed by
dropwise addition of TBDPSCl (800 ꢀL, 3.10 mmol). The solution was stirred at room
temperature for 2 hr. Upon complete conversion to the 6-OTBDPS derivative, as indicated by
TLC (100% EtOAc, starting material Rf ~ 0.1-0.2, product Rf ~0.8-0.9) pyridine was added
(3.3 mL) followed by BzCl (500 ꢀL 4.10 mmol) and DMAP (13 mg, 51 ꢀmol). The solution
was stirred at room temperature for 3 hr before being reduced to dryness. The residue was
taken up in EtOAc (50 mL) and washed with 2.0 M aq. HCl (2 x 25 mL), saturated aqueous
NaHCO3 (2 x 25 mL), deionised water (2 x 25 mL) and brine (25 mL). The organic layer was
dried over MgSO4, filtered and solvent removed under reduced pressure. Silica gel column
chromatography using a gradient elution (0-5% EtOAc in toluene) gave the products as white
foams in 88-96% yields.
4.2.1. Methyl 3,4-O-benzoyl-2-N-carboxybenzyl-6-O-tertbutyldiphenylsilyl-2-deoxy-α-
D-
glucopyranoside (2)
250 mg of 1 gave 2 (560 mg, 88%); White foam was crystallized from petroleum ether and
diethylether to give 2 as white crystals. Rf = 0.7 (toluene/EtOAc, 9/1); [ꢀ]ꢂꢁꢃ= +19.0 (c = 2.0
1
CHCl3); H NMR (400 MHz, CDCl3) δ 7.93 (d, J = 7.2 Hz, 2H, ArH), 7.84 (d, J = 7.2 Hz,
6

2H, ArH), 7.65 (d, J = 6.5 Hz, 2H, ArH), 7.58 (dd, J = 8.0, 1.3 Hz, 2H, ArH), 7.49 (q, J = 7.2
Hz, 2H, ArH), 7.32 (m, 8H, ArH), 7.19 (m, 7H, ArH), 5.68–5.61 (t, J = 10.4 Hz, 1H, H3),
5.54 (t, J = 9.8 Hz, 1H, H4), 5.17 (d, J = 9.9 Hz, 1H, NH), 4.95 (s, 2H, CH2Ph), 4.89 (d, J =
3.5 Hz, 1H, H1), 4.28 (td, J = 10.3, 3.6 Hz, 1H, H2), 4.04 (ddd, J = 9.9, 5.0, 2.4 Hz, 1H, H5),
3.82 (dd, J = 11.5, 5.1 Hz, 1H, H6a), 3.77 (dd, J = 11.4, 2.4 Hz, 1H, H6b) 3.46 (s, 3H,
OCH3), 1.02 (s, 9H, C[CH3]3); 13C NMR (101 MHz, CDCl3) δ 166.9 (C=O), 165.2 (C=O),
156.0 (NC=O), 136.3, 135.8, 135.7, 133.3, 133.2, 130.1, 129.9, 129.7, 129.5, 129.4, 128.5,
128.4, 128.1, 128.0, 127.7, 98.7 (C1), 72.3 (C3), 71.1 (C5), 69.4 (C4), 66.9 (CH2Ph), 63.1
(C6), 55.4 (OCH3), 54.4 (C2), 26.8 (C[CH3]3); HRMS [M+Na]+ calculated for
C45H47NNaO9Si: 796.2912; found: 796.2944.
4.2.2.
Methyl
3,4-O-benzoyl-2-N-phthalimido-6-O-tertbutyldiphenylsilyl-2-deoxy-β-D-
glucopyranoside (9)
780 mg of 8 gave 9 (1.80 g, 96%); Rf = 0.7 (toluene/EtOAc, 9/1); [ꢀ]ꢂꢁꢃ= +36.0 (c = 2.0
CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.88-7.21 (m, 24H, ArH), 6.22 (dd, J = 10.8, 9.2 Hz,
1H, H3), 5.66 (t, J = 9.6 Hz, 1H, H4), 5.44 (d, J = 8.4 Hz, 1H, H1), 4.55 (dd, J = 10.8, 8.4
Hz, 1H, H2), 3.96 (ddd, J = 9.9, 4.4, 2.7 Hz, 1H, H5), 3.94-3.92 (m, 2H, H6a, H6b), 3.51 (s,
3H, OCH3), 1.05 (s, 9H, [C(CH3)3]); 13C NMR (101 MHz, CDCl3) δ 165.9 (C=O), 165.2
(C=O), 135.8, 135.7, 134.9, 134.2, 133.3, 133.2, 130.0, 129.9, 129.8, 129.7, 129.4, 128.9,
128.5, 128.4, 127.9, 127.8, 127.7, 123.7, 99.1 (C1), 75.2 (C5), 71.7 (C3), 70.0 (C4), 63.0
(C6), 56.9 (OCH3), 55.1 (C2), 26.8 (C[CH3])3; HRMS [M+Na]+ calculated for
C45H43NNaO9Si: 792.2599; found: 792.2628.
4.3. General method for TBDPS removal
Silyl protected 2 or 9 (1.0 mmol) was dissolved in CH2Cl2 (10 mL) and to this solution
.
BF3 Et2O (1.2 mL, 10 mmol) was added and the reaction stirred for 6 h. The reaction was
diluted with CH2Cl2 (40 mL) and quenched by addition of saturated aqueous NaHCO3 (100
mL). The biphasic mixture was stirred vigorously until a pH of 7 was achieved for the
organic phase. The organic phase was then separated and reduced to dryness. The products 3
and 10 were isolated by silica gel column chromatography using a gradient elution (0-10 %
EtOAc in toluene) as white foams in 42-46 % yields.
4.3.1. Methyl 3,4-O-benzoyl-2-N-carboxybenzyl-2-deoxy-α-D-glucopyranoside (3)
560 mg of 2 gave 3 (164 mg, 42%); Rf = 0.15 (toluene/EtOAc, 4/1); [ꢀ]ꢂꢁꢃ= +20.0 (c = 2.0
CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.96-7.93 (m, 4H), 7.54-7.47 (m, 2H), 7.39-7.33 (m,
5H), 7.21-7.13 (m, 4H), 5.75 (dd, J = 10.5, 9.8 Hz, 1H, H3), 5.42 (t, J = 9.8 Hz, 1H, H4),
5.17 (d, J = 10.0 Hz, 1H, NH), 4.94 (s, 2H, CH2Ph), 4.90 (d, J = 3.5 Hz, 1H, H1), 4.32 (td, J
= 10.4, 3.6 Hz, 1H, H2), 3.95 - 3.92 (m, 1H, H5), 3.80-3.77 (m, 1H, H6a), 3.70 (dd, J = 12.8,
13
3.5 Hz, 1H, H6b), 3.46 (s, 3H, OCH3), 2.70 (s, 1H, OH); C NMR (101 MHz, CDCl3) δ
166.7 (C=O), 166.5 (C=O), 155.9 (NC=O), 136.2, 133.8, 133.4, 130.1, 130.0, 129.3, 128.8,
128.6, 128.5, 128.5, 128.2, 127.9, 98.9 (C1), 71.6 (C3), 70.4 (C5), 69.7 (C4), 67.0 (CH2Ph),
61.3 (C6), 55.7 (OCH3), 54.2 (C2); HRMS [M+Na]+ calculated for C29H29NNaO9Si:
558.1735; found: 558.1759.
4.3.2. Methyl 3,4-O-benzoyl-2-N-phthalimido-2-deoxy-β-D-glucopyranoside (10)
1
1.5 g of 9 gave 10 (389 mg, 46%); Rf = 0.15 (toluene/EtOAc, 4/1); H NMR (400 MHz,
CDCl3) δ 8.06–7.19 (m, 14H, ArH), 6.32 (dd, J = 10.8, 9.2 Hz, 1H, H3), 5.52 (t, J = 9.6 Hz,
1H, H4), 5.48 (d, J = 8.4 Hz, 1H, H1), 4.55 (dd, J = 10.8, 8.4 Hz, 1H, H2), 3.90 (m, 2H, H5,
13
H6a), 3.82–3.68 (m, 1H, H6b), 2.61 (s, 1H, OH); C NMR (101 MHz, CDCl3) δ 166.3
(C=O), 165.8 (C=O), 134.3, 133.8, 133.4, 130.1, 129.9, 128.8, 128.7, 128.6, 128.5, 123.7,
7

99.4(C1), 74.5 (C5), 71.0 (C3), 70.3 (C4), 61.5 (C6), 57.3 (OCH3), 54.9 (C2); Data matched
those reported previously.39
4.4. General method for 6-O-Sulfation
Compound 3 or 10 (0.5 mmol) was dissolved in pyridine (1.0 mL) in a microwave tube.
.
SO3 pyridine complex (480 mg, 3 mmol) and a magnetic stirer bar were added and the vessel
was sealed. The reaction was heated to 100 °C and irradiated for 1 h. The reaction was
reduced to dryness then taken up in EtOAc (20 mL) and dry loaded on to silica gel. The
products 4 and 11 were subsequently isolated by column chromatography using a gradient
elution (0-20% MeOH in EtOAc then EtOAc/MeOH/33% aq. NH4OH, 7/2/1) to furnish the
products as white solids in 95-96% yields.
4.4.1. Methyl 3,4-O-benzoyl-2-N-carboxybenzyl-6-O-sulfonate-2-deoxy-α-
ammonium salt (4)
D-glucopyranoside
150 mg of 3 gave 4 (168 mg, 95%); Rf = 0.7 (EtOAc/MeOH/33% aq. NH3OH, 7/2/1); [ꢀ]ꢂꢁꢃ=
+37.0 (c = 2.0 CHCl3) 1H NMR (400 MHz; MeOD) δ 7.93-7.85 (m, 4H, ArH), 7.55-7.50 (m,
2H, ArH), 7.37 (m, 4H, ArH), 7.20-7.08 (m, 5H, ArH), 5.73 (dd, J = 10.6, 9.6 Hz, 1H, H3),
5.44 (t, J = 9.8 Hz, 1H, H4), 5.05-4.92 (dd, 2H, CH2Ph), 4.88 (d, J = 3.5 Hz, 1H, H1), 4.38-
4.27 (m, 2H, H2, H5), 4.25-4.14 (m, 2H, H6a, H6b), 3.52 (s, 3H, OCH3); 13C NMR (101
MHz; MeOD) δ 166.2 (C=O), 165.5 (C=O), 157.1 (NC=O), 148.6, 137.2, 136.6, 133.2,
133.1, 129.4, 129.3, 129.2, 129.1, 128.1, 128.0, 127.9, 127.4, 127.0, 98.7 (C1), 72.1 (C3),
70.0 (C4), 68.3 (C5), 66.3 (C6), 66.1 (CH2Ph), 54.7 (OCH3), 53.9 (C2).
4.4.2. Methyl 3,4-O-benzoyl-2-N-phthalimido-6-O-sulfonate-2-deoxy-β-D-glucopyranoside
ammonium salt (11)
370 mg of 10 gave 11 (440 mg, 95%); Rf = 0.7 (EtOAc/MeOH/33% aq. NH3OH, 7/2/1);
1
[ꢀ]ꢂꢁꢃ= +48.0 (c = 2.0 CHCl3); H NMR (400 MHz; CDCl3) δ 7.94 (tt, J = 7.7, 1.7 Hz, 5H,
ArH), 7.86-7.84 (m, 2H, ArH), 7.76-7.74 (m, 2H, ArH), 7.47 (tt, J = 7.4, 1.5 Hz, 1H, ArH),
7.42 (tt, J = 7.5, 1.5 Hz, 1H ArH), 7.34-7.24 (m, 4H, ArH), 6.20 (dd, J = 10.8, 9.2 Hz, 1H,
H3), 5.54 (dd, J = 10.0, 9.2 Hz, 1H, H4), 5.46 (d, J = 8.5 Hz, 1H, H1), 4.47-4.41 (m, 2H, H2,
H6a), 4.35 (dd, J = 11.2, 5.2 Hz, 1H, H6b), 4.26 (ddd, J = 10.1, 5.2, 2.5 Hz, 1H, H5), 3.48 (s,
3H, OCH3); 13C NMR (101 MHz; CDCl3) δ 165.7 (C=O), 165.6 (C=O), 134.1, 134.0, 133.3,
133.1, 131.5, 131.4, 131.3, 129.9, 129.7, 129.6, 128.8, 128.7, 128.3, 128.2, 123.5, 99.5 (C1),
72.31 (C5), 71.5 (C3), 69.4 (C4), 65.6 (C6), 57.9 (OCH3), 54.5 (C2); HRMS [M+Na]+
calculated for C29H25NNaO12S: 634.0995; found: 634.1006.
4.5 Methyl 2-amino-6-O-sulfonate-2-deoxy-α-D-glucopyranoside ammonium salt (5)
6-O-sulfate 4 (166 mg, 0.25 mmol) was dissolved in MeOH (6 mL) and to this Pd/C (67 mg,
0.06 mmol) was added. The reaction was placed under an H2 atmosphere, stirred for 16 h,
filtered through a Celite® pad and reduced to dryness. Crude NMR and LRMS analyses
confirmed loss of the CBz group. The crude material was taken up in MeOH (5 mL) and 1M
NaOMe in MeOH was added until a pH of 10 was achieved. The reaction was then heated to
40 °C for 2 h, cooled to room temperature and Amberlite IR120 H+ resin added. This
suspension was stirred until a pH of 7 was achieved, filtered, washed with MeOH (5 mL) and
water (5 mL) and the filtrate reduced to dryness. The crude product was purified by silica gel
column chromatography (EtOAc/MeOH/ 33% aq. NH3OH, 7/2/1 then EtOH/33% aq.
NH3OH, 9/1). The product-containing fractions were reduced to dryness and lyophilised to
furnish 5 as a white solid (46 mg, 63%). Rf 0.4 (MeCN/33% aq. NH3OH, 4/1); [ꢀ]ꢂꢄ=
=
ꢁ
+79.0 (c = 1.4 H2O); 1H NMR (400 MHz; D2O) δ 4.93 (d, J = 3.4 Hz, 1H, H1), 4.27-4.23 (m,
1H, H6a), 4.19 (dd, J = 11.4, 4.7 Hz, 1H, H6b), 3.86-3.82 (m, 1H, H5), 3.78 (t, J = 9.8 Hz,
8

13
1H, H3), 3.47 (t, J = 9.6 Hz, 1H, H4), 3.38 (s, 3H, CH3), 3.27 (m, 1H, H2); C NMR (101
MHz; D2O) δ 96.2 (C1), 69.9 (C5), 69.8 (C3), 69.1 (C4), 66.7 (C6), 55.3 (OCH3), 53.8 (C2);
HRMS [M-H]- calculated for C7H14NO8S: 272.0440; found: 272.0446. Data matched those
previously reported.29
4.6 Methyl 2-amino-6-O-sulfonate-2-deoxy-β-D-glucopyranoside ammonium salt (12)
6-O-sulfate 11 (370 mg, 0.57 mmol) was dissolved in EtOH (20 mL) and to this ethylene
diamine (760 ꢀL, 11.4 mmol) was added. The solution was heated to 70 °C for 16 h then
reduced to dryness. The crude product was purified by silica gel column chromatography
(EtOAc/MeOH/ 33% aq. NH3OH, 7/2/1 then EtOH/33% aq. NH3OH, 9/1). The product-
containing fractions were reduced to dryness and lyophilised to furnish 12 as a white solid
(92 mg, 56%). Rf = 0.1 (EtOAc/MeOH/33% aq. NH3OH, 7/2/1); [ꢀ]ꢂꢁꢄ= -21.0 (c = 1.6 H2O);
1H NMR (400 MHz; D2O) δ 4.50 (d, J = 8.4 Hz, 1H, H1), 4.27 (dd, J = 11.3, 2.1 Hz, 1H,
H6a), 4.17 (dd, J = 11.2, 4.8 Hz, 1H, H6b), 3.63 (ddd, J = 9.7, 4.8, 2.1 Hz, 1H, H5), 3.56-
3.45 (m, 6H, H3, H4, OCH3), 2.86 (dd, J = 10.2, 8.4 Hz, 1H, H2); 13C NMR (101 MHz; D2O)
δ 101.0 (C1), 73.9 (C5), 72.8 (C3), 69.3 (C4), 66.6 (C6), 57.4 (OCH3), 55.8 (C2); HRMS [M-
H]- calculated for C7H14NO8S: 272.0440; found: 272.0446.
4.7 General procedure for N-acetylation
Compound 5 or 12 (50 ꢀmol) were dissolved in a solution of MeOH/Ac2O (2.20 mL, 10/1).
The reaction was stirred for 16 h then reduced to dryness and purified by silica gel column
chromatography (MeCN/33% aq. NH3OH, 4/1). The product-containing fractions were
reduced to dryness and lyophilised to furnish the products as white powders in 78-83%
yields.
4.7.1. Methyl 2-N-acetamido-6-O-sulfonate-2-deoxy-α-D-glucopyranoside ammonium salt (6)
10 mg of 5 gave 6 (9 mg, 78%). Rf = 0.4 (MeCN/33% aq. NH3OH, 4/1); [ꢀ]ꢂꢁꢄ= +52 (c = 0.7
1
H2O); H NMR (400 MHz; D2O) δ 4.62 (d, J = 3.6 Hz, 1H, H1), 4.19 (dd, J = 11.2, 2.2 Hz,
1H, H6a), 4.11 (dd, J = 11.2, 5.2 Hz, 1H, H6b), 3.81 (dd, J = 10.6, 3.6 Hz, 1H, H2), 3.78-
3.73 (m, 1H, H5), 3.58 (t, J = 9.8 Hz, 2H, H3), 3.39 (dd, J = 10.1, 9.1 Hz, 1H, H4), 3.25 (s,
3H, OCH3), 1.85 (s, 3H, CH3); 13C NMR (peaks extrapolated from HSQC) δ 97.9 (C1), 70.8
(C3), 69.8 (C5), 69.4 (C4), 66.8 (C6), 55.2 (OCH3), 53.3 (C2), 21.1 (CH3); HRMS [M-H]-
calculated for C9H16NO9S: 314.0546; found: 314.0548. Data matched those previously
reported.29
4.7.2. Methyl 2-N-acetamido-6-O-sulfonate-2-deoxy-β-
D
-glucopyranoside ammonium salt
(13)
1
20 mg of 12 gave 13 (19 mg, 83%). Rf = 0.2 (MeCN/33% aq. NH3OH, 4/1); H NMR (400
MHz; MeOD) δ 4.37 (dd, J = 11.0, 2.0 Hz, 1H, H6a), 4.32 (d, J = 8.4 Hz, 1H, H1), 4.16 (dd,
J = 11.0, 5.8 Hz, 1H, H6b), 3.67 (dd, J = 10.1, 8.4 Hz, 1H, H2), 3.52-3.49 (m, 1H, H5), 3.48-
3.44 (m, 4H, OCH3, H4), 3.37 (dd, J = 9.7, 8.8 Hz, 1H, H3), 2.00-1.99 (m, 3H, CH3); HRMS
[M-H]- calculated for C9H16NO9S: 314.0546; found: 314.0548; Data matched those
previously reported.40
4.8. General Procedure for N-Sulfation
Compound 5 or 12 (50 ꢀmol) were dissolved in water (2.0 mL). A portion from both
NaHCO3 (70 mg) and SO3.pyridine (50 mg) was added at 10 min., 1 h, 2 h and 4 h time
points and the suspension was then stirred at room temperature for a further 16 h. The
reaction was reduced to dryness, MeOH (5 mL) was added, the suspension centrifuged and
the liquors were removed and reduced. This process was repeated three times to remove
9

excess salt. The crude residue was then purified by silica gel column chromatography
(MeCN/33% aq. NH3OH, 4/1). The product-containing fractions were combined, reduced to
dryness and lyophilised to furnish the products as white powders in 38-51% yields.
4.8.1 Methyl 6-O-sulfonate-2-deoxy-2-sulfamino-α-D-glucopyranoside ammonium salt (7)
10 mg of 5 gave 7 (5 mg, 38%). Rf = 0.3 (MeCN/33% aq. NH3OH, 4/1); [ꢀ]ꢂꢁꢄ= +28 (c = 0.3
1
H2O); H NMR (400 MHz; D2O) δ 4.94 (d, J = 3.6 Hz, 1H, H1), 4.25 (dd, J = 11.0, 1.9 Hz,
1H, H6a), 4.16 (dd, J = 11.5, 5.0 Hz, 1H, H6b), 3.81-3.77 (m, 1H, H5), 3.51 (dd, J = 9.8, 9.3
Hz, 1H, H3), 3.44 (t, J = 9.5 Hz, 1H, H4), 3.35 (s, 3H, OCH3), 3.19 (ddd, J = 10.1, 3.7, 0.8
13
Hz, 1H, H2); C NMR (peaks extrapolated from HSQC) δ 98.1 (C1), 71.1 (C3), 69.3 (C4),
69.1 (C5), 66.7 (C6), 57.2 (C2), 55.0 (OCH3); HRMS [M-H]2- calculated for C7H13NO11S2:
175.4970; found: 175.4975. Data matched those previously reported.29
4.8.2 Methyl 6-O-sulfonate-2-deoxy-2-sulfamino-β-D-glucopyranoside ammonium salt (14)
20 mg of 12 gave 14 (13.5 mg, 51%). Rf = 0.3 (MeCN/33% aq. NH3OH, 4/1); [ꢀ]ꢂꢁꢄ= -15.0 (c
= 1.2 H2O) 1H NMR (400 MHz; D2O) δ 4.41 (d, J = 8.4 Hz, 1H, H1), 4.27 (dd, J = 11.2, 2.1
Hz, 1H, H6a), 4.15 (dd, J = 11.2, 5.2 Hz, 1H, H6b), 3.60-3.56 (m, 2H, H3, H5), 3.47 (s, 3H,
13
OCH3), 3.42 (t, J = 9.4 Hz, 1H, H4), 2.94 (dd, J = 10.1, 8.4 Hz, 1H, H2); C NMR (101
MHz; D2O) δ 102.5 (C1), 74.5 (C3 or C5), 73.4 (C3 or C5), 69.5 (C4), 67.0 (C6), 59.9 (C2),
57.2 (OCH3); HRMS [M-H]2- calculated for C7H13NO11S2: 175.4970; found: 175.4973.
4.9. Cell based assays
Murine NIH 3T3 fibroblast cells and African green monkey Vero kidney epithelial
cells were purchased from ATCC. Murine BaF3 lymphocytes transfected with human
FGFR1c were a kind gift from Prof. David Ornitz (Washington University, St. Louis, USA).
Vero and 3T3 cells were maintained at 50-75% confluence in DMEM supplemented with
10% foetal bovine serum, 20 mM
L-glutamine, 100 U/ml penicillin-G and 100U/ml
streptomycin sulfate (all purchased from Gibco/ThermoFisher, UK). BaF3 cells were
maintained at no greater than 1 x 106 cells/mL in RPMI supplemented with 10% foetal
bovine serum, 20 mM L-glutamine, 100 U/ml penicillin-G, 100U/ml streptomycin sulfate
(all purchased from Gibco/ThermoFisher, UK) and 1 ng/ml recombinant murine Il-3 (R&D
Systems, UK). Cells were maintained at 37 °C, 5% CO2.
4.9.1 Cell toxicity assays
Vero and 3T3 cells were plated into 96-well cell culture plates at 1000 cells/well in
100 µl of maintenance medium. Cells were allowed to adhere overnight. Medium was
replaced with 100 µl maintenance medium +/- 100 µg/ml of saccharides or 0.1% Triton X-
100 (Roche/Sigma). Cells were incubated for 48 hours then 250 µg/ml MTT (final
concentration, Roche/Sigma) was added and the cells were incubated a further 4 hours.
Formazan product was solubilised with the addition of 50 µl 10% SDS + 0.1N HCl (both
from Roche/Sigma). Absorbance was read at 570 nm on a Tecan Infinite M200Pro plate
reader. The procedure for BaF3 cells was identical, with the following exceptions. Cells
were plated at 10000 cells/well, incubated with saccharides for 72 hours and removal of Il-3
served as a control for cell death, rather than Triton X-100. Results are representative of 3
independent experiments for each cell type with triplicate wells for each condition.
4.9.2 FGF-2 Cell proliferation assays
BaF3 cells were maintained as per 4.9. For FGF-2 proliferation assays cells were
washed 2 x with 10 mL of maintenance medium without Il-3 to remove Il-3. Cells were
plated at 10000 cells/ml in 96 well cell culture plates with 100 µl of maintenance medium
10

supplemented with 1nM recombinant human FGF-2 (R&D Systems) and 1 µg/ml porcine
intestinal mucosa heparin (Celsus) alone or in combination with 100 µg of the indicated
saccharides. Cells were incubated for 72 hours then cell number was determined by MTT as
in 4.9.1.
Acknowledgements
Science Foundation Ireland (Award 13/IA/1959) and EPSRC (EP/P000762/1) are
thanked for project grant funding. We also thank the EPSRC UK National Mass
Spectrometry Facility (NMSF) at Swansea University.
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13

•
•
Chemical synthesis of a sulphated D-glucosamine library
Access to α- and β-O-methyl glycosides of
variant N-substitution
D-glucosamine with O6 sulfation and
•
Evaluation of compound series in cell toxicity assays

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: