Influence of the reducing-end anomeric configuration of the Man9epitope on DC-SIGN recognition

Article abstract of DOI:10.1039/d0ob01380c

High-mannose (Man9GlcNAc2) is the main carbohydrate unit present in viral envelope glycoproteins such as gp120 of HIV and the GP1 of Ebola virus. This oligosaccharide comprises the Man9 epitope conjugated to two terminal N-acetylglucosamines by otherwise

Full text of DOI:10.1039/d0ob01380c

Organic &
Biomolecular Chemistry
View Article Online
View Journal
PAPER
Influence of the reducing-end anomeric
configuration of the Man9 epitope on DC-SIGN
recognition†
Cite this: DOI: 10.1039/d0ob01380c
Noelia de la Cruz,a Javier Ramos-Soriano,*a José J. Reina,a José L. de Paz,a
a
Michel Thépaut,b Franck Fieschi,b Ana Sousa-Hervesa and Javier Rojo
*
High-mannose (Man9GlcNAc2) is the main carbohydrate unit present in viral envelope glycoproteins such
as gp120 of HIV and the GP1 of Ebola virus. This oligosaccharide comprises the Man9 epitope conjugated
to two terminal N-acetylglucosamines by otherwise rarely-encountered β-mannose glycosidic bond.
Formation of this challenging linkage is the bottleneck of the few synthetic approaches described to
prepare high mannose. Herein, we report the synthesis of the Man9 epitope with both alpha and beta
configurations at the reducing end, and subsequent evaluation of the impact of this configuration on
binding to natural receptor of high-mannose, DC-SIGN. Using fluorescence polarization assays, we
demonstrate that both anomers bind to DC-SIGN with comparable affinity. These relevant results there-
fore indicate that the more synthetically-accesible Man9 alpha epitope may be deployed as ligand for
DC-SIGN in both in vitro and in vivo biological assays.
Received 6th July 2020,
Accepted 27th July 2020
DOI: 10.1039/d0ob01380c
rsc.li/obc
Specific ICAM-3 Grabbing Non-integrin) receptor, found in
lipid patches at the dendritic cell (DC) surface.7 In particular,
Introduction
Carbohydrates are ubiquitous in nature and participate in gp120 of HIV-1 has an average of 24 N-linked glycans clustered
many biological events relevant to health and disease, such as on the protein surface, of which 53–76% are high-mannose
cell growth and differentiation, fertilization, inflammation, derivatives.8 This cluster presentation is fundamental to the
tumour progression and metastasis, viral infection, and many efficient interaction with DC-SIGN.
others.1 The function of carbohydrates in these processes
results from interactions with specific receptors, mainly pro-
teins known as lectins.2 Carbohydrate–protein interactions are
characterised by a high selectivity and a low affinity, the latter
of which is compensated by multivalent interactions resulting
from the presentation of multiple copies of the carbohydrate
epitopes and receptors in nature.3 Understanding carbo-
hydrate-mediated processes therefore requires the accessibility
of multivalent carbohydrate platforms capable of intervening
with these processes.4–6
High-mannose (Man9GlcNAc2) is
a complex relevant
N-glycan present in several viral envelope glycoproteins
(Fig. 1). This sugar plays a crucial role during the attachment
of pathogens to cells in the first stages of the infection process
through the interaction with the DC-SIGN (Dendritic Cell-
aGlycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ),
CSIC – Universidad de Sevilla, Av. Américo Vespucio 49, Seville 41092, Spain.
E-mail: javiramossoriano@gmail.com, javier.rojo@iiq.csic.es
bUniv. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale, 38000 Grenoble,
France
†Electronic supplementary information (ESI) available: 1H and 13C NMR spectra
and selected HSQC and MS spectra. See DOI: 10.1039/D0OB01380C
Fig. 1 Chemical structure of high-mannose and its constitutive parts,
the beta Man9 and the GlcNAc2 epitopes.
This journal is © The Royal Society of Chemistry 2020
Org. Biomol. Chem.

View Article Online
Paper
Towards better understanding the recognition process
Organic & Biomolecular Chemistry
between high-mannose and DC-SIGN, several multivalent
systems have been envisaged. However, the structural complex-
ity of the constituent sugars and the difficulty of obtaining
sufficient quantities of pure material from natural sources
renders the preparation of multivalent systems carrying the
natural beta epitope rather challenging. In fact, very few
examples are reported in the literature describing the synthesis
of Man9 and high-mannose multivalent systems.9–11 In this
context, different approaches have been developed for the
preparation of simpler compounds that mimic the multivalent
presentation of the sugar found in nature. Small fragments of
the Man9 epitope have been synthesised and evaluated in
different glycomimetic systems in pursuit of sugars simpler
than Man9 but with reasonable binding affinities for
DC-SIGN.12–15 Recently, we have developed a straightforward
synthetic strategy for the preparation of the Man9 epitope with
the natural beta configuration at the reducing end.16 Although
the synthesis that we have described is competitive with those
published previously by other laboratories, the preparation of
the β-mannose unit remains the limiting step. The synthesis of
the appropriately-functionalised beta-mannose monosacchar-
ide building block, required to perform the necessary glycosy-
lation steps towards the preparation of the nonasaccharide,
demands eleven synthetic steps with a global yield of 43%
from S-tolyl-α-D-mannopyranoside.16 As a possible solution,
we considered whether the more synthetically-accessible
α-mannose analogue would provide a similar affinity to
Fig. 2 Retrosynthetic scheme for the preparation of αMan9 and βMan9.
Scheme 1 Consecutive synthesis of mannose derivative 6.
DC-SIGN than the beta anomer, therefore circumventing the subjecting 5 to benzaldehyde dimethyl acetal and camphorsul-
requirement for the complicated β-mannose building block, in fonic acid (CSA). Subsequently, position 3 was protected as the
the preparation of the corresponding carbohydrate multivalent p-methoxybenzy ether, via the initial formation of a tin acetal
systems.
between position 2 and 3, followed by reaction with p-methoxy-
To evaluate our proposed strategy, we have synthesised the benzyl chloride (PMBCl) and tetrabutyl ammonium iodine
alpha and beta anomers of the Man9 epitope and the corres- (TBAI). The free OH in position 2 was then protected by ben-
ponding trivalent glycocluster to examine the differences in zoylation with benzoyl anhydride in the presence of triethyl
binding affinities to the DC-SIGN receptor using a fluorescence amine and a catalytic amount of dimethylamino pyridine
polarization assay.
(DMAP). Finally, the position 3 was selectively deprotected
with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) to yield
the target mannose derivative 6 in 26% overall yield from
mannose 5 following final chromatographic purification.
With the monosaccharide 6 in hand, glycosylation with the
previously synthesised trisaccharide 7 and pentasaccharide
Results and discussion
Preparation of Man9 epitopes and glycoclusters
The synthesis of beta Man9 using a convergent straightforward 8,16 enabled the formation of the protected Man9 nonasacchar-
synthetic strategy was previously described by our laboratory.16 ide 11 (Scheme 2).
In this work, we used the same strategy to prepare the corres-
ponding alpha anomer.
The glycosylation between trisaccharide 7 and mannose
derivative 6 was performed using N-iodo succinimide and tri-
The di-, penta-, and trisaccharides building blocks were fluoromethanesulfonic acid as the glycosylation promotor to
common intermediates for both anomers, (Fig. 2) being the obtain tetrasaccharide 9 in 88% yield. Next, the benzylidene
mannose of the reducing end with the alpha configuration in acetal was removed in acidic media using p-TsOH to afford the
the anomeric position prepared as described in Scheme 1.
tetrasaccharide 10 (bearing unprotected hydroxyl groups at
The synthesis of the appropriately-protected reducing end positions 4 and 6 of the terminal mannose at the reducing
mannose was achieved as depicted in Scheme 1, starting from end) in 78% yield. The final glycosylation between tetrasac-
the previously described mannosyl derivative 5.17 Mannose 5 charide 10 and pentasaccharide 8 took place only at the posi-
was orthogonally protected using a consecutive approach, per- tion 6 hydroxyl group of the acceptor, since this is more reac-
forming purification only at the final step. Positions 4 and 6 tive than the sterically hindered hydroxyl group at position 4.
were protected via the formation of the benzylidene acetal by This glycosylation was carried out using the same conditions
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Organic & Biomolecular Chemistry
Paper
Scheme 3 Synthesis of the trivalent glycoclusters 17 and 18.
focal position of the scaffold. This reaction was performed
using an excess of NaN3 in DMF at 70 °C for two days furnish
azido-functionalised glycoclusters 17 and 18 in excellent yields.
Finally, it was necessary to introduce a chromophore for the
Scheme 2 Synthesis of the αMan9 12.
as above to yield the protected nonasaccharide 11 in 73% fluorescence polarization assays. An alkynyl derivative of fluor-
1
yield. The nonasaccharide 11 was characterised by H and 13C escein, the commercially available FAM-alkyne 6-isomer 19,
NMR and ESI-MS. Finally, global deprotection step with was therefore conjugated to the αMan9 12, the βMan9 13, and
NaOMe and 2 M NaOH in MeOH and toluene cleaved all their corresponding trivalent glycoclusters 17 and 18, respect-
O-benzoyl groups affording the αMan9 12 in excellent yield ively, by a CuAAC click reaction promoted by CuBr and TBTA
(Scheme 2). In this way, the synthesis of the alpha anomer of in DMSO at room temperature (Scheme 4).
Man9 was achieved in an expedient manner through a conver-
The compounds were treated with Quadrasil MP resin and
gent strategy. Both Man9 epitopes (alpha and beta) were pre- then, submitted to a LH20 Sephadex chromatography to
pared with a short spacer at the anomeric position functiona- deliver the fluorescently-labelled compounds 20, 21, 22 and 23
lised with a terminal azido group. This group permits conju- in excellent yields.
gation to multivalent scaffolds using the Cu(I) azide–alkyne
With the fluorescent tool compounds prepared, we evalu-
ated their capacity to interact with DC-SIGN, the natural recep-
cycloaddition (CuAAC) reaction.20,21
To examine the effect that the multivalent presentation has tor of this Man9 ligand.
on the binding DC-SIGN to both anomers, we prepared the tri-
valent glycoclusters with the alpha and beta Man9 epitopes
(Scheme 3).
Fluorescence polarization assays
Fluorescence polarization is widely used to study binding
For this purpose, we employed a trialkynylated pentaerythri- events owing to several advantages of this technique.22,23 The
tol scaffold, frequently used for the preparation of carbo- assay requires only small quantities of ligands and receptors,
hydrate multivalent systems by our group. This scaffold 14 was is well suited for high-throughput screening and does not
prepared in two steps from pentaerythritol as previously require the attachment of any of the partners to a surface,
reported.18 The coupling of αMan9 and βMan9 ligands was allowing ligands and receptors diffuse freely in solution. In
then carried out via a click chemistry19 CuAAC reaction pro- particular, fluorescence polarization experiments have been
moted by CuSO4, sodium ascorbate and tris[(1-benzyl-1H-1,2,3- successfully employed in the analysis of carbohydrate–protein
triazol-4-yl)methyl]amine (TBTA) under mild conditions to interactions.24–27 Thus, we selected this assay to evaluate the
obtain the corresponding trivalent glycoclusters. These multi- binding affinity of our alpha and beta epimers of Man9 (20
valent systems were purified by treatment with Quadrasil MP and 21) and their corresponding multivalent systems (22 and
resin to remove the copper catalyst and by G50 Sephadex 23) for DC-SIGN using the tetravalent Extracellular Domain
chromatography to afford the glycoclusters 15 and 16 in good (ECD) of the lectin. For this, we have used a microtiter plate
yields (Scheme 3). The final step was the substitution of the where different concentrations of the protein (DC-SIGN ECD),
chlorine atom for an azido group at the end of the linker at the ranging from 75 nM to 28 µM in Tris buffer, were added to the
This journal is © The Royal Society of Chemistry 2020
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 3 Langmuir isothermal curves of binding between DC-SIGN ECD
and fluorescence compounds: (a) αMan9 (20); (b) βMan9 (21); (c) trivalent
glycocluster 22; and (d) trivalent glycocluster 23.
In order to evaluate the influence of the anomer configur-
ation when the Man9 ligand is presented in a multivalent
scaffold, fluorophore labelled glycoclusters 22 and 23 were
tested in the fluorescence polarization assays (Fig. 3). In this
case, the KD were one order of magnitude lower than the data
found for the monovalent systems, indicating a clear multi-
valent effect (0.53 0.09 and 0.37 0.04 μM for the trivalent
αMan9 and βMan9, respectively). Again, no significant differ-
ences between the dissociation constants of the two anomers
were found as in the case of the monovalent ligands.
Conclusions
The stereochemical configuration of sugars is fundamental to
their selective recognition by lectins. Indeed, just a simple
difference in the configuration of one position can drive the
recognition by one specific lectin, e.g. glucose (with all OH
groups in equatorial disposition) versus galactose (which bears
an axial OH at C4). Furthermore, the configuration of the gly-
cosidic bonds between the monosaccharidic units that form
an oligosaccharide are relevant for their recognition and func-
tion. In the case of high mannose, all the mannoses of the
Man9 epitope display the alpha configuration of the glycosidic
Scheme 4 Synthesis of fluorescence labelled compounds 20, 21, 22
and 23.
wells containing a fixed final 10 nM concentration of appropri- bonds, as usual for this type of sugar. However, the linkage
ate fluorescent ligands (20–23). We observed an increase in the between the Man9 epitope and the GlcNAc disaccharide exhi-
fluorescence polarization value with increasing protein con- bits a beta configuration. This glycosidic bond configuration is
centrations, demonstrating that the fluorescence ligands rare for mannoses and presents a significant hurdle towards
bound to DC-SIGN ECD. The resulting Langmuir isotherm addressing this bond configuration via chemical synthesis. All
curves are represented in Fig. 3. From these curves, the KD of N-glycans have this moiety as connection with the corres-
the binding process for each ligand was calculated. For the α ponding Asn in the N-glycan proteins. This specific arrange-
and β anomers of Man9, similar values of KD, (5.2 1.2 and 4.6 ment is critical for the proper orientation of the mannosyl
1.3 μM, respectively) were found demonstrating that the con- cluster, providing a rigid structure on the protein surface to
figuration of the anomer in the mannosyl unit at the reducing optimize the binding to DC-SIGN. However, when new glyco-
end of the oligosaccharide does not play a critical role in the multivalent systems are designed, the glycan epitope is nor-
binding process.
mally conjugated to the multivalent scaffold with a flexible
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Organic & Biomolecular Chemistry
Paper
linker and therefore the specific orientation provided by the tion mixture was kept under reflux at 110 °C for 1 h. After
beta-mannose linkage is likely not necessary. To demonstrate removal the solvent, the crude was diluted with CH2Cl2 and
this hypothesis, we have synthesised the Man9 epitope of high washed with water. The organic layer was dried over anh.
mannose with alpha and beta configuration in the terminal MgSO4, filtered and concentrated under vacuum. To the result-
mannose at the reducing end (20 and 21) and the corres- ing mixture containing 4,6-O-benzylidene-3-O-p-methoxybenzyl
ponding trivalent glycoclusters (22 and 23). These ligands were derivative in CH2Cl2 (15 mL), Bz2O (1.5 g, 6.42 mmol), Et3N
labelled with a fluorescein derivative and their binding to (1.3 mL, 9.63 mmol) and a catalytic amount of DMAP were
DC-SIGN evaluated by fluorescence polarization assays. Our subsequently added and the reaction was stirred at rt for 1 h.
findings have shown that the configuration, alpha or beta, of The reaction mixture was washed with NaHCO3 sat. aq. soln.
the anomeric position of the terminal mannose does not influ- The organic phase was dried over anh. MgSO4, filtered and
ence in the binding affinity for the natural receptor DC-SIGN concentrated under vacuum. Finally, to a stirred solution of
and therefore they can be used interchangeably, meaning the the α-D-mannoside derivative in CH2Cl2 (70 mL) and water
more synthetically-accessible alpha-linked epimers can be (2.7 mL), DDQ (2.23 g, 9.63 mmol) was added at rt. After 1 h,
deployed instead of the more challenging natural beta ana- NaHCO3 sat. aq. soln. was added, and the mixture was
logues. We anticipate this knowledge is of key relevance in the extracted with CH2Cl2. The extract was washed several times
design of new glycosystems of high mannose, since notably with NaHCO3 sat. aq. soln. and then dried over anh. MgSO4,
reduces the complexity, the time and the cost of the glycan filtered and concentrated under vacuum. The crude was puri-
synthesis by circumventing the synthetic bottleneck of the beta fied by column chromatography on silica gel (EtOAc/n-hexane
mannose preparation.
1 : 3) to give the compound 6 (320 mg, 0.72 mmol, 26%) as a
colourless oil. [α]D = −37 (c 1.00, CHCl3). H-NMR (400 MHz,
1
CDCl3) δ: 8.11 (m, 2H, H–Ar), 7.60 (m, 1H, H–Ar), 7.56–7.45
(m, 4H, H–Ar), 7.43–7.35 (m, 3H, H–Ar), 5.67 (s, 1H, Hacetal),
5.52 (dd, J2,3 = 3.6, J2,1 = 1.6 Hz, 1H, H-2), 5.01 (d, J1,2 = 1.6 Hz,
1H, H-1), 4.41 (dt, J3,4 = 9.6, J3,2 = 3.9 Hz, 1H, H-3), 4.33 (dd,
J6a,6b = 9.9, J6a,5 = 4.4 Hz, 1H, H-6a), 4.06 (t, J4,3 = J4,5 = 9.5 Hz,
1H, H-4), 4.03–3.84 (m, 3H, H-5, H-6b, H-1′a), 3.73–3.63 (m,
1H, H-1′b), 3.55–3.40 (m, 2H, H-2′), 2.32 (d, JOH,3 = 4.2 Hz, 1H,
OH). 13C-NMR (100 MHz, CDCl3) δ: 166.0, 137.1, 133.5, 129.9,
129.5, 129.3, 128.5, 128.3, 126.3, 102.2, 98.7, 79.2, 72.5, 68.7,
67.1, 66.9, 63.8, 50.4. ESI-MS m/z calcd for C22H23N3O7: 441.2;
found: 464.2 [M + Na]+.
Tetrasaccharide 9. To a solution of acceptor 6 (49 mg,
0.11 mmol), donor 7 (275 mg, 0.17 mmol) and 4 Å molecular
sieves in anh. CH2Cl2 (5.8 mL) were added and the mixture
was stirred at −20 °C for 30 min. Then, NIS (39 mg,
0.17 mmol) and TfOH (2.9 μL, 0.03 mmol) were added and the
reaction was stirred at −20 °C for 15 min. The reaction was
quenched with NaHCO3 sat. aq. soln. The reaction mixture was
filtered through Celite and washed several times with CH2Cl2.
Experimental
Materials and methods
Solvents were HPLC grade and used as received unless other-
wise stated. Size exclusion chromatography was performed
with Sephadex LH20, G-25 and G-50 (GE Healthcare). Thin-
layer chromatography (TLC) was performed on silica plates
(Merck). Reagents and QuadraSil® MP were purchased from
Sigma Aldrich. 2-Azidoethyl α-D-mannopyranoside17 and trialk-
ynylated scaffold 14 18 were synthesised as previously
described. NMR experiments were performed using a Bruker
Advance DRX 400 instrument. NMR chemical shifts are
reported in ppm (δ units) downfield from the CDCl3 signal or
the HOD peak (D2O). 2D experiments (COSY and HSQC) were
performed when necessary. NMR spectra were analysed with
MestreNova software.
Synthesis of ligands
2-Azidoethyl 2-O-benzoyl-4,6-O-benzylidene-α-D-mannopyra- The organic layer was washed with Na2S2O3 sat. aq. soln. and
noside (6). To a solution of 2-azidoethoxy-α-D-mannopyranose then dried over anh. MgSO4, filtered and concentrated under
(5) (800 mg, 3.21 mmol) in CH3CN (8 mL), CSA (186 mg, vacuum. The crude was purified by column chromatography
0.8 mmol) and benzaldehyde dimethyl acetal (0.5 mL, on silica gel (EtOAc/n-hexane 1 : 2 to 1 : 1.25) to give the tetra-
3.55 mmol) were added at rt. The reaction mixture was stirred saccharide 9 (193 mg, 0.10 mmol, 88%) as a white solid. [α]D
=
overnight and the mixing became solidified during addition of −22 (c 1.00, CHCl3). 1H-NMR (400 MHz, CDCl3) δ: 8.22 (m, 1H,
the reagent, which indicates the progress of the reaction. The H–Ar), 8.19–8.12 (m, 2H, H–Ar), 8.09–7.92 (m, 7H, H–Ar),
reaction mixture was quenched with Et3N (250 μL) and dis- 7.92–7.81 (m, 7H, H–Ar), 7.76–7.68 (m, 2H, H–Ar), 7.64–7.19
solved in excess of EtOAc and water. The reaction mixture was under CDCl3 (m, 36H, H–Ar), 7.14–6.95 (m, 4H), 6.78 (m, 1H),
extracted twice with EtOAc and the combined organic layers 6.05–5.81 (m, 5H), 5.79–5.69 (m, 2H), 5.64 (dd, J = 9.2, J = 3.1
were dried over anh. MgSO4, filtered and concentrated under Hz, 1H), 5.57 (s, 1H, H-1), 5.49 (br s, 1H, Hacetal), 5.35 (s, 1H,
vacuum. After being dissolved in anh. toluene (29 mL), dibutyl- H-1), 5.05 (d, J = 1.5 Hz, 1H, H-1), 4.91 (s, 1H, H-1), 4.72–4.50
tin oxide (879 mg, 3.55 mmol) was added to the resulting (m, 4H), 4.47 (t, J = 2.8 Hz, 1H), 4.38 (m, 1H), 4.35–4.13 (m,
mixture containing the 4,6-O-benzylidene derivative. The reac- 4H), 4.07–3.94 (m, 3H), 3.94–3.79 (m, 3H), 3.65 (m, 1H), 3.35
tion mixture was kept under reflux at 110 °C for 4 h, cooled to (m, 2H). 13C-NMR (100 MHz, CDCl3) δ: 166.4, 166.0, 165.9,
rt and PMBCl (0.5 mL, 3.55 mmol) and TBAI (370 mg, 165.8, 165.5, 165.3, 165.3, 165.2, 165.2, 165.0, 164.8, 137.0,
3.55 mmol) were added to it under Ar atmosphere. The reac- 133.7, 133.5, 133.3, 133.2, 133.1, 133.1, 133.1, 133.0, 132.8,
This journal is © The Royal Society of Chemistry 2020
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
130.1–129.6, 129.3, 129.2, 129.2, 129.1, 128.9–128.3 101.9, [M + 2Na]2+ and 1473.5 [M + 3Na]3+. ESI-HRMS m/z calcd for
99.8, 90.1, 79.1, 71.9, 71.4, 70.4, 70.1, 69.8, 69.7, 69.5, 68.8,
67.6, 67.2, 67.0, 66.3, 64.1, 63.6, 63.5, 62.9, 62.5, 50.4. ESI-MS
C
245H203N3O73Na [M + 2Na]2+: 2189.1115; found: 2189.1088.
αMan9 (12). To a solution of compound 11 (166 mg,
m/z calcd for C110H93N3O32: 1967.6; found: 1990.4 [M + Na]+. 0.04 mmol) in MeOH/toluene (4 : 1, 1.7 mL), NaOMe (11 mg,
ESI-HRMS m/z calcd for C110H93N3O32Na [M + Na]+: 1990.5634; 0.20 mmol) and a NaOH 2 M solution (0.7 mL) were added
found: 1990.5609
and the reaction was stirred at 50 °C for 5 h. After neutraliz-
Tetrasaccharide 10. p-Toluenesulfonic acid monohydrate ation with Amberlite IR-120H+, the solution was filtered and
(25 mg, 0.13 mmol) was added to a stirred solution of tetrasac- concentrated. The crude was purified by size-exclusion chrom-
charide 9 (173 mg, 0.09 mmol) in CH3CN (4.4 mL) at rt. After atography (Sephadex G-25, H2O/MeOH 9/1), giving Man9 (12)
24 h, the reaction mixture was quenched with Et3N (30 μL) and (55 mg, 0.04 mmol, 93%) as a white amorphous solid.
concentrated under vacuum. The residue was purified by 1H-NMR (400 MHz, D2O) δ: 5.40 (s, 1H, H-1), 5.33 (s, 1H, H-1),
column chromatography on silica gel (acetone/toluene 1 : 5) to 5.30 (s, 1H, H-1), 5.15 (s, 1H, H-1), 5.08–5.01 (m, 3H, 3 × H-1),
give the tetrasaccharide 10 (130 mg, 0.07 mmol, 78%) as a 4.88 (s, 1H, H-1), under D2O (1H, H-1), 4.19–3.60 (m, 58H).
white amorphous solid. [α]D = −15 (c 1.00, CHCl3). 1H-NMR 13C-NMR (100 MHz, D2O) δ: 102.2, 100.8, 100.6, 100.0, 99.5,
(400 MHz, CDCl3) δ: 8.14 (m, 2H, H–Bz), 8.07–7.93 (m, 13H, 98.0, 78.8–78.5, 73.2, 73.2, 72.7, 71.1, 71.1, 70.3–70.0, 69.6,
H–Bz), 7.90–7.82 (m, 4H, H–Bz), 7.70 (m, 2H, H–Bz), 7.61–7.18 66.9, 66.8, 66.6, 65.7, 65.5, 65.2, 61.2–61.0, 50.2. ESI-MS m/z
under CDCl3 (m, 34H, H–Bz), 6.13 (t, J = 10.2 Hz, 1H), calcd for C56H95N3O46: 1545.5; found: 1568.2 [M + Na]+, 795.5
6.03–5.90 (m, 3H), 5.84–5.72 (m, 2H), 5.67–5.57 (m, 3H, 1H-1), [M + 2Na]2+.
5.52 (d, J = 1.6 Hz, 1H, H-1), 5.09 (br s, 1H, H-1), 5.00 (d, J = 1.7
D3-αMan9-Cl (15). Nonasaccharide 12 (22.0 mg, 14.23 μmol)
Hz, 1H, H-1), 4.73–4.31 (m, 12H), 4.31–4.15 (m, 2H), 4.01 (m, and [2-[2-(2-chloroethoxy)ethoxy]ethoxy]ethoxymethyl trikis(2-
2H), 3.95–3.78 (m, 3H), 3.59 (dt, J = 10.4, 4.8 Hz, 1H), 2.34 (m, propyniloxymethyl)-methane (14) (1.6 mg, 3.59 μmol) were dis-
2H). 13C-NMR (100 MHz, CDCl3) δ: 166.4, 166.3, 166.0, 165.9, solved in H2O/DMSO (1 : 1, 0.5 mL). Fresh solutions of
165.7, 165.5, 165.2, 164.9, 133.6, 133.5, 133.4, 133.3, 133.2, CuSO4·5H2O (1.80 μmol), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)
133.0, 130.2–129.8, 129.6, 129.5, 129.4, 129.3, 129.1, 129.0, methyl]amine (TBTA) (3.59 μmol) and sodium ascorbate
128.9, 128.6–128.4, 100.8, 99.4, 98.1, 76.4, 73.3, 72.4, 70.5, (5.39 μmol) were added to a sealed microwave vial. The solu-
70.3, 69.9, 69.7–69.6, 69.4, 68.2, 67.8, 67.0, 66.6, 63.9–63.8, tion was heated at 60 °C in a microwave oven for 30 min. A
62.6, 62.3, 50.4 (C-2′). ESI-MS m/z calcd for C103H89N3O32
1879.5; found: 962.5 [M + Na]+. ESI-HRMS m/z calcd for solution and stirred for 20 min at rt. After that, the mixture
103H89N3O32Na [M + Na]+: 1902.5321; found: 1902.5307.
was filtered and the resulting solution was purified by size-
αMan9 protected (11). To a solution of acceptor 10 (110 mg, exclusion chromatography (Sephadex G-50, H2O/MeOH 9 : 1)
:
metal scavenger resin, QuadrasilMP, was added to the reaction
C
0.06 mmol), donor 8 (228 mg, 0.09 mmol) and 4 Å molecular yielding the glycocluster 15 (12 mg, 2.37 μmol, 66%) as a white
sieves in anh. CH2Cl2 (4.1 mL) were added and the mixture amorphous solid. 1H-NMR (400 MHz, D2O) δ: 8.06 (s, 3H,
was stirred at −20 °C for 30 min. Then, NIS (42 mg,
0.09 mmol) and TfOH (3.1 μL, 17.55 μmol) were added and the (br s, 3H, 3H-1), 5.09–5.02 (m, 9H, 9H-1), under D2O (6H,
reaction was stirred at −20 °C for 15 min. The reaction was 6H-1), 4.67 (m, 3H, CH2CHHN), 4.57 (br s, 3H,
Htriazole), 5.41 (br s, 3H, 3H-1), 5.37–5.27 (m, 6H, 6H-1), 5.15
O
quenched with NaHCO3 sat. aq. soln. The reaction mixture was OCHHCtriazole), 4.14–3.62 (m, 198H). 13C-NMR (100 MHz, D2O)
filtered through Celite and washed several times with CH2Cl2. δ: 144.2, 125.4, 102.2, 100.7, 99.8, 99.5, 98.0, 78.8–78.4,
The organic layer was washed with Na2S2O3 sat. aq. soln. and 73.3–73.2, 72.7, 72.0, 71.1–71.0, 70.8, 70.3, 70.0, 70.0, 69.7,
then dried over anh. MgSO4, filtered and concentrated under 69.6, 69.5, 66.9, 66.8, 65.9, 65.8, 65.6, 65.4, 65.2, 64.8, 63.4,
vacuum. The crude was purified by column chromatography 62.5, 61.0, 50.1, 43.2. ESI-MS m/z calcd for C191H320N9O145Cl:
on silica gel (EtOAc/n-hexane 4 : 5 → 1 : 1) to give the nonasac- 5080.7; found: 2562.9 [M + 2Na]2+, 1715.5 [M + 3Na]3+, and
charide 11 (185 mg, 0.04 mmol, 73%) as a white solid. [α]D
=
1295.7 [M + 4Na]4+.
D3-αMan9-N3 (17). To a solution of glycocluster 15 (9.0 mg,
−19 (c 1.00, CHCl3). 1H-NMR (400 MHz, CDCl3) δ: 8.30–7.67
(m, 52H, HBz), 7.56–7.18 (m, 80H, HBz), 6.99 (m, 2H, HBz), 1.77 μmol) in DMF (1 mL) sodium azide (1.2 mg, 17.71 μmol)
6.81 (t, J = 7.5, 1H, HBz), 6.22–5.80 (m, 16H), 5.55 (s, 1H, H-1), was added. The mixture was stirred at 70 °C for 2 days. After
5.46 (m, 2H, 2H-1), 5.32 (s, 1H, H-1), 5.18 (s, 1H, H-1), 5.05 (m, that time, the solvent was evaporated under vacuum and the
2H, 2H-1), 4.74–4.02 (m, 36H, H-1, H-1), 3.84–3.72 (m, 2H), crude was purified using Amicon Ultra-15 centrifugal filters
3.49–3.32 (m, 3H). 13C-NMR (100 MHz, CDCl3) δ: 166.5, 166.4, (MWCO 3 kDa), yielding the glycocluster 17 (9 mg, 1.77 μmol,
1
166.4, 166.3, 166.1, 166.0, 165.8, 165.6, 165.6, 165.4, 165.4, quant.) as a white amorphous solid. H-NMR (400 MHz, D2O)
165.3, 165.0, 165.0, 164.8, 164.8, 164.6, 133.5, 133.4, 133.4, δ: 8.06 (s, 3H, Htriazole), 5.41 (br s, 3H, 3H-1), 5.35–5.29 (m, 6H,
133.2, 133.2, 133.1, 130.2–129.5, 129.5, 129.5, 129.3, 129.2, 6H-1), 5.15 (br s, 3H, 3H-1), 5.07–5.03 (m, 9H, 9H-1), under
129.2, 129.0, 129.0, 128.9, 128.9, 128.7–128.3, 101.1, 100.9, D2O (6H, 6H-1), 4.67 (m, 3H, OCHHCH2N), 4.58 (br s, 3H,
100.3, 99.9, 99.5, 98.5, 97.7, 97.5, 78.2, 77.9, 75.6, 72.4, 72.2, OCHHCtriazole), 4.14–3.60 (m, 198H). 13C-NMR (100 MHz, D2O)
71.9, 71.1, 70.8, 70.5, 70.1–69.4, 68.9, 68.1, 68.0, 67.7, δ: 144.2, 125.5, 102.3, 100.7, 99.8, 99.5, 98.0, 78.8–78.4,
67.3–67.0, 66.6, 66.4, 66.1, 63.7, 63.5, 63.1, 62.6, 62.4, 50.4. 73.3–73.2, 72.0, 71.1–71.0, 70.3–69.9, 69.7–69.5, 66.9–66.8,
ESI-MS m/z calcd for C245H203N3O73: 4354.2; found: 2200.1 65.9, 65.6, 65.4, 65.2, 63.4, 62.5, 61.1–61.0, 50.1, 50.1, 44.6.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Organic & Biomolecular Chemistry
Paper
ESI-MS m/z calcd for C191H320N12O145Cl: 5087.2; found: 2567.0 HSQC, D2O) δ: 131.2, 128.2, 128.2, 127.9, 121.7, 103.0, 102.2,
[M + 2Na]2+, 1781.1 [M + 3Na]3+, and 1294.6 [M + 4Na]4+.
100.8, 100.6, 100.0, 99.5, 98.0, 78.8–78.5, 73.2, 73.2, 72.7, 71.1,
D3-βMan9-Cl (16). Nonasaccharide 13 (22.0 mg, 14.23 μmol) 71.1, 70.3–70.0, 69.6, 66.9, 66.8, 66.6, 65.7, 65.5, 65.2,
and 14 (1.6 mg, 3.59 μmol) were dissolved in H2O/DMSO (1 : 1, 61.2–61.0, 50.2. ESI-MS: m/z calcd for C80H110N4O52: 1958.6,
0.5 mL). Fresh solutions of CuSO4·5H2O (1.80 μmol), TBTA found: 978.1 [M − 2H]2−
.
(3.59 μmol) and sodium ascorbate (5.39 μmol) were added to a
sealed microwave vial. The solution was heated at 60 °C in a 6-isomer 19 (6.6 mg, 10.36 μmol) and the epitope β-Man9 13
microwave oven for 30 min. metal scavenger resin, (8 mg, 5.18 μmol) in DMSO (300 μL), another solution of CuBr
βMan9-fluorescein (21). To a solution of the FAM-alkyne
A
QuadrasilMP, was added to the reaction solution and stirred (5.18 μmol) and TBTA (10.36 μmol) in the same solvent
for 20 min at rt. After that, the mixture was filtered and the (150 μL) was added. The reaction mixture was stirred on at rt.
resulting solution was purified by size-exclusion chromato- Thereafter, a metal scavenger resin, QuadrasilMP, was added
graphy (Sephadex G-50, H2O/MeOH 9 : 1) yielding the glyco- to the reaction solution and stirred for 20 min at rt. After that,
cluster 16 (14.8 mg, 2.91 μmol, 81%) as a white amorphous the mixture was filtered and the resulting solution was purified
1
solid. H-NMR (400 MHz, D2O) δ: 8.05 (s, 3H, 3Htriazole), 5.42 by size-exclusion chromatography (Sephadex LH-20, H2O/
(br s, 1H, 3H-1), 5.34–5.28 (m, 6H, 6H-1), 5.15 (br s, 3H, 3H-1), MeOH 9 : 1), yielding the fluorescent probe 21 (10.1 mg,
5.09–5.03 (m, 9H, 9H-1), 4.87 (br s, 3H, 3H-1), 4.67 (m, 3H, quant.) as an orange amorphous solid. 1H-NMR (400 MHz,
OCH2CHHN), 4.61–4.56 (br s, 6H, H-1, OCHHCtriazole), D2O) δ: 8.05–7.98 (m, 2H), 7.94 (m, 1H), 7.57 (br s, 1H),
4.18–3.56 (m, 198H). 13C-NMR (100 MHz, D2O) δ: 144.1, 7.15–7.07 (m, 2H), 6.69–6.63 (m, 4H), 5.37 (br s, 1H, H-1),
125.5, 102.3, 102.2, 100.8, 100.7, 100.6, 99.9, 99.5, 98.0, 5.31–5.26 (m, 2H, 2H-1), 5.10 (br s, 1H, H-1), 5.06–5.00 (m, 4H,
80.9, 78.9–78.5, 74.0, 73.3–73.2, 72.7, 71.1, 70.8, 70.3–69.9, 4H-1), 4.65 (m, 2H, CH2Ctriazole), 4.59 (m, 2H, CH2CH2N), 4.49
69.7–69.5, 68.3–67.7, 66.9–66.8, 65.6–65.2, 63.5, 62.5, (br s, 1H, H-1), 4.21 (m, 1H), 4.12–4.05 (m, 6H), 4.03–3.92 (m,
61.1–61.0, 50.3, 44.7, 43.2. ESI-MS m/z calcd for 6H), 3.90–3.60 (m, 42H), 3.41 (m, 1H). 13C-NMR (100 MHz,
C
191H320N9O145Cl: 5080.7; found: 2561.1 [M + 2Na]2+, 1715.2 selected data obtained from HSQC, D2O) δ: 131.0, 128.2, 128.2,
[M + 3Na]3+, and 1293.5 [M + 4Na]4+.
127.9, 121.7, 103.0, 102.3, 102.1, 100.7, 100.5, 99.9, 99.5, 97.9,
D3-βMan9-N3 (18). To a solution of glycocluster 16 (20.0 mg, 81.0, 78.9, 78.6, 78.5, 78.4, 74.0, 73.2–73.1, 72.6, 71.1,
3.94 μmol) in DMF (1 mL) sodium azide (2.6 mg, 39.36 μmol) 70.3–69.9, 69.4, 68.4, 66.9–66.8, 65.6–65.4, 61.1–60.9, 50.3.
was added. The mixture was stirred at 70 °C for 2 days. After ESI-MS: m/z calcd for C80H110N4O52: 1958.6, found: 1982.3
that time, the solvent was evaporated under vacuum and the [M − H]−, and 1002.1 [M − 2H]2−
.
crude was purified using Amicon Ultra-15 centrifugal filters
D3-αMan9-fluorescein (22). To a solution of the FAM-alkyne
(MWCO 3 kDa), yielding the glycocluster 18 (20 mg, 3.94 μmol, 6-isomer 19 (1.3 mg, 3.14 μmol) and the trivalent glycocluster
1
quant.) as a white amorphous solid. H-NMR (400 MHz, D2O) of αMan9 17 (8 mg, 1.57 μmol) in DMSO (400 μL), another
δ: 8.05 (s, 3H, 3Htriazole), 5.41 (br s, 1H, 3H-1), 5.34–5.29 (m, solution of CuBr (1.57 μmol) and TBTA (3.14 μmol) in the
6H, 6H-1), 5.15 (br s, 3H, 3H-1), 5.07–5.03 (m, 9H, 9H-1), same solvent (200 μL) was added. The reaction mixture was
under D2O (3H, 3H-1), 4.68 (m, 3H, OCHHCtriazole), 4.60–4.55 stirred on at rt. Thereafter,
a metal scavenger resin,
(m, 6H, H-1, OCH2CHHN), 4.12–3.46 (m, 198H). 13C-NMR QuadrasilMP, was added to the reaction solution and stirred
(100 MHz, D2O) δ: 144.1, 125.5, 102.3, 102.2, 100.8, 100.7, for 20 min at rt. After that, the mixture was filtered and the
100.6, 99.9, 99.5, 98.0, 80.9, 78.9–78.4, 74.0, 73.3–73.2, 72.7, resulting solution was purified by size-exclusion chromato-
72.0, 71.1, 70.3–69.9, 69.7–69.5, 69.2, 68.3, 67.7, 66.9–66.8, graphy (Sephadex LH-20, H2O/MeOH 9 : 1), yielding the fluo-
65.6–65.2, 63.5, 62.4, 61.1–61.0, 50.2, 50.1, 44.7. ESI-MS m/z rescent probe 22 (8.7 mg, quant.) as an orange amorphous
calcd for C191H320N12O145: 5087.2; found: 2566.5 [M + 2Na]2+, solid. 1H-NMR (400 MHz, D2O) δ: 8.06–7.78 (m, 6H), 7.57 (br s,
1717.9 [M + 3Na]3+, and 1293.9 [M + 4Na]4+.
1H), 7.07–6.88 (m, 2H), 6.62–6.42 (m, 4H), 5.35–5.16 (m, 9H,
αMan9-fluorescein (20). To a solution of the FAM-alkyne 9H-1), 5.06 (br s, 3H, 3H-1), 4.96 (br s, 9H, 9H-1), under D2O
6-isomer 19 (2.67 mg, 6.48 μmol) and the epitope αMan9 12 (6H, 6H-1), 4.55–4.29 (m, 6H), 4.07–3.11 (m, 213H). 13C-NMR
(5 mg, 3.24 μmol) in DMSO (300 μL), another solution of CuBr (100 MHz, selected data obtained from HSQC, D2O) δ: 126.8,
(3.24 μmol) and TBTA (6.48 μmol) in the same solvent (150 μL) 122.9, 102.9, 102.3, 100.7, 99.8, 99.5, 98.0, 78.8–78.4,
was added. The reaction mixture was stirred on at rt. 73.3–73.2, 72.0, 71.1–71.0, 70.3–69.9, 69.7–69.5, 66.9–66.8,
Thereafter, a metal scavenger resin, QuadrasilMP, was added 65.9, 65.6, 65.4, 65.2, 63.4, 62.5, 61.1–61.0, 50.1. ESI-MS: m/z
to the reaction solution and stirred for 20 min at rt. After that, calcd for C215H335N13O151: 5514.9, found: 2749.4 [M − 2H]2−
the mixture was filtered and the resulting solution was purified and 1833.1 [M − 3H]3−
by size-exclusion chromatography (Sephadex LH-20, H2O/ D3-βMan9-fluorescein (23). To a solution of the FAM-alkyne
,
.
MeOH 9 : 1), yielding the fluorescent probe 20 (6.3 mg, quant.) 6-isomer 19 (2.3 mg, 5.5 μmol) and the trivalent glycoclusters
as an orange amorphous solid. 1H-NMR (400 MHz, D2O) δ: of βMan9 18 (14 mg, 2.75 μmol) in DMSO (180 μL), another
8.22–7.87 (m, 3H), 7.68 (br s, 1H), 7.30–7.07 (m, 2H), 6.87–6.54 solution of CuBr (2.75 μmol) and TBTA (5.5 μmol) in the same
(m, 4H), 5.40–5.24 (m, 3H, 3H-1), 5.11 (br s, 1H, 1H-1), 5.04 (br solvent (90 μL) was added. The reaction mixture was stirred on
s, 3H, 3H-1), under D2O (2H, 2H-1), 4.64 (m, 2H), 4.12–3.57 at rt. Thereafter, a metal scavenger resin, QuadrasilMP, was
(m, 58H). 13C-NMR (100 MHz, selected data obtained from added to the reaction solution and stirred for 20 min at rt.
This journal is © The Royal Society of Chemistry 2020
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
After that, the mixture was filtered and the resulting solution
was purified by size-exclusion chromatography (Sephadex
Acknowledgements
LH-20, H2O/MeOH 9 : 1), yielding the fluorescent probe 23 This work was financially supported by MINECO (CTQ2017-
(15.0 mg, quant.) as an orange amorphous solid. 1H-NMR 86265-P, PGC2018-099497-B-100 and IJCI-2015-23272), and
(400 MHz, D2O) δ: 8.02–7.80 (m, 6H), 7.54 (br s, 1H), 7.01–6.87 ISCIII RETICS ARADyAL (RD16/0006/0011). Grants were co-
(m, 2H), 6.58–6.46 (m, 4H), 5.40–5.30 (m, 6H, 6H-1), 5.21 (m, funded by the European Regional Development Fund
6H, 6H-1), 5.06 (br s, 3H, 3H-1), 4.96 (m, 9H, 9H-1), 4.53–4.41 (ERDF). A. S.-H. and N. d. l. C thank MINECO for their Juan de
(m, 12H, 3H-1), 4.29 (br s, 6H), 4.07–3.05 (m, 204H). 13C-NMR la Cierva-Incorporacion and FPI contracts, respectively. This
(100 MHz, selected data obtained from HSQC, D2O) δ: 131.0, work used the Multistep Protein Purification Platform for
129.6, 128.5, 124.0, 122.4, 103.8, 102.3, 102.2, 100.8, 100.7, human CLRs production of the Grenoble Instruct-ERIC Center
100.6, 99.9, 99.5, 98.0, 80.9, 78.9–78.4, 74.0, 73.3–73.2, 72.7, (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble
72.0, 71.1, 70.3–69.9, 69.7–69.5, 69.2, 68.3, 67.7, 66.9–66.8, Partnership for Structural Biology (PSB), supported by FRISBI
65.6–65.2, 63.5, 62.4, 61.1–61.0, 50.1 (OCH2CH2N). ESI-MS: m/z (ANR-10-INBS-05-02) and GRAL, financed within the University
calcd for C215H335N13O151: 5514.9, found: 2748.5 [M − 2H]2−
and 1830.8 [M − 3H]3−
,
Grenoble Alpes graduate school (Ecoles Universitaires de
Recherche) CBH-EUR-GS (ANR-17-EURE-0003). F. F. also
acknowledge the French Agence Nationale de la Recherche
(ANR) PIA for Glyco@Alps (ANR-15-IDEX-02). We acknowledge
Dr Michael P. O’Hagan for the revision of the English version
of the manuscript.
.
DC-SIGN ECD expression
DC-SIGN ECD expression and production was performed as
previously described. Briefly, the DC-SIGN ECD protein is
expressed as inclusion bodies in E. coli, refolded and purified
using a mannose affinity chromatography step followed by a
size exclusion.28,29 Both steps ensure respectively the selection
of correctly folded and oligomerized protein.
References
1 A. Varki, Glycobiology, 2017, 27, 3–49.
DC-SIGN binding assays by fluorescence polarization
2 H. Kaltner, J. Abad-Rodríguez, A. P. Corfield, J. Kopitz and
H.-J. Gabius, Biochem. J., 2019, 476, 2623–2655.
3 J. J. Lundquist and E. J. Toone, Chem. Rev., 2002, 102, 555–
578.
4 N. Jayaraman, Chem. Soc. Rev., 2009, 38, 3463–3483.
5 V. Wittmann, Curr. Opin. Chem. Biol., 2013, 7, 982–989.
6 B. Lepenies, J. Lee and S. Sonkaria, Adv. Drug Delivery Rev.,
2013, 65, 1271–1281.
7 Y. van Kooyk and T. B. H. Geijtenbeek, Nat. Rev. Immunol.,
2003, 3, 697–709.
8 L. Mathys and J. Balzarini, PLoS One, 2015, 10, e0130621,
DOI: 10.1371/journal.pone.013062.
9 V. Y. Dudkin, M. Orlova, X. Geng, M. Mandal, W. C. Olson
and S. J. Danishefsky, J. Am. Chem. Soc., 2004, 126, 9560–
9562.
10 L. X. Wang, J. Ni, S. Singh and H. Li, Chem. Biol., 2004, 11,
127–134.
The fluorescence polarization measurements were performed
in 384-well microplates (black polystyrene, non-treated,
Corning), using a TRIAD multimode microplate reader (from
Dynex) with excitation and emission wavelengths of 485 and
535 nm, respectively. Fluorescent compounds 20–23 and
DC-SIGN-ECD were dissolved in Tris buffer (25 mM, pH 8,
150 mM NaCl, 4 mM CaCl2). 15 μL of a 20 nM fluorescent
ligand solution were transferred to each well. Then, 15 μL of
protein solutions, with concentrations ranging from 75 nM to
28 μM, were added to the microplate wells. Therefore, the total
sample volume in each well was 30 μL. The microplate was
shaken in the dark for 5 min, before reading. Blank wells con-
tained 15 μL of the DC-SIGN-ECD solution and 15 μL of Tris
buffer, and their measurements were subtracted from all
values. All experiments on samples were performed in repli-
cates of three.
Wells containing 15 μL of the 20 nM fluorescence com-
pound solution and 15 μL of Tris buffer afforded the back-
ground polarization of the fluorescent molecule, in the
absence of protein. This value was subtracted from the polariz-
ation values of all the samples, giving the increment in the
fluorescence polarization (ΔP). The average ΔP values of three
replicate wells were plotted against the concentration of
DC-SIGN-ECD, and the resulting curve was fitted to the
equation for a one-site binding model: y = ΔPmaxx/[KD + x]
where ΔPmax is the maximal value of ΔP and KD is the dis-
sociation constant of the interaction.
11 S.-K. Wang, P.-H. Liang, R. D. Astronomo, T.-L. Hsu,
S.-L. Hsieh, D. R. Burton and C.-H. Wong, Proc. Natl. Acad.
Sci. U. S. A., 2008, 105, 3690–3695.
12 F. Chiodo, P. M. Enríquez-Navas, J. Angulo, M. Marradi and
S. Penadés, Carbohydr. Res., 2015, 405, 102–109.
13 H.-C. Wen, C.-H. Lin, J.-S. Huang, C.-L. Tsai, T.-F. Chen and
S.-K. Wang, Chem. Commun., 2019, 55, 9124–9127.
14 H. Feinberg, R. Castelli, K. Drickamer, P. H. Seeberger and
W. I. Weis, J. Biol. Chem., 2007, 282, 4202–4209.
15 J. Ramos-Soriano, J. J. Reina, B. M. Illescas, N. de la Cruz,
L. Rodríguez-Pérez, F. Lasala, J. Rojo, R. Delgado and
N. Martín, J. Am. Chem. Soc., 2019, 141, 15403–15412.
16 J. Ramos-Soriano, M. C. de la Fuente, N. de la Cruz,
R. C. Figueiredo, J. Rojo and J. J. Reina, Org. Biomol. Chem.,
2017, 15, 8877–8882.
Conflicts of interest
There are no conflicts to declare.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Organic & Biomolecular Chemistry
Paper
17 J. J. Reina and J. Rojo, Tetrahedron Lett., 2006, 47, 2475– 24 P. Sörme, B. Kahl-Knutsson, M. Huflejt, U. J. Nilsson and
2578. H. Leffler, Anal. Biochem., 2004, 334, 36–47.
18 R. Ribeiro-Viana, M. Sánchez-Navarro, J. Luczkowiak, 25 S. Maza, M. Mar Kayser, G. Macchione, J. Lopez-Prados,
J. R. Koeppe, R. Delgado, J. Rojo and B. G. Davis, Nat.
Commun., 2012, 3, 1303.
J. Angulo, J. L. de Paz and P. M. Nieto, Org. Biomol. Chem.,
2013, 11, 3510–3525.
19 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., 26 C. Solera, G. Macchione, S. Maza, M. M. Kayser, F. Corzana,
Int. Ed., 2001, 40, 2004–2021. J. L. de Paz and P. M. Nieto, Chem. – Eur. J., 2016, 22, 2356–2369.
20 C. W. Tornøe, C. Christensen and M. Meldal, J. Org. Chem., 27 I. Joachim, S. Rikker, D. Hauck, D. Ponader, S. Boden,
2002, 67, 3057–3062.
21 V. V. Rostovtsev, L. G. Green, V. V. Fokin and
R. Sommer, L. Hartmann and A. Titz, Org. Biomol. Chem.,
2016, 14, 7933–7948.
K. B. Sharpless, Angew. Chem., Int. Ed., 2002, 41, 2596– 28 J. J. Reina, I. Díaz, P. M. Nieto, N. E. Campillo, J. A. Páez,
2599.
G. Tabarani, F. Fieschi and J. Rojo, Org. Biomol. Chem.,
2008, 6, 2743–2754.
29 G. Tabarani, M. Thépaut, D. Stroebel, C. Ebel, C. Vivès,
P. Vachette, D. Durand and F. Fieschi, J. Biol. Chem., 2009,
284, 21229–21240.
22 W. A. Lea and A. Simeonov, Expert Opin. Drug Discovery,
2011, 6, 17–32.
23 K. Kakehi, Y. Oda and M. Kinoshita, Anal. Biochem., 2001,
297, 111–116.
This journal is © The Royal Society of Chemistry 2020
Org. Biomol. Chem.