Chemo-Enzymatic Synthesis of S. mansoni O-Glycans and Their Evaluation as Ligands for C-Type Lectin Receptors MGL, DC-SIGN, and DC-SIGNR

Article abstract of DOI:10.1002/chem.202000291

Due to their interactions with C-type lectin receptors (CLRs), glycans from the helminth Schistosoma mansoni represent promising leads for treatment of autoimmune diseases, allergies or cancer. We chemo-enzymatically synthesized nine O-glycans based on th

Full text of DOI:10.1002/chem.202000291

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doi.org/10.1002/chem.202000291
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&
Carbohydrates
Chemo-Enzymatic Synthesis of S. mansoni O-Glycans and Their
Evaluation as Ligands for C-Type Lectin Receptors MGL, DC-SIGN,
and DC-SIGNR
Julie Pham,[a] Alvaro Hernandez,[a, b] Anna Cioce,[a] Silvia Achilli,[c, d] Giulio Goti,[e]
Corinne Vivꢀs,[c] Michel Thepaut,[c] Anna Bernardi,[e] Franck Fieschi,[c] and Niels-
Christian Reichardt*[a, f, g]
Abstract: Due to their interactions with C-type lectin recep-
tors (CLRs), glycans from the helminth Schistosoma mansoni
represent promising leads for treatment of autoimmune dis-
eases, allergies or cancer. We chemo-enzymatically synthe-
sized nine O-glycans based on the two predominant O-
glycan cores observed in the infectious stages of schistoso-
miasis, the mucin core 2 and the S. mansoni core. The O-gly-
cans were fucosylated next to a selection of N-glycans di-
rectly on a microarray slide using a recombinant fucosyl-
transferase and GDP-fucose or GDP-6-azidofucose as donor.
Binding assays with fluorescently labelled human CLRs DC-
SIGN, DC-SIGNR and MGL revealed the novel O-glycan O8 as
the best ligand for MGL from our panel. Significant binding
to DC-SIGN was also found for azido-fucosylated glycans.
Contrasting binding specificities were observed between the
monovalent carbohydrate recognition domain (CRD) and the
tetravalent extracellular domain (ECD) of DC-SIGNR.
Introduction
nents in the immune system acting as pattern recognition re-
ceptors (PRRs).[1,2] Their interaction with pathogen associated
molecular patterns (PAMPs) at the pathogen surface can lead
to a series of signalling events ultimately resulting in a skewed
immune response.[3,4] The glycans on the surface of S. mansoni
are thought to be responsible for the characteristic TH2/Treg
response observed during chronic schistosomiasis.[5] Concomi-
tantly, reduced disease related inflammation was observed in
autoimmune pathologies such as multiple sclerosis, rheuma-
toid arthritis, type I diabetes and inflammatory bowel diseases
for patients infected with helminths or treated with parasite
extracts.[6]
S. mansoni glycans play an important role in host infection by
helminths and mounting evidence suggests that they contrib-
ute to the parasite evasion of the host immune system by hi-
jacking C-type lectin receptors (CLRs). CLRs are crucial compo-
[a] Dr. J. Pham, Dr. A. Hernandez, Dr. A. Cioce, Dr. N.-C. Reichardt
CIC biomaGUNE, Glycotechnology Group
Paseo Miramꢀn 182, 20014 San Sebastian (Spain)
E-mail: nreichardt@cicbiomagune.es
[b] Dr. A. Hernandez
Asparia Glycomics S.L.
Mikeletegi 83, 20009 San Sebastian (Spain)
Although yet to be fully assigned, the S. mansoni glycome
displays a rich array of immunogenic glycan epitopes such as
LeX and LDNF which are known to interact with host CLRs.
Moreover, interesting similarities can be observed between hel-
minth and host glycans. Indeed, the S. mansoni specific O-
glycan core, which is predominantly found in the cercariae,
closely resembles an O-glycan core found in the intermediate
snail host Biomphalaria glabrata.[7,8] On the other hand, the
mucin core 2, which is abundantly found in mammalian host
mucosal tissues, is predominantly found in the helminth
eggs.[9] Thus, S. mansoni is strongly suspected to employ
glycan mimicry as a strategy to subvert the host immune
system to allow for parasitic survival.[10]
[c] Dr. S. Achilli, Dr. C. Vivꢁs, Dr. M. Thepaut, Prof. Dr. F. Fieschi
CNRS, CEA, Institut de Biologie Structurale
Universitꢂ Grenoble Alpes
38100 Grenoble (France)
[d] Dr. S. Achilli
Present address: DCM, UMR 5250
Universitꢂ Grenoble Alpes, CNRS
38000 Grenoble (France)
[e] Dr. G. Goti, Prof. Dr. A. Bernardi
Dipartimento di Chimica, Universitꢃ degli Studi di Milano
via Golgi 19, 20133 Milano (Italy)
[f] Dr. N.-C. Reichardt
CIBER-BBN
Paseo Miramꢀn 182, 20014 San Sebastian (Spain)
[g] Dr. N.-C. Reichardt
The immunomodulatory properties of S. mansoni O-glycans
place them as potentially interesting lead compounds for the
development of carbohydrate-based drugs to treat immuno-
compromised patients with autoimmune diseases, allergies or
cancer. However, most studies have focused on parasite N-gly-
Basque Research and Technology Alliance (BRTA)
Paseo Miramꢀn 182, 20014 San Sebastian (Spain)
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/chem.202000291.
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cans or the antigenic motifs alone and only few describe the
relevance of parasite O-glycans and their interactions with
CLRs.[11,12] This is mainly due to the challenge of isolating pure
compounds in sufficient amounts for functional and diagnostic
tests.[13–15]
all acetates without affecting the base-sensitive N-Troc
group.[18] Subsequent acetal protection of O-4 and O-6 with
benzaldehyde dimethyl acetal under acid catalysis provided 4
in 70%. Glycosylation of the remaining free O-3 with the galac-
tose imidate 7 under TMSOTf catalysis then afforded disacchar-
ide 5 in 62% yield, as a common intermediate in the synthesis
of both core structures. Reductive ring opening of the benzyli-
dene acetal in 5 with BH3·THF and catalytic TMSOTf furnished
the 4-benzyl ether 6 in a moderate 61% yield. Finally, glycosy-
lation at O-6 either with galactose donor 7 or GlcNAc donor 8
afforded trisaccharides 9 and 10 in 40% and 73% yield, respec-
tively.
The aim of our study was to investigate the interaction of S.
mansoni O-glycans with key CLRs and identify lead structures
for improved targeting of immune cells via specific CLR-glycan
interactions. To this end, we report, here for the first time, the
chemoenzymatic synthesis of several S. mansoni O-glycans and
evaluate their interactions with fluorescently labeled human
CLRs DC-SIGN, DC-SIGNR and MGL.
For the enzymatic diversification of the core structures with
recombinant glycosyltransferases both O-glycan cores were
partially deprotected. The benzyl protection was left un-
touched at this stage of the synthesis to aid in the separation
of products by HPLC after enzymatic modifications. Deprotec-
tion of the N-Troc functionality under standard conditions with
LiOH or zinc/acetic acid amalgam failed to provide clean prod-
ucts but refluxing the compounds 9 and 10 in 1m TBAF in THF
afforded the clean S. mansoni core 11 in 95% and the mucin
core 2 trisaccharide 12 in 78% yield after three steps.[19–22]
Both core structures were then diversified by enzymatic gly-
cosylations to access the Gal1-4GlcNAc (LN) and GalNAc1-
4GlcNAc (LDN) motifs typically observed in the cercarial and
egg glycomes of S. mansoni. Towards this end, we employed
three recombinant glycosyltransferases, a bovine beta-1,4-gal-
actosyltransferase (b4Gal-T1), a double mutant (Y289LC342T)
conferring beta-1,4-N-galactosaminyltransferase (DMGalT1) ac-
tivity and a Neisseira meningitidis beta-1,3-N-acetylglucosami-
Results and Discussion
The retrosynthetic analysis of the O-glycan cores O1 and O2
(Scheme 1) suggested a convergent strategy involving three
building blocks. After conjugation of the GalNAc donor 1 with
a protected amine linker 2, glycosylation in O-3 with galactose
donor 7 would provide the Galb1-3GalNAc disaccharide 5
common to both cores. A second glycosylation in O-6 with gal-
actose (Gal) 7 or N-acetylglucosamine (GlcNAc) donor 8 would
furnish the S. mansoni core O1 or mucin 2 core O2, respective-
ly.
To facilitate the immobilization of final compounds as li-
gands on glycan arrays or to carrier proteins, the known galac-
tosamine trichloroacetimidate donor 1[16] (Scheme 2) was con-
jugated with N-benzyl-benzylcarbamate protected aminopentyl
linker 2[17] to obtain glycoconjugate 3 in 73%. Treatment of 3
with a guanidine/guanidium solution quantitatively hydrolyzed
Scheme 1. Retrosynthetic analysis for the S. mansoni core and core 2 trisaccharides O1 and O2.
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Scheme 2. Convergent synthesis of the partially protected S. mansoni and mucin 2 cores 11 and 12. Reagents and conditions: a) Linker 2, TMSOTf, DCM,
ꢀ408C, 73%; b) i) G/GHNO3, MeOH/DCM; ii) PhCH(OMe)2, cat. CSA, DCM, 70%; c) Donor, TMSOTf, DCM, ꢀ408C, 62%; d) 1m BH3.THF, TMSOTf, DCM, 61%;
e) Donor 7, TMSOTf, DCM, ꢀ418C, 40%; f) Donor 8, TMSOTf, DCM, ꢀ408C, 73%; g) i) 1m TBAF, THF, reflux; ii) Ac2O, pyridine; iii) 0.5m NaOMe, MeOH, 70–90%.
nyltransferase (LgtA X) construct prepared exclusively for this
purpose (Scheme 3).[23–25] The new LgtA X construct contains
two polyhistidine tags and a thioredoxin domain to increase
LgtA purity and solubility, respectively.
Before enzymatic introduction of an acid-labile fucose resi-
due we decided to remove remaining benzyl and Cbz protect-
ing groups by hydrogenation. Protected glycans 11–17 were
dissolved in a mixture of H2O/MeOH/AcOH, and hydrogenated
under Pd/C(10%) catalysis under atmospheric pressure of
H2(g), which afforded the deprotected final glycans O1–O7 in
40–100% yield (Figure 1).[27]
The general substrate and donor specificity of LgtA, includ-
ing a comparison with the human homologue B3GnT2, has
previously been described by Blixt et al. Employing N-glycan
G5 (Figure 1) we confirmed the enzymatic activity of LgtA X to
be similar to LgtA in solution, that is, for the elongation of ter-
minal galactose with GlcNAc. To evaluate the usefulness of the
enzyme for diversification of the S. mansoni core type trisac-
charide we assessed the enzyme selectivity and activity on tri-
saccharide 11. Incubation of the O-glycan core with 2 potential
acceptor sites afforded 57% of a b1-6 monosubstituted prod-
uct 13, next to only 7% of the bis-substituted product.[26]
For the synthesis of the proposed Galb1-4[Fuca1-3]GlcNAc
(LeX)-type and GalNAcb1-4[Fuca1-3]GlcNAc (LDNF) structures
we employed a recombinant a1,3-fucosyltransferase from Cae-
norhabditis elegans (CeFUT6) and a commercial Heliobacter
pylori a-1,3 fucosyltransferase (HP-FucT). CeFUT6 has a known
Lewis-X fucosylation activity which was employed in this study
to yield O8 from O7 in 73% yield.[28] HP-FucT has a broad sub-
strate and donor specificity including the C-6 azido-fucose sur-
rogate (FucZ).[29,30] Introducing an azide function as a biorthog-
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Scheme 3. Enzymatic diversification of cores structures 11 and 12 yielding glycans 13 (57%), 14 (28%), 15 (49%), 16 (61%) and 17(59%).
Figure 1. a) Selection of ligands immobilized on the microarray; b) surface functionalization of the ITO slide.
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Figure 2. Optimization of reaction conditions for on-chip fucosylation; a) MALDI-TOF MS of fucosylation of O7; b) Effect of enzyme concentration; c) Fucose
donor concentration; D) Azido fucose donor concentration on product conversion.
onal handle in the molecule opened the possibility of generat-
ing glycomimetics by copper-catalyzed cycloaddition with al-
kynes to afford a library of novel glycomimetics with potential-
ly improved affinity towards CLRs.[31–34]
version than a single exposure with 2 mm donor concentration
(Figure 2C). While for glycosylation with the natural GDP-
fucose donor, two incubation cycles were sufficient to reach a
maximum conversion between 65–90%, similar conversion
yields employing the GDP- 6-azido-fucose required 3 cycles.
Product conversion yields showed a certain degree of struc-
ture-dependent variability but were altogether above 80% for
the fucose addition and above 75% for the azido-fucose addi-
tion (see Supporting Information, Figure S5).
We decided to employ a microarray platform for the rapid
preparation of fucosylated and azido-fucosylated compounds.
Performing the reactions in parallel and on a microscale de-
creases the need of expensive azido-donor by several orders of
magnitude compared to the solution phase synthesis of indi-
vidual compounds. The synthetic parasite O-glycans O1-O8
were printed alongside a set of N-glycans which were chosen
as additional fucose acceptors from a glycan library available
in our laboratory onto NHS activated indium tin oxide (ITO)
slides (Figure 1).[35,36] The bio-functionality of the printed gly-
cans was confirmed by incubating the array with the plant
lectin peanut agglutinin (PNA) ECD recognizing Galb1-3GalNAc
residues (see Supporting Information, Figure S4).[37,38]
As a general note to the reader unfamiliar with glycan array
binding experiments, the binding data between different
arrays were only compared in relative terms that is, by compar-
ing ligand binding profiles. Apart from the underlying binding
affinity between carbohydrate ligand and lectin, the absolute
fluorescence intensities given as RFU values depend on the
degree of protein labelling, the incubation time, the washing
conditions and image acquisition parameters among others.
Some of these parameters are more easily controlled than
others and efforts are under way in many labs to improve re-
producibility of glycan arrays to allow for a comparison of data
between arrays and different immobilization platforms. Unless
an internal reference is used to normalize fluorescence values
between different slides or arrays, or extreme measures are
put in to place to reproduce incubation and analysis condi-
tions for all slides, absolute fluorescence values between differ-
ent arrays should not be compared.
Initially, we screened donor and HP-FucT enzyme concentra-
tions to optimize on-chip fucosylation for printed glycans.
Yields for the enzymatic on-chip glycosylation were deter-
mined by MALDI-TOF MS as ratios of the sum of product
signal intensities (glycan#-F for fucosylation and glycan#-Z for
azido-fucosylation) to remaining starting material signal inten-
sities (Figure 2A). Plotting the product ratios for the different
glycans showed no increase in product formation beyond an
enzyme concentration of 189 mUmLꢀ1 (Figure 2B) possibly
due to enzyme precipitation at higher concentration.
The effect of the spatial organization of carbohydrate bind-
ing domains on glycan interaction was first assessed by com-
paring the fluorescent binding profiles of the monovalent CRD
Repeated glycosylation with freshly prepared enzyme solu-
tion and 1 mm of GDP-fucose produced higher product con-
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Figure 3. Relative fluorescence intensities and fluorescence image for the unmodified array after incubation with DC-SIGNR CRD (10 mgmLꢀ1, orange bars)
and DC-SIGNR ECD (50 mgmLꢀ1, blue bars). Each bar in the histogram represents the average of fluorescence from 3 spots which were normalized to G10
and the propagation of error is shown as an error bar.
and the tetrameric ECD of DC-SIGNR on the unmodified array
(Figure 3). DC-SIGNR is an endothelial tetrameric C-type lectin
receptor that binds to high mannose and complex glycans
found on surface glycoproteins of Ebola, HIV or hepatis
virus.[39] It has also been suggested as an entry path for S. man-
soni larvae that begin their migration towards the intestine
and liver.[40] The glycan binding profile of DC-SIGNR has been
previously studied by glycan array and frontal affinity chroma-
tography. Strong binders include high mannose and hybrid
structures and to a lesser extent galactosylated complex gly-
cans with terminal GlcNAc residues.[41–43] Lacking high mannose
and hybrid structures on our array, we found strongest binding
to the ECD domain for glycans with terminal GlcNAc (G4, O2
and O3). The CRD showed preferential binding towards gly-
cans with a S. mansoni core and galactose residues G10, G14
and O4, while mucin core derivatives O2 and O3 which strong-
ly bound the ECD construct did not show interaction with the
isolated carbohydrate recognition domain CRD. In addition,
and contrary to the ECD, fucosylated glycans (O8, G11, G13
and G7) displayed enhanced lectin binding compared to their
non-fucosylated counterparts for the CRD (O7 and G5).
The observed differences in binding profiles towards mono-
valent CRD and tetravalent ECD receptor organization are strik-
ing and have been observed previously for other systems.[44]
Based on our array binding data, we can only speculate on the
underlying molecular basis determining the observed change
in specificity. Rapid rebinding to multiple equivalent binding
sites in close proximity could improve binding to some struc-
tures, while others could face additional binding penalties due
to steric clashes in the tetrameric lectin organization. In addi-
tion, chelation could favor binding to compounds which are
capable of interacting with more than one binding site. This
observation could have practical implications for lectin binding
studies as for simplicity and practical reasons these are often
carried out on the monomeric CRD, which is easier to express
and purify. The natural presentation of DC-SIGNR, however, is
determined by the organization via the stem region into de-
fined tetramers. Any development of multivalent inhibitors
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Figure 4. Binding profile of the array immobilized glycans with DC-SIGN ECD (10 mgmLꢀ1). Histogram bars represent the average fluorescence from 3 spots
and error bars represent the standard deviation.
should therefore take the natural receptor organization into
account. DC-SIGNR, unlike DC-SIGN, binds to neoglycoconju-
gates presenting all Lewis antigens except Lewis X epitopes.[42]
We therefore turned our attention to DC-SIGN. Recently, we re-
ported complementary binding selectivity towards one of the
isomeric N-glycans G3/G8 and G4/G9 by DC-SIGN and DC-
SIGNR.[43]
bind to DC-SIGN with similar strength as LDNF structures.[43]
Among the strongest binders in our previously reported
screen were bi-antennary glycans with terminal GlcNAc resi-
dues and high mannose structures, not included in the present
array.
After on-chip fucosylation with HP-FucT, DC-SIGN bound to
all enzymatically extended structures, though the spot-to-spot
variability increased considerably, possibly due to partial re-
moval of the non-covalently attached glycoconjugates during
prolonged washing procedures (Figure 5).
DC-SIGN is found on dendritic cells and macrophages as a
tetrameric lectin and displays specificity for mannose, fucose
and GlcNAc residues. N-glycans bearing LeX and LDNF epitopes
in the soluble egg antigens have been shown to bind to DC-
SIGN and inhibit DC activation.[45,46] Incubation of the unmodi-
fied initial array with fluorescently labeled DC-SIGN ECD
showed strongest binding to glycans presenting LeX (G10, O8)
or LDNF (G11-G13) glycan determinants but not to the bis-
core fucosylated N-glycan G7 (Figure 4) possibly due to the
impact of core fucosylation on glycan conformation and hence
lectin recognition.[47] Glycans G3, G4 and O3 presenting a free
GlcNAc residue on the 6-arm barely showed interaction with
the lectin. However, in a previous screen on a glycan array
with different surface architecture they have been shown to
Nevertheless, the population of fucosylated O-glycans in our
array bound with intensity similar to the fucosylated N-glycans,
making them equally as valuable in the analyses of CLR-glycan
interaction, as previously stated by Van Diepen et al.[48]
Lewis X binding to DC-SIGN CRD is thought to be favored
by Van der Waals interactions between the fucose C-2 and the
neighboring V351.[49] However, given the limitations in glycan-
lectin interaction predictions, it was difficult to predict whether
the azide modification at C-6 would affect glycan binding.[50,51]
Despite also showing an increased degree of variability, DC-
SIGN bound to all azido-fucosylated structures of our array, in-
Figure 5. Glycan binding of glycan array with tetravalent DC-SIGN ECD (10 mgmLꢀ1) after on-chip fucosylation with HP-FucT. Each bar in the histogram repre-
sents the average of fluorescence from 3 spots and the standard deviation is given as an error bar.
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Figure 6. Glycan binding profile for DC-SIGN ECD (10 mgmLꢀ1) after modification of the glycan array with the C6-azido-fucose. Each bar in the histogram rep-
resents the average fluorescence for 3 replicate spots and the standard deviations are given as an error bar.
dicating that the C-6 modification does not significantly con-
tribute to glycan-CLR interaction (Figure 6). Additionally, it was
interesting to note that glycans G11Z and G13Z containing an
LDNF and azido-LDNF epitope were observed to be the high-
est binders.
dues with MGL. A similar binding strength was seen for the
isolated LDN epitope G16 and glycans presenting the LDN
motif on one or both antennae.[43] Glycan O5 with an asym-
metrically extended S. mansoni core, presenting a single
GalNAc residue, was the strongest binding ligand on the array.
Molecular modeling studies suggest that the presence of a
a1,3 fucose residue would disrupt the chelation of the GalNAc
3-OH with calcium at the binding site but our MGL binding
profile results does not support this generalized rule.[55] Al-
though lower binding was observed for the LDNF glycan G12
than for the isolated LDN epitope G16, both mono-fucosylated
bi-antennary glycans G11 and G13 showed higher fluorescence
intensities than the non-fucosylated compound G6. In addi-
tion, we were surprised to observe binding of the LeX epitope
G10 with a similar binding strength as for the LDNF epitope
G12, neither of which was anticipated to bind MGL as fucosyla-
tion should inhibit binding. These results suggest that fucosy-
lation may indeed, induce alternate binding modes for non-
GalNAc ligands in MGL CRD.
Finally, we looked at macrophage galactose lectin (MGL)
binding to our collection of immobilized O- and N-glycans.
MGL is a PRR expressed at the cell surface of macrophages
and dendritic cells that recognizes predominantly terminal
GalNAc residues found on Tn antigen glycosylated mucins
overexpressed on cancer cells or the GalNAcb1-4GlcNAc (LDN)
disaccharide motif of the parasite S.mansoni.[52] Targeting MGL
with high affinity glycans or glycomimetics has been suggest-
ed as a strategy for macrophage targeting in cancer immune
therapy and drug delivery.[53,54] In line with its reported binding
specificity, we found strong interactions of MGL with all gly-
cans presenting terminal GalNAc residues of the unmodified
array (Figure 7). We observed a low degree of oligosaccharide
context dependent alteration of the interaction of GalNac resi-
Figure 7. Glycan binding profile with MGL ECD (10 mgmLꢀ1). Histogram bars represent the average fluorescence from 3 spots and the standard deviation is
given as an error bar.
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Figure 8. Fluorescence intensities for A) fucosylated array and B) the azido-fucosylated array after incubation with MGL ECD (10 mgmLꢀ1). Histogram bars rep-
resent the average fluorescence from 3 spots and the standard deviation is given as an error bar.
On-chip enzymatic fucosylation led to a generally reduced
binding to MGL with the exception of O5F and G2F which
maintained high affinities (Figure 8A).
study highlights the potential of employing glycan arrays to
identify leads for the further development of glycomimetics
with improved CLR targeting properties. In addition, our com-
parison of the binding profiles of CRD and ECD domains of
DC-SIGNR underscores the importance of multivalent receptor
presentation for inducing binding with an additional level of
glycan selectivity.
Despite the spot-to-spot variability increasing considerably
here too, the comparison of relative binding profiles showed
that the elongation with 6-azido-fucose decrease binding to
most of the immobilized glycans with the exception of G2Z,
G4Z and G6Z which remained relatively unchanged.
Experimental Section
Conclusions
Chemical Synthesis
We have chemo-enzymatically synthesized eight novel O-gly-
cans based on the S. mansoni specific core and the mammalian
mucin core 2. The unexpected regioselectivity for the 6-arm of
the S. mansoni core, observed for the recombinant glycosyl-
transferase LgtA X provided a simple entry into products with
enzymatic elongations on the 6-arm. The eight synthesized O-
glycans were printed alongside a number of N-glycans and
were enzymatically modified in good to excellent yields with
fucose and non-natural 6-azido-fucose. Glycan-array-based pre-
liminary binding studies with the human C-type lectin recep-
tors MGL, DC-SIGNR and DC-SIGN showed interesting novel in-
sights into the fine specificity of CLR-glycan binding. This
5-(Benzyl (benzyloxycarbonyl)amino) pentyl 3,4,6-tri-O-acetyl-2-
deoxy-2- ((2,2,2-trichloroethoxycarbonylamino) b-d-galactopyra-
noside 3: To a solution of 1[16](11.8 g, 18.9 mmol) in dry DCM
(30ml) on activated molecular sieves and under argon was added a
solution
of
N-benzyl-N-(5-hydroxypentyl)carbamate
(7.42 g,
22.7 mmol) 2 in dry DCM (200 mL). The solution was placed at 08C
before TMSOTf (0.690 mL, 3.8 mmol) was added dropwise. The re-
action was allowed to warm to RT and stirred for 2hours after
which TLC showed full consumption of starting material. The reac-
tion mixture was quenched with Et3N and filtered through celite.
The crude sample was purified by flash column chromatography
1
(15!40% EtOAc:Tol) to yield 3 as a white gum, 11 g, 73%. H NMR
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Chemistry—A European Journal
(500 MHz, CDCl3) d=7.41–7.23 (m, 9H, Ar), 7.17 (d, J=8.1 Hz, 1H,
Ar), 5.36 (d, J=3.3 Hz, 1H, H-4), 5.25–5.09 (m, 3H, H-3, CH2Bn), 4.79–
4.59 (m, 2H, CH2Troc), 4.59–4.40 (m, 3H, H-1,CH2Bn), 4.14 (qd, J=
11.2, 6.7 Hz, 2H, 2H-6), 3.94–3.70 (m, 3H, H-5, H-2, CHlinker), 3.50–
3.25 (m, 1H, CHlinker), 3.23–3.12 (m, 2H, CH2), 2.14 (s, 3H, OCH3),
2.05 (s, 3H, OCH3), 1.99 (s, 3H, OCH3), 1.59–1.43 (m, 4H, 2CH2linker),
1.39–1.18 ppm (m, 2H, CH2linker).13C NMR (126 MHz, CDCl3) d=
170.45, 170.37, 156.77, 156.30, 154.38, 137.79, 136.74, 128.57,
128.44, 127.92, 127.81, 127.73, 127.35, 127.20, 101.25, 95.67, 74.30,
70.50, 69.86, 69.73, 67.21, 66.76, 61.48, 52.73, 50.48, 50.37, 47.28,
46.10, 29.04, 28.66, 27.76, 26.99, 22.96, 20.69, 20.65 ppm. HRMS
(MALDI-Tof): m/z calcd for C35H43Cl3N2O12 [M+Na]+ : 811.1778,
found: 811.1728. [a]D20 =ꢀ11.88 (c=1, CHCl3)
(126 MHz, CDCl3) d=170.31, 170.08, 169.34, 156.77, 156.22, 154.05,
137.86, 136.86, 136.74, 128.84, 128.55, 128.47, 128.11, 127.93,
127.80, 127.30, 127.19, 126.26, 101.86, 100.69, 99.74, 95.61, 76.01,
74.23, 70.87, 70.82, 69.69, 69.41, 69.19, 68.87, 67.17, 67.08, 66.45,
61.56, 53.82, 50.35, 47.26, 45.95, 29.68, 29.08, 28.81, 27.77, 27.27,
23.31, 23.06, 20.71, 20.56 ppm. HRMS [M+Na]+ (MALDI-Tof) m/z
calcd for C50H63Cl3N2O18 [M+Na]+: 1103.2724 found: 1103.2716.
[a]D20 = +13.98 (c=1, CHCl3).
5-(Benzyl (benzyloxycarbonyl)amino) pentyl 2,3,4,6-tetra-O-
acetyl-b-d-galactopyranosyl-(1!3)-4-O-benzyl-2-deoxy-2-
((2,2,2-trichloroethoxycarbonylamino) b-d-galactopyranoside 6:
To a solution of 5 (5.6 g, 5.2 mmol) in dry DCM (35ml) under Ar at
08C was added 1m BH3.THF (20.7 mL, 20.7 mmol) dropwise. The
solution was allowed to cool before adding TMSOTf (0.467 mL,
2.59 mmol) dropwise and the reaction was stirred at 08C under Ar
for 1.5 hours after which full conversion was observed by TLC. The
ice bath was removed and the solution was quenched with a solu-
tion of MeOH:Et3N (10:1) until effervescence ceased. The reaction
mixture was concentrated in vacuo and purified by FCC (50!
5-(Benzyl (benzyloxycarbonyl)amino) pentyl 4,6-O-benzylidene-
2-deoxy-2- ((2,2,2-trichloroethoxycarbonylamino) b-d-galacto-
pyranoside 4: Compound 3 (10.2 g, 12.9 mmol) was dissolved in
[18]
G/GHNO3 (700 mL) and the solution was stirred at RT for 15 mins
until full consumption of starting material was observed by TLC.
The reaction mixture was concentrated in vacuo then washed thor-
oughly with DCM and filtered through celite. The filtrate was con-
centrated to yield a pale pink residue. This was dissolved in dry
acetonitrile (100 mL) under argon and benzaldehyde dimethyl
acetal (3.4 mL, 2.25 mmol) and camphor sulfonic acid (600 mg,
2.6 mmol) were added. The reaction was stirred at RT overnight
after which successful conversion was observed by TLC. Et3N was
added and the reaction mixture was concentrated in vacuo. The re-
sulting residue was purified by flash column chromatography (3!
1
100% EtOAc:Hex) to yield 6 as a white solid, 3.4 g, 61%. H NMR
(500 MHz, CDCl3) d=7.43 (d, J=6.8 Hz, 2H, Ar), 7.39–7.13 (m, 13H,
Ar), 5.43–5.38 (S, 1H, H-4’), 5.27 (dd, J=10.5, 8.0 Hz, 1H, H-2’), 5.17
(d, J=10.9 Hz, 2H, OCH2Bn), 4.99 (dd, J=10.5, 3.4 Hz, 1H, H-3’), 4.92
(d, J=11.8 Hz, 1H, 4-OCH2Bn), 4.84–4.64 (m, 5H, CH2Troc, 4-OCH2Bn
,
H-1, H-1’), 4.47 (d, J=8.8 Hz, 3H, H-3, NCH2Bn ), 4.17 (ddq, J=15.9,
11.1, 6.1, 4.7 Hz, 2H, H-6’), 3.98–3.65 (m, 4H, H5’, H-4, CHlinker, H-6a),
3.53–3.14 (m, 6H, H-6b, H-2, H-5, CHlinker, CH2linker), 2.14 (s, 3H, CH3),
2.10 (s, 3H, CH3), 2.02 (s, 3H, CH3), 1.99 (s, 3H, CH3), 1.53 (d, J=
18.6 Hz, 4H, 2CH2linker), 1.31 ppm (s, 2H, CH2linker). 13C NMR (126 MHz,
CDCl3) d=170.41, 170.15, 170.07, 169.51, 138.31, 137.82, 129.09,
128.55, 128.46, 128.35, 128.03, 127.94, 127.79, 127.30, 127.19,
102.45, 99.95, 95.60, 78.73, 74.53, 74.42, 74.22, 73.97, 73.85, 70.74,
70.68, 69.88, 69.53, 69.04, 67.20, 67.09, 61.79, 61.27, 54.89, 50.37,
47.24, 45.97, 29.12, 28.75, 27.78, 27.19, 23.28, 23.07, 20.72, 20.63,
20.56 ppm. HRMS (MALDI-Tof) m/z calcd for C50H61Cl3N2O18
[M+Na]+: 1105.2881, found: 1105.2839. [a]D20 =ꢀ18.48 (c=1,
CHCl3).
1
10% MeOH:DCM) to yield 4 as a white solid, 6.78 g, 70%. H NMR
(500 MHz, CDCl3) d=7.51 (d, J=5.2 Hz, 2H, Ar), 7.42–7.22 (m, 11H,
Ar), 7.17 (d, J=7.0 Hz, 2H, Ar), 5.57 (s, 1H, CHPh), 5.23–5.13 (m, 2H,
CH2Ph), 4.72 (d, J=11.7 Hz, 2H, CH2Troc), 4.50 (m, 3H, H-1, CH2Bn),
4.32 (d, J=12.4 Hz, 1H, H-6a), 4.20 (d, J=3.6 Hz, 1H, H-4), 4.08 (dd,
J=12.4, 1.9 Hz, 1H, H-6b), 4.03–3.82 (m, 2H, H-3, CH2), 3.65(m, 1H,
H-2), 3.5 (s, 1H, H-5), 3.43–3.25(m, 1H, CHlinker), 3.25–3.15(m, 2H,
CH2), 1.62–1.41 (m, 2H, 2CH2linker), 1.39–1.16 ppm (m, 2H,
CH2linker).13C NMR (126 MHz, CDCl3) d 152.34, 151.88, 150.76, 150.49,
134.39, 134.36, 134.34, 134.15, 133.47, 133.32, 126.34, 125.72,
125.69, 125.60, 125.43, 125.16, 125.10, 124.98, 124.88, 124.57,
124.54, 124.39, 123.71, 99.78, 99.53, 99.33, 94.60, 75.14, 75.07,
74.49, 70.66, 70.37, 69.68, 69.62, 69.52, 67.75, 67.72, 66.93, 56.43,
51.84, 51.71, 48.87, 47.78, 31.72, 31.33, 30.52, 29.75, 26.02,
25.90 ppm. HRMS (MALDI-Tof) m/z calcd for C36H41Cl3N2O9 [M+Na]+
: 773.1774, found: 773. 1772. [a]D20 =ꢀ3.58 (c=1, CHCl3).
5-(Benzyl (benzyloxycarbonyl)amino) pentyl 2,3,4,6-tetra-O-
acetyl-b-d-galactopyranosyl-(1!3)-[2,3,4,6-tetra-O-acetyl-b-d-
galactopyranosyl-(1!6)]-4-O-benzyl-2-deoxy-2- ((2,2,2-trichlor-
oethoxycarbonylamino) b-d-galactopyranoside 9: To a solution
of 6 (470 mg, 0.434 mmol) and 7 (256 mg, 0.520 mmol) in dry DCM
(5ml) on activated molecular sieves, under argon and at ꢀ608C
was added TMSOTf (12 ml, 65.1 mmol) dropwise. The reaction was
stirred between ꢀ408C and ꢀ208C for 1 h, as monitored by TLC.
Triethylamine was added and the reaction was filtered through
celite then concentrated in vacuo before purification by FCC (40!
100% EtOAc:Hex) to yield 9 as a white foam, 252 mg, 41%.
1H NMR (500 MHz, CDCl3) d=7.43–7.21 (m, 14H, Ar), 7.16 (d, J=
7.4 Hz,1H, Ar), 5.99 (s, 0.5H, NHTroc), 5.42–5.35 (m, 2H, H-4’, H-4“),
5.31 (d, J=10.4 Hz, 0.5H, NHTroc), 5.24 (dd, J=10.5, 7.9 Hz, 1H, H-
2’), 5.21–5.11 (m, 3H, H-2’, OCH2Bn), 5.01–4.89 (m, 3H, H-3, H-3’,
CHTroc), 4.83–4.62 (m, 6H, H-1, H-1’, 4-OCH2Bn, CHTroc), 4.58–4.40 (m,
4H, H-1“, H-3, NCH2Bn), 4.21–4.09 (m, 4H, H-6’, H-6“), 3.96–3.61 (m,
6H, 2H-6, H-4, H-5’, H-5“, CHlinker), 3.60–3.54 (m, 1H, H-5), 3.52–3.14
(m, 4H, H-2, 3CHlinker), 2.15–1.95 (m, 24H, 8CH3), 1.52 (d, J=26.0 Hz,
4H, 2CH2linker), 1.36–1.26 ppm (m, 2H, CH2linker).13C NMR (126 MHz,
CDCl3) d=170.49, 170.43, 170.34, 170.27, 170.22, 170.19, 169.57,
169.30, 156.35, 154.23, 138.35, 137.96, 129.07, 128.66, 128.58,
128.28, 128.07, 127.97, 127.90, 127.42, 127.30, 102.46, 101.35,
99.88, 78.38, 75.15, 74.36, 70.95, 70.82, 70.78, 70.75, 69.14, 69.06,
67.29, 67.15, 67.03, 61.32, 61.08, 55.02, 50.50, 47.45, 29.81, 29.24,
28.84, 27.94, 27.42, 23.60, 23.28, 20.94, 20.85, 20.79, 20.76, 20.68.
5-(benzyl (benzyloxycarbonyl)amino) pentyl 2,3,4,6-tetra-O-
acetyl-b-d-galactopyranosyl-(1!3)-4,6-O-benzylidene-2-deoxy-2-
((2,2,2-trichloroethoxycarbonyl amino) b-d-galactopyranoside 5:
To a solution of 4 (6.26 g, 8.3 mmol) and 7[56](5.0 g, 10.1 mmol) in
dry DCM (120 mL) on activated molecular sieves at ꢀ408C and
under argon was added TMSOTf (0.301 mL, 1.7 mmol) and the re-
action was stirred at ꢀ20 for 1 h as monitored by TLC. Triethyla-
mine was added and the reaction was filtered through celite then
concentrated to be purified by FCC (20!70% EtOAc:Hex) yielding
1
5 as a white foam, 5.6 g, 62%. H NMR (500 MHz, CDCl3) d=7.55
(d, J=7.6 Hz, 2H, Ar), 7.40–7.20 (m, 12H, Ar), 7.16 (d, J=7.3 Hz,
1H, Ar), 5.57 (s, 1H, CHPh), 5.36 (d, J=3.4, 1.2 Hz, 1H, H-4’), 5.21
(dd, 1H, H-2’), 5.16 (s, 2H, CO2CH2Bn), 4.97 (dd, J=10.4, 3.5 Hz, 1H,
H-3’), 4.90–4.80 (m, 2H, H-1, CHCCl3), 4.77 (d, J=8.0 Hz, 1H, H-1’),
4.65–4.55 (m, 1H, CHCCl3), 4.47 (s, 3H, H-3, NCH2Bn), 4.34–4.28 (m,
2H, H-6a, H-4), 4.15 (ddd, 2H, 2H-6’), 4.06 (dd, J=12.4, 1.8 Hz, 1H,
H-6b), 3.92 (s, 1H, CHlinker), 3.87 (td, 1H, H-5’), 3.53 (s, 1H, H-2), 3.44
(s, 1H, H-5), 3.39 (s, 1H, CHlinker), 3.23 (s, 2H, CH2linker), 2.16 (s, 3H,
Ac), 2.04 (d, J=5.7 Hz, 6H, 2CH3), 1.97 (s, 3H, CH3), 1.52 (d, J=
28.7 Hz, 4H, CH2linker), 1.36–1.23 ppm (m, 2H, CH2linker).13C NMR
&
&
Chem. Eur. J. 2020, 26, 1 – 14
www.chemeurj.org
10
ꢁ 2020 Wiley-VCH GmbH
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Full Paper
doi.org/10.1002/chem.202000291
Chemistry—A European Journal
HRMS (MALDI-Tof) m/z calcd for C64H79Cl3N2O27 [M+Na]+: 1435.383,
found: 1435.3923. [a]D20 =ꢀ11.38 (c=1, CHCl3)
5-(Benzyl (benzyloxycarbonyl)amino) pentyl 2,3,4,6-tetra-O-
acetyl-b-d-galactopyranosyl-(1!3)-[3,4,6-tri-O-acetyl-2-deoxy-2-
((2,2,2-trichloroethoxycarbonylamino)-b-d-glucopyranosyl-(1!
6)]-4-O-benzyl-2-deoxy-2- ((2,2,2-trichloroethoxycarbonylamino)
0.5m NaOMe (1.5 mL, 750 mmol) was added. The RM was stirred at
RT for 1 hour after which it was quenched with AmberliteꢂIR 120
(H). The filtrate was concentrated, purified by Sephadex LH-20 (D=
3.5 cm, H=45 cm, MeOH) and lyophilized to yield 11 as a white
powder, 87.5 mg, 99%. 1H NMR (500 MHz, CD3OD) d=7.46–7.16
(m, 15H,Ar), 5.15 (d, J=16.3 Hz, 2H, OCH2Bn), 4.99 (d, J=11.6 Hz,
1H, 4-OCHBn), 4.70 (d, J=11.6 Hz, 1H, 4-OCHBn), 4.50 (s, 2H,
NCH2Bn), 4.47 (s, 1H, H-1), 4.30 (d, J=7.6 Hz, 1H, H-1’), 4.24 (d, J=
7.5 Hz, 1H, H-1“), 4.08 (dd, J=15.4, 6.2 Hz, 2H, H-2, H-4), 3.90 (dd,
J=10.9, 2.9 Hz, 1H, H-3), 3.87–3.69 (m, 10H, H-4’, H-4“, 2H-6, 2H-6’,
2H-6“, H-5, CHlinker), 3.58 (dd, J=9.7, 7.6 Hz, 1H, H-2’), 3.48 (tdd, J=
20.9, 10.2, 7.3 Hz, 7H, H-2“, H-3’, H-3“, H-5’,H-5“,H-2“, CHlinker), 3.22
(dd, J=14.3, 6.9 Hz, 2H, CH2linker), 1.97–1.89 (m, 3H, CH3), 1.57–1.42
(m, 4H, 2CH2linker), 1.28–1.21 ppm (m, 2H, CH2linker). 13C NMR
(126 MHz, CD3OD) d=174.30, 140.51, 129.59, 129.55, 129.05,
128.36, 107.18, 105.20, 102.59, 81.67, 77.15, 76.96, 76.54, 75.63,
75.13, 74.97, 74.56, 72.63, 72.54, 70.28, 70.09, 69.83, 68.47, 62.85,
b-d-galactopyranoside 10: To
a
solution of
6
(420 mg,
0.387 mmol) and with the imidate 8 (291 mg, 0.465 mmol) in dry
DCM (5ml) on activated molecular sieves, under argon and at
ꢀ608C was added TMSOTf (10.5 mL, 58.1 mmol). The reaction was
stirred between ꢀ408C and ꢀ208C for 1 h until full reaction was
observed as monitored by TLC. Triethylamine was added and the
reaction was filtered through celite. The reaction crude was then
concentrated in vacuo and purified by FCC (40!60% EtOAc:Hex)
yielding 10 as a white solid, 438 mg, 73%. 1H NMR (500 MHz,
CDCl3) d=7.42–7.13 (m, 15H, Ar), 5.98 (s, 0.5H, N-HTroc), 5.45 (s,
0.5H, N-HTroc), 5.39 (s, 1H, H-4’), 5.30–5.13 (m, 4H, H-2’, H-3“,
OCH2Bn), 5.06 (t, J=9.6 Hz, 1H, H-4“), 4.98 (dd, J=10.5, 3.5 Hz, 1H,
H-3’), 4.91 (d, J=11.3 Hz, 1H, CHTroc), 4.84–4.59 (m, 6H, CHTroc, 4-
OCH2Bn,H-1,H-1’,H-1“) 4.46 (dt, J=22.7, 12.8 Hz, 3H, NCH2Bn, H-3),
4.27 (dd, J=12.3, 4.7 Hz, 2H, H-6’a,H-6“a), 4.12 (ddd, J=26.1, 11.8,
4.5 Hz, 2H, H-6’b,H6“b), 3.96–3.25 (m, 10H, H-4,H-5,H-5’, H-5“, 2H-6,
CH2linker, H-2, H-2“), 3.19 (s, 2H, CH2linker), 2.16–1.95 (m, 21H, 7CH3),
1.65–1.44 (m, 4H, 2CH2linker), 1.38–1.28 ppm (m, 2H,
CH2linker).13C NMR (126 MHz, CDCl3) d=170.73, 170.58, 170.22,
170.12, 169.52, 156.32, 154.11, 138.38, 137.83, 136.67, 128.90,
128.58, 128.49, 128.18, 127.97, 127.79, 127.34, 127.23, 102.31,
101.03, 100.07, 95.63, 78.22, 75.09, 74.40, 74.23, 72.18, 71.82, 70.79,
70.70, 69.00, 68.50, 67.24, 67.09, 62.03, 61.18, 56.38, 54.80, 50.39,
47.35, 28.83, 27.81, 27.20, 23.37, 20.75, 20.68, 20.65, 20.59 ppm.
HRMS (MALDI-Tof) m/z calcd for C65H79Cl6N3O27 [M+Na]+:
1566.2928, found: 1566.2886. [a]D20 =ꢀ6.28 (c=1, CHCl3)
62.26, 53.74, 49.85, 30.24, 24.24, 23.27 ppm. HRMS (MALDI-Tof) m/z
calcd for C47H64N2O23 [M+Na]+: 967.4052, found: 967.408, [a]D
ꢀ5.7(c=1, MeOH).
=
20
5-(Benzyl (benzyloxycarbonyl)amino) pentylb-d-galactopyrano-
syl-(1!3)-[ 2-deoxy-2-acetamido-b-d-glucopyranosyl-(1!6)]-4-
O-benzyl-2-deoxy-2-acetamido-b-d-galactopyranoside 12: To a
solution of 10 (390 mg, 0.253 mmol) in THF (6ml) was added TBAF
(1m in THF, 0.607 mL, 0.607 mmol) dropwise. The yellow reaction
mixture was refluxed for 1.5 hours after which it was placed on ice
and quenched with MeOH. The reaction mixture was concentrated
to dryness in vacuo before being redissolved in anhydrous pyridine
(3ml). Acetic anhydride (0.6 mL, 6.32 mmol) was added and the re-
action was left to stir overnight. MeOH was added and the reaction
mixture was concentrated in vacuo. The crude was purified by FCC
(0!5% MeOH:DCM) to yield 5- (benzyl (benzyloxycarbonyl)amino)
pentyl 2,3,4,6-tetra-O-acetyl-b-d-galactopyranosyl-(1!3)-[3,4,6-tri-
O-acetyl-2-deoxy-2-acetamido-b-d-glucopyranosyl-(1!6)]-4-O-
5-(Benzyl (benzyloxycarbonyl)amino) pentylb-d-galactopyrano-
syl-(1!3)-[ b-d-galactopyranosyl-(1!6)]-4-O-benzyl-2-deoxy-2-
acetamido-b-d-galactopyranoside 11: To a solution of 9 (154 mg,
0.109 mmol) in THF (3ml) was added TBAF (1m in THF, 0.163 mL,
0.163 mmol) dropwise. The yellow reaction mixture was refluxed
for 1.5 h after which it was placed on ice and quenched with
MeOH. The reaction mixture was concentrated to dryness in vacuo
before being redissolved in anhydrous pyridine (1 mL). Acetic an-
hydride (0.3 mL, 3.27 mmol) was added and the reaction was left
to stir overnight. MeOH was added and the reaction mixture was
concentrated in vacuo. The crude was purified by FCC (50!100%
EtOAc:Hex) to yield 5- (benzyl (benzyloxycarbonyl)amino) pentyl
2,3,4,6-tetra-O-acetyl-b-d-galactopyranosyl-(1!3)-[2,3,4,6-tetra-O-
acetyl-b-d-galactopyranosyl-(1!6)]-4-O-benzyl-2-deoxy-2- acetami-
benzyl-2-deoxy-2-acetamido-b-d-galactopyranoside as
a brown
solid, 226 mg, 70%. 1H NMR (500 MHz, CDCl3) d=7.43–7.13 (m,
15H, Ar), 5.39 (d, J=3.5 Hz, 1H, H-4’), 5.26–5.10 (m, 4H, H-2’, H-3“,
OCH2Bn), 5.08–4.98 (m, 2H, H-4“, H-3’), 4.87 (d, J=11.4 Hz, 1H, 4-
OCHBn), 4.82 (d, J=8.3 Hz, 1H, H-1), 4.64 (dt, J=27.6, 9.0 Hz, 4H, 4-
OCHBn, H-1’, H-1“, H-3), 4.49 (m, J=17.7, 16.6 Hz, 2H,NCH2Bn), 4.30–
4.03 (m, 4H, 2H-6’, 2H-6“), 3.97–3.71 (m, 5H, H-2“, H6a, H-4, H-5’,
CHlinker), 3.70–3.57 (m, 3H, H6b, H-5“, H-5), 3.52–3.25 (m, 3H, H-2,
CH2linker), 3.24–3.11 (m, 2H, CH2linker), 2.15–1.84 (m, 24H), 1.64–1.42
(m, 4H, CH2linker), 1.36–1.26 ppm (m, 2H, CH2Linker).13C NMR
(126 MHz, CDCl3) d=170.88, 170.75, 170.22, 170.12, 169.44, 169.23,
138.52, 137.76, 128.86, 128.60, 128.51, 128.12, 127.72, 127.68,
127.38, 127.27, 101.87, 100.95, 99.53, 75.34, 74.33, 73.63, 72.62,
71.76, 70.88, 70.77, 69.66, 69.26, 68.96, 68.53, 67.23, 67.18, 62.08,
61.17, 54.88, 50.35, 47.32, 28.82, 27.34, 23.66, 23.30, 20.85, 20.79,
do-b-d-galactopyranoside
a
pale yellow solid, 125 mg,
90%.1H NMR (500 MHz, CDCl3) d=7.43–7.13 (m, 15H,Ar), 5.38 (dd,
J=12.7, 3.5, 1.1 Hz, 2H, H-4’,H-4“), 5.24 (t, J=9.2 Hz, 1H,H-2’), 5.19–
5.11 (m, 3H, OCH2Bn, H-2“), 5.02 (dd, J=10.4, 3.4 Hz, 1H, H-3’), 4.95
(dd, J=10.5, 3.4 Hz, 1H, H-3“), 4.88 (dd, 2H, H-1, 4-OCHBn), 4.75–
4.63 (m, 3H, H-1’, H-3, 4-OCHBn), 4.60–4.38 (m, 3H, H-1“,NCH2Bn),
4.22–4.09 (m, 4H, 2H-6’, 2H-6“), 3.96–3.55 (m, 7H, H-4, H-5, H-5’, H-
5“, 2H-6, CHlinker), 3.43–3.28 (m, 2H, H-2, CHlinker), 3.16 (s, 2H,
CH2linker), 2.15–1.93 (m, 27H, 9CH3), 1.54 (s, 4H, 2CH2linker), 1.37–
1.21 ppm (m, 2H, CH2linker).13C NMR (126 MHz, CDCl3) d=170.37,
170.31, 170.25, 170.17, 170.10, 170.07, 169.18, 138.26, 137.74,
101.95, 101.18, 99.26, 77.88, 75.12, 74.13, 70.83, 70.58, 69.88, 69.21,
68.91, 67.18, 67.06, 67.02, 61.26, 60.98, 55.21, 50.30, 47.29, 45.91,
29.63, 29.10, 28.71, 27.41, 23.59, 23.12, 20.81, 20.64, 20.61,
20.54 ppm. HRMS (MALDI-Tof) m/z calcd for C63H80N2O26 [M+Na]+:
1303.4893, found: 1303.5106, [a]D20 =ꢀ20,5(c=1 CHCl3). The prod-
uct (120 mg, 94 mmol) was redissolved in dry MeOH (4 mL) and
20.75, 20.71, 20.68, 20.65, 20.59 ppm. HRMS (MALDI-Tof) m/z calcd
for C63H81N3O25 [M+Na]+: 1302.5057, found: 1302.5106, [a]D
=
20
ꢀ18.28 (c=1, CHCl3). The product (218 mg, 0.170 mmol) was redis-
solved in dry MeOH(9 mL) and 0.5m NaOMe (2.5 mL, 1.24 mmol)
was added. The RM was stirred at RT for 1 hour after which it was
quenched with AmberliteꢂIR 120 (H). The filtrate was concentrated,
purified by Sephadex LH-20 (D=3.5 cm, H=45 cm, MeOH) and
lyophilized to yield 12 as a white powder, 153 mg, 100%.1H NMR
(500 MHz, CD3OD) d=7.45–7.40 (m, 2H, Ar), 7.40–7.20 (m, 12H,
Ar), 7.18 (s, 1H, Ar), 5.15 (d, J=15.8 Hz, 2H, OCH2Bn), 4.98 (d, J=
11.6 Hz, 1H, 4-OCHBn), 4.67 (d, J=11.5 Hz, 1H, 4-OCHBn), 4.50 (s, 2H,
NCH2Bn), 4.41 (brs, 1H, H-1), 4.36 (d, J=8.4 Hz, 1H, H-1“), 4.30 (d,
J=7.6 Hz, 1H, H-1’), 4.06 (brs, 1H, H-2), 4.02 (d, J=3.1 Hz, 1H, H-4),
3.91–3.73 (m, 6H,H6’a, H6“b,H6c, H-3, H-4’), 3.71–3.54 (m, 5H,H6’a,
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C49H67N3O18 [M+Na]+: 1008.4317, found: 1008.437, [a]D20 =ꢀ6.3
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J.P., S.A., A.C., N.C.R, and F.F. were supported by the EU Horizon
2020 Research and Innovation Program (Marie Skłodowska-
Curie Grant 642870, ETN-Immunoshape). N.C.R additionally ac-
knowledges funding from the Ministry of Science and Educa-
tion (MINECO) Grant No. CTQ2017-90039-R and RTC-2017-
6126-1 and the Maria de Maeztu Units of Excellence Program
from the Spanish State Research Agency-Grant No.MDM-2017-
0720. F.F. acknowledges also the French Agence Nationale de
la Recherche (ANR) PIA for Glyco@Alps (ANR-15-IDEX-02).The
Multistep Protein Purification Platform (MP3) was exploited for
human DC-SIGN/R and MGL ECD production with support
from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-
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Keywords: carbohydrates
lectins · microarrays
· enzymes · glycoconjugates ·
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Manuscript received: January 17, 2020
Revised manuscript received: April 7, 2020
Version of record online: && &&, 0000
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FULL PAPER
&
Carbohydrates
J. Pham, A. Hernandez, A. Cioce, S. Achilli,
G. Goti, C. Vivꢁs, M. Thepaut, A. Bernardi,
F. Fieschi, N.-C. Reichardt*
&& – &&
Chemo-Enzymatic Synthesis of S.
mansoni O-Glycans and Their
Evaluation as Ligands for C-Type
Lectin Receptors MGL, DC-SIGN, and
DC-SIGNR
A series of parasite O-glycans struc-
tures have been prepared by chemoen-
zymatic synthesis and their affinity to-
wards several C-type lectins screened
using glycan arrays.
&
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Chem. Eur. J. 2020, 26, 1 – 14
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ÝÝ These are not the final page numbers!