Synthesis of Asparagine Derivatives Harboring a Lewis X Type DC-SIGN Ligand and Evaluation of their Impact on Immunomodulation in Multiple Sclerosis

Article abstract of DOI:10.1002/chem.202004076

The protein myelin oligodendrocyte glycoprotein (MOG) is a key component of myelin and an autoantigen in the disease multiple sclerosis (MS). Post-translational N-glycosylation of Asn₃₁ of MOG seems to play a key role in modulating the immune response towards myelin. This is mediated by the interaction of Lewis-type glycan structures in the N-glycan of MOG with the DC-SIGN receptor on dendritic cells (DCs). Here, we report the synthesis of an unnatural Lewis X (Le^X)-containing Fmoc-SPPS-compatible asparagine building block (SPPS=solid-phase peptide synthesis), as well as asparagine building blocks containing two Le^X-derived oligosaccharides: LacNAc and Fucα1-3GlcNAc. These building blocks were used for the glycosylation of the immunodominant portion of MOG (MOG_31-55) and analyzed with respect to their ability to bind to DC-SIGN in different biological setups, as well as their ability to inhibit the citrullination-induced aggregation of MOG_31-55. Finally, a cytokine secretion assay was carried out on human monocyte-derived DCs, which showed the ability of the neoglycopeptide decorated with a single Le^X to alter the balance of pro- and anti-inflammatory cytokines, inducing a tolerogenic response.

Full text of DOI:10.1002/chem.202004076

Accepted Article
Accepted Article
Title: Synthesis of asparagine derivatives harboring a Lewis X type DC-
SIGN ligand and evaluation of their impact on immunomodulation
in multiple sclerosis
Authors: Ward Doelman, Mikkel H. S. Marqvorsen, Fabrizio Chiodo,
Sven C. M. Bruijns, Gijsbert A. van der Marel, Yvette van
Kooyk, Sander Izaak van Kasteren, and Can Araman
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.202004076
Link to VoR: https://doi.org/10.1002/chem.202004076
01/2020

10.1002/chem.202004076
Chemistry - A European Journal
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Synthesis of asparagine derivatives harboring a Lewis X type DC-
SIGN ligand and evaluation of their impact on immunomodulation
in multiple sclerosis
Ward Doelman[a], Mikkel H. S. Marqvorsen[a], Fabrizio Chiodo[b], Sven C. M. Bruijns[b], Gijsbert A. van
der Marel[a], Yvette van Kooyk[b], Sander I. van Kasteren[a]*, Can Araman[a]*
[a]
W.Doelman, Dr. M.H.S. Marqvorsen, Prof. G. van der Marel, Dr. S.I. van Kasteren, Dr. C. Araman
Leiden Institute of Chemistry, Leiden University
Einsteinweg 55, 2333 CC Leiden (The Netherlands)
E-mail: m.c.araman@lic.leidenuniv.nl
s.i.van.kasteren@chem.leidenuniv.nl
[b]
Dr. F. Chiodo, S.C.M. Bruijns, Prof. Y van Kooyk
Amsterdam UMC-Location Vrije Universiteit Amsterdam, Department of Molecular Cell Biology and Immunology
De Boelelaan 1108 1081 HZ Amsterdam
Supporting information for this article is given via a link at the end of the document.
Abstract: The protein myelin oligodendrocyte glycoprotein (MOG) is
a key component of myelin and an autoantigen in the disease multiple
sclerosis (MS). The posttranslational N-glycosylation of Asn31 of MOG
seems to play a key role in modulating the immune response towards
myelin. This is mediated by the interaction of Lewis type glycan
structures on the N-glycan of MOG to the DC-SIGN receptor on
dendritic cells (DCs). Here, we report the synthesis of an unnatural
Lewis X (LeX) containing Fmoc-SPPS compatible asparagine building
block, as well as asparagine building blocks containing two LeX
derived oligosaccharides: LacNAc and Fucα1-3GlcNAc. These
building blocks were utilized for the synthesis of glycosylated MOG
(MOG31-55) and were analyzed with respect to their ability to bind to
DC-SIGN in different biological setups, as well as their ability to inhibit
the citrullination induced aggregation of MOG31-55. Finally, a cytokine
secretion assay was carried out on human moDCs, showing the ability
of a neoglycopeptide decorated with a single LeX to alter the balance
of pro- and antiinflammatory cytokines, inducing a tolerogenic
response.
against MOG are circulating in the bloodstream of patients
suffering from various demyelinating diseases such as MS and N-
methyl-D-aspartate receptor-encephalitis[9], and that a peptide
fragment comprising AAs 35-55, MOG35-55, is a key T-cell epitope
in EAE.[10,11]
We have recently discovered
a potential reason for the
pathogenicity of this MOG35-55-peptide in EAE: after post-
translational citrullination (deimination of guanidine on arginine),
the peptide can form amyloid-like aggregates intracellularly,
where they appear to be cytotoxic.[12,13] Citrullination of myelin
proteins is considered to be critical in MS. For example, another
antigenic myelin protein, myelin basic protein (MBP), has been
shown to have increased citrullination in myelin samples from MS
patients.[14] Together, these advances led to the hypothesis that
post-translational citrullination of MOG could be in part
responsible for the disease pathogenesis in EAE to be shifted
towards neurodegeneration rather than autoimmunity.
In light of the above findings, we wanted to explore whether the
native N-glycan at position 31 has an effect on the aggregation
behavior of the citrullinated peptide, as O-glycosylation of serine
or threonine residues has previously been shown to be inhibitory
on the aggregation of a tau derived peptide, a highly aggregation-
prone protein family involved in Alzheimer’s disease.[15] The
introduction of N-glycans and mimics thereof on peptides derived
from prion protein[16] and the full-length prion protein[17] itself, has
also been shown to decrease or even abrogate aggregation.
Furthermore, previous studies on the glycosylation of MOG
suggest that the nature of the carbohydrate structures of the N-
glycan plays an important role in the modulation of immunological
tolerance through glycan interaction with the dendritic cell-specific
intercellular adhesion molecule-3–grabbing nonintegrin (DC-
SIGN) receptor.[18] This receptor has been shown to recognize the
Introduction
Multiple Sclerosis (MS) is
a
group of auto-immune
neurodegenerative diseases, characterized by the formation of
lesions in the patient’s brain that lead to loss of functions.[1] The
pathology of MS is not fully understood, but degradation of myelin
sheath seems to be a critical step in the process.[2] Myelin sheaths
are comprised of myelin, an insulating substance consisting of
lipids, proteins and other molecules, and are responsible for fast
information transfer through axons.[3] Indeed, some proteinogenic
components of myelin sheath have been shown to become
antigenic upon their degradation.[4] For example, myelin
oligodendrocyte glycoprotein (MOG), an exclusively CNS-
resident protein found on the surface of oligodendrocytes and
myelin sheaths, acts as an autoantigen in an MS-like animal
model, experimental autoimmune encephalomyelitis (EAE).[5]
MOG is a glycoprotein, decorated with an N-glycan[6] on Asn31,
with a molecular mass of 26-28 kDa.[7,8] It comprises 245 amino
acids (AA) and belongs to the immunoglobulin superfamily (Ig).
Over the last few decades, it has been shown that antibodies
fucose-containing Lewis-type glycans[19]
,
especially the
trisaccharide Galβ1-4(Fucα1-3)GlcNAc, better known as LewisX
(LeX), which has been shown to be highly abundant on natively
glycosylated MOG.[20] Hence, studies using synthetic
neoglycopeptides bearing DC-SIGN binding N-glycan mimics
may shed light on the role of a putative interaction between DC-
SIGN and MOG in MS. We synthesized MOG31-55-peptides
decorated with LeX and LeX derived oligosaccharides (LacNAc
and Fucα1-3GlcNAc) on the N-terminal asparagine (Asn31) and
1
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Chemistry - A European Journal
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assess the effect of these modifications on the aggregation
proneness of the peptides. It was our aim to link the glycans to
the peptides via amide linkages, to minimize artefacts stemming
from various non-native linkers.[21–23] To achieve this, we
extended our recently published method for the synthesis of
inflammatory) and IL-12p70 (pro-inflammatory) production by LeX
decorated peptides.
The outcome of these biochemical and immunological studies
suggested that i) aggregation behavior of citrullinated MOG31-55
can be halted or abrogated depending on the glycan; ii) our amide
linked LeX ligand indeed binds to DC-SIGN in an ELISA and iii)
the LeX decorated neoglycopeptide has an in vitro tolerogenic
effect (cytokine secretion assay) thus potentially prevents
inflammation. Hence, we report the first synthesis of MOG31-55
derivatives that are site-specifically decorated with DC-SIGN
ligands and analyze the immunological consequences of
exposure of moDCs to the LeX decorated peptide.
glycosylated
asparagine
derivatives
using
larger
oligosaccharides.[24] By using these asparagine building blocks
with our previously established model peptide, MOG31-55[12], we
were able to evaluate the effect of glycosylation on citrullination-
dependent aggregation of MOG. Subsequently, using the binding
of LeX decorated neoglycopeptides to DC-SIGN was confirmed by
solid-phase immunoassays. Finally, a cytokine secretion assay in
monocyte derived dendritic cells (moDCs) from human donors
was utilized to analyze the degree of modulation for IL-10 (anti-
Scheme 1. A) Structures of glycosylated asparagine derivatives 1-4 B) Retrosynthetic analysis of the synthesis of LeX decorated MOG31-55 peptides. X = NH (Arg) or X = O (Cit).
developed by von dem Bruch and Kunz.[36] We designed and
synthesized three glycosyl amide derivatives of asparagine as
Fmoc-SPPS (solid phase peptide synthesis) compatible building
blocks, containing LeX and two LeX derivatives, LacNAc and
Results and Discussion
Fucα1-3GlcNAc, attached to the asparagine side chain via the
reducing ends of respective sugars (Scheme 1A, 1-4). The
LacNAc construct (3) served as a negative control for DC-SIGN
binding, as the interaction of LeX with the receptor has been
shown to be fucose dependent.[32]
We chose to base our synthesis on acid labile para-
methoxybenzyl (PMB) and para-methoxybenzylidene groups,
which would be removed during global peptide deprotection in
standard Fmoc-based solid phase peptide synthesis, and on
esters, which can be selectively removed using hydrazine in
methanol after the acidic global deprotection of the peptide. By
including these protecting groups from the start of the
oligosaccharide synthesis, late-stage protecting group
manipulation could mostly be avoided.
While N-glycosylation of asparagine is of prime importance for a
variety of protein functions such as signaling and folding[25], the
typical size and complexity of an N-glycan poses a considerable
synthetic challenge. N-glycosylated peptides have been
generated using semisynthetic methods involving synthesis
and/or isolation of carbohydrate segments which can be linked
covalently using endohexosaminidases[26,27], or extended via
specific glycosyltransferases as recently demonstrated by Boons
and colleagues.[28,29] Synthetic preparation of an entire peptide
bearing a natural N-glycan has also been reported.[30]
Previous work from our group and others[31–35] has
demonstrated that fucosylated glycans interact with DC-SIGN
without the need for an N-glycan core structure. This inspired us
to synthesize a LeX N-glycan derivative similar to the one
2
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Scheme 2. Synthesis of Lewis X azide (A) 11 and LacNAc azide 14 (B)
The linkages between the oligosaccharides and the asparagine
side chain were installed using our recently developed two-step
one-pot approach for the synthesis of glycosylated asparagine
derivatives.[24] Here, we combine a Staudinger reduction to
transform a glycosyl azide into a glycosyl amine, as reported by
many others[37–39], followed by aspartic anhydride ring-opening,
generating a protected glycosyl asparagine derivative (Scheme
1B). The synthesis of the protected LeX glycosyl azide 11
(Scheme 2) was initiated from the para-methoxy benzylidene
protected glycosyl azide 5 (synthesis in SI) by means of a
treated with HF·pyridine for removal of the tert-butyldimethylsilyl
(TBS) group, followed by acetylation to afford the desired
peracetylated glycosyl azide 14 in 85% yield over 2 steps.
Asparagine derivatives 1-4 were prepared following a general
synthetic strategy involving the Staudinger reduction of a glycosyl
azide followed by direct ligation of the resulting glycosyl amine
with Fmoc aspartic anhydride, a sequence reported recently by
us (Scheme 3A).[24] Accordingly, Fmoc-Asn(GlcNAc)-OH (1) was
synthesized from easily obtained glycosyl azide 15[46] in three
steps by PMe3-mediated azide reduction, followed by addition of
H2O to the crude iminophosphorane to obtain the intermediate
glycosyl amine. The desired asparagine derivative was formed by
redissolving the crude glycosyl amine in DMSO followed by
addition of Fmoc aspartic anhydride. Precipitation directly
afforded the desired SPPS building block 1 in 69% yield.
The above sequence proved similarly useful for the
preparation of the other desired glycosylated asparagine building
blocks (2-4, Scheme 3A). However, precipitation or extraction
were found to be less efficient for small scale purification of the
more complex carbohydrates, and therefore we subjected these
compounds to silica gel chromatography for purification. Using
this approach, the fucosylated glycosyl azide 7 was converted to
its corresponding SPPS building block 2 in 65% yield, while
lactosyl compound 14 was similarly converted to compound 3 in
63% yield. (Scheme 3A).
NIS/TMSOTf-promoted
fucosylation
reaction
with
the
thioglycoside 6 (synthesis in SI) to afford disaccharide 7 in 71%
yield. The presence of the acetamido group was detrimental to
the results of the following glycosylation, an often encountered
problem with N-acetyl-glucosamine derived acceptors.[40]
Accordingly, disaccharide 7 was treated with an excess of acetyl
chloride and diisopropylethylamine (DiPEA) to convert the amide
functionality to the less interfering imide in 89% yield.[41] Reductive
opening of the para-methoxybenzylidene with BH3/Bu2BOTf was
performed as described[42], affording compound 8 in 81% yield.
Finally, galactosylation with the trichloroacetimidate donor 9
(synthesis in SI) yielded the desired protected trisaccharide 10 in
77% yield. Chemoselective deacetylation of 10 using N,N-
dimethylaminopropylamine (DMAPA)[43] afforded 11 in 87% yield.
The protected lactosaminyl azide 14 was prepared using a
literature protocol for regioselective glycosylation of 1,6-protected
GlcNAc derivatives.[44,45] Silyl ether protected glycosyl azide 12
was subjected to BF3·Et2O promoted galactosylation with
trichloroacetimidate donor 9, affording the partially protected
disaccharide 13 in a 56% yield (Scheme 2). This compound was
For the trisaccharide glycosyl azide, transformation of the
NAc2 functionality to the acetamide was required, as Staudinger
reduction of 10 afforded conversion to an unknown side product.
3
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Scheme 4. Synthetic strategy employed for the synthesis of MOG31-55 glycopeptides. X
= Arg (17a-20a) or Cit (17b-20b)
The peptides were cleaved from the solid support under acidic
conditions (95:2.5:2.5 TFA:TIS:H2O mixture for 2 hours) for the
non-fucosylated peptides, and more dilute acidic conditions
(50:2.5:2.5:45 TFA:TIS:H2O:DCM mixture for 4 hours) for the
fucose containing ones, to prevent hydrolysis of the acid labile α-
fucosyl bond.[49] The reaction time under these less acidic
conditions had to be extended to ensure complete removal of the
Scheme 3: A) Synthesis of glycosylated Fmoc-asparagine derivatives 1-4 via the two
step Staudinger reduction/aspartic anhydride coupling approach. B) observed
reaction when performing Staudinger reduction of diacetylimide 16
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
(Pbf)
protecting groups, which are more acid stable than the usual side
chain protecting groups (Boc/tBu) in Fmoc-SPPS.[50]
Acetyl migration is a likely explanation, as Staudinger reduction of
the more simple NAc2 protected glycosyl azide 16 afforded
clean conversion to the more readily assignable glycosyl
acetamide 16a (Scheme 3B). Glycosyl azide 11 was coupled to
Fmoc aspartic anhydride yielding the desired LeX SPPS building
block 4 as an inseparable 10:1 mixture with its corresponding iso-
asparagine isomeric product. It has been shown that
dimethylacetamide (DMA) gives similar regioselectivity as DMSO
when used as solvent for aspartic anhydride ring opening
reactions.[47] However, the lower melting point of this solvent
allows for aspartic anhydride ring-opening at 0°C, potentially
increasing regioselectivity. Indeed, this solvent and temperature
change resulted in the desired LeX asparagine 4 being formed in
74% yield with complete regioselectivity.
The syntheses of the desired glycopeptides were initiated with
automated SPPS of the MOG32-55 peptide on Tentagel®S-RAM
resin, using HCTU as the coupling reagent. These peptides where
then manually elongated at the N-terminus with glycosylated
asparagines 1-4 using DEPBT as the coupling reagent to prevent
aspartimide formation, as described by Yamamoto et al.[48] The
general synthetic strategy used for the synthesis of the
glycopeptides is outlined in Scheme 4.
To remove the remaining ester protecting groups on the
carbohydrates, the crude peptides were treated with 10%
hydrazine monohydrate in methanol. Crude glycopeptides were
purified by preparative reverse-phase (RP) HPLC. All four
different glycosylated asparagine building blocks exhibited good
coupling efficiencies under the conditions used here (Scheme 4)
and the neoglycopeptides 17a-20a were isolated in moderate to
good yields after RP-HPLC (Table 1).
In order to test whether glycosylation has an impact on the
aggregation behavior of citrullinated MOG-derived peptides,
we prepared MOG31-55 peptides carrying both post-translational
modifications, namely citrullination and glycosylation. For the
citrullination pattern we chose to replace both Arg41 and Arg46 with
citrullines, since we have previously shown that this citrullination
pattern enhances aggregation behavior.[12] Furthermore, the
location of the modifications in the putative MHC-I restricted non-
[51]
human primate epitope MOG40-48
was interesting, as
citrullination of one of these positions was demonstrated to
exacerbate ongoing EAE.[52]
4
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susceptibility of all glycopeptides to amyloid-like aggregation
using the previously described ThT fluorescence assay.[12] In this
assay, a fluorogenic substrate, Thioflavin T, with a selectivity
towards cross β-sheet structures as found in amyloid-like
aggregates, is used to detect whether such aggregation occurs.
Non-citrillunated peptides did not show aggregation at
physiologically relevant concentrations (10 µM, Figure 1A). For
the citrullinated peptides, the effect of glycosylation seemed to be
structure dependent.
Whilst all forms of glycosylation had an inhibitory effect on
aggregation (Figure 1B), the inclusion of a single GlcNAc
modification (17b) was sufficient to completely abrogate the
aggregation, displaying the powerful effect glycosylation can have
on peptide aggregation. The DC-SIGN ligand LeX (20b) showed
a similar inhibition of aggregation to that of GlcNAc suggesting the
potential in controlling immune household and not the
neurodegenerative mechanism in MS. However, other
glycosylation patterns tested, Fucα1-3GlcNAc (18b) and LacNAc
(19b), did not fully inhibit aggregation delaying only its onset
(Figure 1B). In our previous studies[12] , we have shown that
citrullinated MOG35-55 peptides are cytotoxic to murine bone
marrow derived dendritic cells (BMDCs). Citrullinated MOG31-55
however was not yet tested. To analyze whether native,
glycosylated or citrullinated MOG31-55 variants show similar
cytotoxicity to those of citrullinated MOG35-55, we conducted cell
Table 1. Yields of glycopeptides obtained using the synthetic strategy outlined
in Scheme 4 after preparative HPLC. The number for each compound is given
together with the HPLC yield based on crude mass.
Amino acid
X = Arg
X = Cit
GlcNAc (
1
)
17a (4.0%)
18a (5.6 %)
19a (5.8 %)
20a (4.1 %)
17b (8.6%)
Fucα1-3GlcNAc (
2
)
18b (2.1 %, 5.7 %[a])
19b (5.6 %)
LacNAc (
Lewis X (
3
4
)
)
20b (6.1 %, 4.8%[a])
[a] The product containing methionine oxidation was isolated separately.
The citrullinated peptides (17b-20b) were synthesized using
the same methodology as for their non-citrullinated counterparts,
using Fmoc-citrulline as the 41st and 46th amino acid. Similar
levels of glycosyl amino acid incorporation and similar RP-HPLC
yields were achieved during the synthesis of these glycopeptides
(Table 1).
viability
assays
using
3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium Bromide (MTT) as described previously
(Figure S1).[12] BMDCs were treated with citrullinated peptides
18b-20b as well as their non-glycosylated counterpart
(cit_MOG31-55) at four different concentrations (40, 20, 10 and 5
µM). None of the tested peptides showed significant decrease in
viability of BMDCs at any concentration tested. Remaining
glycosylated MOG31-55 derivatives as well as the native variant did
also not exhibit any significant drop in cell viability in BMDCs.
Figure 1. ThT aggregation assay of non-citrullinated (A) and citrullinated (B)
glycosylated MOG31-55 peptides 17a-20b. Peptides were tested at a concentration of
10 µM. Positive control (black diamonds) is nonglycosylated MOG31-55 citrullinated at
positions 41 and 46. All data were recorded at an excitation wavelength of 444 ± 9
nm and an emission wavelength of 485 ± 9 nm. All samples were used at a pH of 5.0
and aggregation assays were performed at least three times and with experimental
triplicates.
To assess the influence of glycosylation on immune-
relevant MOG31-55
, we opted for different biophysical and
biochemical experiments. First, we determined the secondary
structure in solution via circular dichroism (CD). All peptides
showed a pre-dominantly random-coiled structure. The effect of
addition of the α-helix stabilizer TFE (50% v/v in PBS) or SDS at
non-micellar concentrations (4 mM) was also evaluated (Figure
S1). These results indicate the peptides are not prone to β-sheet
formation.
Figure 2. In vitro DC-SIGN binding assay (A) and moDC cytokine profiling upon
exposure to 20a (B,C). A) DC-SIGN-FC ELISA. Lewis X decorated polymer (PAA-LeX)
was used as the positive control, while for the negative control no peptide was
added, meaning they are fully blocked with BSA. The DC-SIGN ELISA has been
performed three times showing similar results. The graph shows data of one
representative experiment out of three independent experiments performed in
duplicate. Error bars represent standard deviation. B) Ratio of IL10/IL12p70
secretion measured upon moDC stimulation with either 20a or non-glycosylated
control in the presence of 10 ng/mL of LPS. This graph is a representative plot from
one donor (N=3). C) Normalized ratios for IL-10 and IL-12p70 secretion between
non-glycosylated peptide MOG31-55 and peptide 20a harboring LeX incubated with
Next, inspired by the recently published aggregation
behavior of citrullinated MOG31-55 peptides, we evaluated the
5
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moDCs at different concentrations in the presence 10 ng/mL LPS. Here a ratio of 1
means cytokine production is the same for both peptides, while a ratio of 0.5
means cytokine production is halved for 20a compared to non-glycosylated
peptide. The results are the average of three experiments performed using cells
from three separate donors, each measured in duplicate.
we plotted the ratio of cytokine secretion between stimulation of
moDCs with 20a and non-glycosylated MOG31-55 for all donors
(N=3). A reduction in secretion of pro-inflammatory cytokine IL-
12p70 is observed, while secretion of anti-inflammatory IL-10
remains unchanged. Since the DC-SIGN-Fc binding ELISA
shows a binding interaction between the LeX decorated peptide
and not the non-glycosylated peptide, a DC-SIGN driven process
is strongly suggested.
Overall, it could be concluded that glycosylation of MOG31-
does not alter its biophysical properties as measured by CD,
55
whereas GlcNAc and LeX modifications abrogate amyloid like
behavior of MOG31-55. Moreover, no major cytotoxic effects were
observed for citrullinated and glycosylated MOG31-55 derivatives in
BMDCs, which renders them useful for subsequent studies to
explore the impact of DC-SIGN binding on moDCs.
Conclusion
Next, we investigated the physiological relevance of our
simplified N-glycan structures. To assess the ability of the model
N-glycans to bind DC-SIGN, a DC-SIGN binding ELISA was
carried out.[53,54] Briefly, peptides were coated on the bottom of
high-binding plates. DC-SIGN binding was assessed by
incubating with recombinant DC-SIGN-Fc construct (N-terminally
truncated extracellular domain (K62-A404) of human DC-SIGN
expressed with the fused Fc region of human IgG1 at the N-
terminus) followed by an HRP-conjugated secondary antibody for
qualitative readout of binding. The results of this assay are
displayed in Figure 2.
As expected[19], LeX peptides 20a (Figure 2A) and 20b
(Figure S3) were recognized by DC-SIGN-Fc, while the other
glycopeptides were recognized to a lesser extent (18a-b, Figure
S3) or not at all (19a-b, Figure 2A and Figure S3). Note that we
observed an increase in binding affinity of GlcNAcylated peptide
17a to DC-SIGN (Figure S3). This can be explained by GlcNAc
being a weak binder to DC-SIGN with an IC50 of 5 mM in vitro.[55]
Citrullination of this peptide, 17b, inhibited the increase in binding
affinity (Figure S3).
Finally, we investigated the downstream effects of stimulation of
human monocytes-derived dendritic cells (moDCs) with LeX
decorated peptide 20a. Since DC-SIGN is absent on murine
DCs[56], human dendritic cells, derived from donor blood, are an
useful alternative. Furthermore, the pathophysiology of MS is not
completely mimicked by murine EAE[57], further necessitating the
use of human model systems when feasible. We utilized a well-
established assay[58] measuring the release of anti- and pro-
inflammatory cytokines, IL-10 and IL-12p70 respectively. It has
been shown that stimulation of DC-SIGN with fucosylated glyco-
conjugates (in presence of TLR4 ligands) induce an upregulation
of IL-10 and a down-regulation of IL-12p70, switching the immune
response towards tolerance instead of inflammation. In this assay
moDCs, derived from peripheral blood monocytes (PBMCs) of
three donors, are stimulated with peptide 20a or non-glycosylated
MOG31-55 at distinct concentrations (14, 7 and 3.5 µM) in presence
or absence of the TLR4 ligand LPS (from E. coli at 10 ng/mL), and
their cytokine secretion levels are measured.[59] As expected, no
cytokine production was observed upon stimulation of moDCs
with peptide in the absence of LPS (Figure S4). However, upon
co-stimulation with LPS, we observed a LeX-dependent effect for
MOG31-55 on IL-12p70 secretion at all concentration tested for
peptide 20a. In Figure 2B the ratio of IL-10/IL-12p70 secretion is
plotted for a single donor (representative for three independent
experiments, N=3), showing an increase for the LeX decorated
neoglycopeptide 20a over the non-glycosylated control at all
concentrations tested. This increase in IL10/IL12p70 ratio shows
that stimulation with peptide 20a leads to a more tolerogenic
response compared to non-glycosylated MOG31-55. In Figure 2C
We have developed a synthetic route for three novel SPPS
compatible glycosylated Fmoc-asparagine building blocks,
including an asparagine derivative of the important DC-SIGN
ligand LeX. These building blocks have been synthesized from the
glycosyl-azides
using
our
Staudinger-reduction/aspartic
anhydride ring-opening approach. By careful choice of protecting
groups during the oligosaccharide assembly, the amount of
protecting group manipulations could be kept to a minimum, while
glycopeptide deprotection was accomplished in a straightforward
manner. To demonstrate this, we have synthesized glycosylated
derivatives of the peptide MOG31-55 in good yields and purity, as
well as derivatives that are both glycosylated and citrullinated.
Using these synthetic neoglycopeptides, we have demonstrated
that glycosylation has a powerful effect on citrullination driven
aggregation of this model peptide. Interestingly, the effect
glycosylation has on citrullination driven aggregation seems to be
also dependent on oligosaccharide structure. Furthermore, we
have shown that LeX, while linked to asparagine directly via an
amide bond, is capable of binding to DC-SIGN, via ELISA. Finally,
we showed that a peptide, decorated with LeX on asparagine, was
able to elicit a tolerogenic response (reduced IL12p70 secretion
compared to non-glycosylated counterpart), when used to
stimulate moDCs.
Experimental Section
General methods for SPPS An automated synthesizer (PTI Tribute UV-
IR synthesizer, Gyros Protein Technologies) was utilized. If not stated
otherwise, peptides were synthesized on Tentagel S RAM resin (Rapp
Polymere GmbH, Germany) on a 100 µmol scale using 5.0 equiv of each
amino acid (AA) with respect to the resin loading. Fmoc protected amino
acids were purchased from either Novabiochem or Sigma-Aldrich. For the
amino acids that require sidechain protection, the following protecting
groups were used: tBu for Ser, Thr and Tyr; OtBu for Asp and Glu; Trt for
Asn, Gln and His; Boc for Lys and Trp; Pbf for Arg; An equimolar quantity
of
2-(6-Chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate (HCTU) was used as activator. Coupling cycles of 1
h were utilized, and unreacted amines were capped after each cycle using
a solution of 500 μL of acetic anhydride, 250 μL of DIPEA, and 4.25 mL of
DMF for 5 min at room temperature twice. Fmoc deprotection was
accomplished with 20% piperidine in DMF (3 x 5 min). Cleavage of non-
glycosylated peptides was accomplished using a 95:2.5:2.5 mixture of
TFA:TES:H2O for 3 hours, followed by precipitation from cold diethyl ether
and recovery of the precipitate by centrifugation. Peptides were
characterized using electrospray ionization mass spectrometry (ESI-MS)
on a Thermo Finnigan LCQ Advantage Max LC-MS instrument with a
Surveyor PDA plus UV detector on an analytical C18 column
(Phenomenex, 3 μm, 110 Å, 50 mm × 4.6 mm) in combination with buffers
A (H2O), B (MeCN), and C (1% aq TFA). Quality of crude peptides was
evaluated with a linear gradient of 10-50% B with a constant 10% C over
6
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10.1002/chem.202004076
Chemistry - A European Journal
FULL PAPER
10 minutes, while final peptide quality was evaluated using a linear
gradient of 5-65% B with a constant 10% C over 30 minutes.
Nγ-[3,4-di-O-benzoyl-2-O-(4-methoxybenzyl)-α-l-fucopyranoside-
(1→3)-4,6-O-(4-methoxybenzylidene)-2-deoxy-2-acetamido-β-d
glucopyranosyl]-Nα-fluorenylmethoxycarbonyl-l-asparagine
-
(2)
Glycosyl azide 7 (168 mg, 0.2 mmol) was dissolved in dry THF (2 mL) and
trimethylphosphine was added as a 1 M solution in THF (1.1 eq, 220 µL,
0.22 mmol). The reaction was stirred for 10 minutes at room temperature
and H2O (50 eq, 180 µL, 10 mmol) was added. After stirring for 1 hour at
room temperature, the reaction was concentrated and the residue was
dissolved in DMSO (2 mL). Fmoc-aspartic anhydride[60] (1.0 eq, 67 mg, 0.2
mmol) was added and the reaction mixture was stirred for 1 hour at room
temperature. The solvent was removed in vacuo and the crude was
subjected to silica gel column chromatography (0 → 8% MeOH in DCM, Δ
Incorporation of glycosylated amino acids Synthesis of glycopeptides
was carried out at 25 µmol scale. Fmoc group was removed from the resin
bound peptide using 2 x 2 mL of 20% piperidine in DMF (3 + 7 min). After
Fmoc deprotection, the resin was washed five times with DMF (5 x 5 mL).
Fully protected glycosylated asparagine (2 eq, 50 µmol) was dissolved in
500 µL of
a 0.3 M solution of 3-(diethoxyphosphoryloxy)-1,2,3-
benzotriazin-4(3H)-one (DEPBT) in DMF by the addition of DIPEA (8.7 µL,
2 eq, 50 µmol). The mixture was agitated for at least 5 minutes or until all
amino acid had been dissolved. The solution containing the activated
amino acid was added to the resin and the resin was incubated overnight
under mild agitation. After overnight coupling, the resin was washed with
DMF (5 x 5 mL) and a small portion was deprotected to confirm
incorporation of the glycosylated amino acid. Fmoc deprotection was
carried out as normal using a freshly prepared piperidine solution. Full
cleavage of the peptide was achieved using 2 mL of 95:2.5:2.5 mixture of
TFA:TES:H2O for 2 hours or 50:2.5:2.5:45 mixture of TFA:TES:H2O:DCM
for 4 hours for fucose containing peptides. The deprotected peptide was
precipitated in cold diethyl ether (10 mL) and the resin was washed with
DCM (1 mL) which was added to the ether phase. After centrifugation, the
pellet was washed with a small amount of diethyl ether (3-5 mL) and
centrifugated again. To facilitate the removal of the ester protection groups,
the peptide was suspended in methanol (2.25 mL) in a roundbottom flask
and placed under N2 atmosphere, followed by the addition of hydrazine
monohydrate (0.25 mL). After stirring overnight, the reaction progress was
checked by LC-MS. When complete deprotection was confirmed the
volatiles were removed in vacuo to yield the crude glycopeptide.
Preparative reverse phase HPLC on a Waters AutoPurification system
(eluent A: H2O + 0.2% TFA; eluent B: ACN) with a preparative Gemini C18
column (5 µm, 150 x 21.2 mm) yielded the final products.
25
= 1%). This yielded the title compound (150 mg, 0.13 mmol, 65%). [α]D
= -73.3 (c 1.00 in CHCl3). 1H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 9.4
Hz, 1H, NγH), 8.16 (d, J = 9.0 Hz, 1H, NHC(O)CH3), 7.87 (t, J = 8.0 Hz, 3H,
CHarom), 7.81 – 7.64 (m, 5H, CHarom), 7.64 – 7.47 (m, 5H, CHarom), 7.47 –
7.26 (m, 8H, NαH, CHarom), 7.18 – 7.04 (m, 2H, CHarom), 6.97 – 6.89 (m,
2H, CHarom), 6.73 – 6.62 (m, 2H, CHarom), 5.71 (s, 1H, PMP-CHacetal), 5.42
– 5.33 (m, 2H, H1’, H3’), 5.23 (d, J = 3.5 Hz, 1H, H4’), 5.15 (t, J = 9.5 Hz,
1H, H1), 4.55 – 4.43 (m, 2H, H5’, PMB-CHH), 4.39 – 4.30 (m, 2H, PMB-
CHH, Asn-CH), 4.30 – 4.17 (m, 4H, Fmoc-CH2, H5, Fmoc-CH), 4.13 (t, J
= 9.5 Hz, 1H, H3), 3.99 (dd, J = 10.7, 3.5 Hz, 1H, H2’), 3.95 – 3.84 (m, 1H,
H2), 3.76 – 3.60 (m, 9H, H6, H4, OCH3, OCH3), 2.66 (dd, J = 16.1, 5.6 Hz,
1H, Asn-CHH), 1.82 (s, 3H, NHC(O)CH3), 0.46 (d, J = 6.4 Hz, 3H, H6’).
13C NMR (101 MHz, DMSO-d6) δ = 170.2 (C=O), 169.7 (C=O), 165.6
(C=O), 164.8 (C=O), 159.7 (Cq), 158.8 (Cq), 155.9 (C=O), 143.9 (Fmoc-
Ar), 143.8 (Fmoc-Ar), 140.7 (Fmoc-Ar), 133.7 (CHarom), 133.5 (CHarom),
130.0 (Cq), 129.2 (CHarom), 129.1 (Cq), 129.0 (CHarom), 128.8 (CHarom),
128.5 (CHarom), 127.8 (CHarom), 127.8 (CHarom), 127.7 (CHarom), 127.1
(CHarom), 125.3 (CHarom), 120.1 (CHarom), 113.4 (CHarom), 100.9(PMP-CH),
96.2 (C1’), 79.4 (C1, C4), 75.3 (C3), 72.3 (C4’), 71.5 (C2’), 70.0 (PMB-
CH2), 69.6 (C3’), 68.0 (C5), 67.8 (C6), 65.8 (Fmoc-CH2), 63.9 (C5’), 55.1
(OCH3, C2), 55.0 (OCH3), 50.4 (Asn-CH), 46.6 (Fmoc-CH), 37.3 (Asn-CH2),
23.1 (NHC(O)CH3), 15.2, (C6’). HRMS (ESI) m/z: [M + H+] calcd for
C63H63N3O18H 1150.41794, found 1150.41741.
Nγ-[3,4,6-tri-O-acetyl-2-deoxy-2-acetamido-β-d-glucopyranosyl]-Nα-
fluorenylmethoxycarbonyl-l-asparagine (1) Glycosyl azide 15 (200 mg,
0.54 mmol) was dissolved in THF (0.76 mL) and the solution was cooled
in an icebath. 0.54 mL of a 1 M solution of trimethylphosphine (1.0 eq, 0.54
mmol) in THF was added dropwise over 2 minutes, during which gas
evolution was observed. The icebath was removed, and the reaction was
stirred for 5 minutes before H2O (10 eq, 97 µL, 5.4 mmol) was added. The
reaction was stirred at room temperature for 1.5 hours, after which it was
concentrated. The residue containing the crude glycosyl amine was
redissolved in DMSO (1.8 mL) and Fmoc-aspartic anhydride[60] (1.0 eq,
181 mg, 0.54 mmol) was added. The reaction was stirred for 2 hours at
room temperature. The DMSO solution was added dropwise to a
centrifuge tube containing 30 mL of a 2:1 mixture of diethyl ether and ethyl
acetate and a precipitate started to form. The compound was left to fully
precipitate for 16 hours at room temperature, after which it was collected
by centrifugation. The supernatant was discarded and the pellet was
washed with a small amount of the diethyl ether/ethyl acetate mixture. After
removing the volatiles under reduced pressure, the title compound was
obtained as a white amorphous solid (255 mg, 0.37 mmol, 69%). 1H NMR
(500 MHz, DMSO-d6) δ 8.60 (d, J = 9.8 Hz, 1H, NγH), 7.99 – 7.78 (m, 3H,
NHC(O)CH3), 7.71 (d, J = 7.5 Hz, 2H, Fmoc-Ar), 7.51 (d, J = 8.5 Hz, 1H,
NαH), 7.41 (t, J = 7.5 Hz, 2H, Fmoc-Ar), 7.32 (t, J = 7.4 Hz, 2H, Fmoc-Ar),
5.18 (t, J = 9.8 Hz, 1H, H1), 5.10 (t, J = 9.8 Hz, 1H, H3), 4.82 (t, J = 9.8 Hz,
1H, H4), 4.38 (q, J = 7.5 Hz, 1H, Asn-CH), 4.33 – 4.13 (m, 4H, Fmoc-CH2,
Fmoc-CH, H6a), 3.94 (d, J = 11.3 Hz, 1H, H6b), 3.88 (q, J = 9.8 Hz, 1H,
H2), 3.84 – 3.78 (m, 1H, H5), 2.66 (dd, J = 16.3, 5.4 Hz, 1H), 1.99 (s, 3H,
OC(O)CH3), 1.96 (s, 3H, OC(O)CH3), 1.90 (s, 3H, OC(O)CH3), 1.72 (s, 3H,
NHC(O)CH3). 13C NMR (126 MHz, DMSO-d6) δ = 173.0 (C=O), 170.1
(C=O), 169.9 (C=O), 169.6 (C=O), 169.6 (C=O), 169.4 (C=O), 155.9 (C=O),
143.8 (Fmoc-Ar), 143.8 (Fmoc-Ar), 140.7 (Fmoc-Ar), 127.7 (Fmoc-Ar),
127.1 (Fmoc-Ar), 125.3 (Fmoc-Ar), 120.2 (Fmoc-Ar), 78.1 (C1), 73.4 (C3),
72.3 (C5), 68.4 (C4), 65.8 (Fmoc-CH2), 61.9 (C6), 52.2 (C2), 50.0 (Asn-
CH), 46.6 (Fmoc-CH), 36.9 (Asn-CH2), 22.6 (NHC(O)CH3), 20.6
(OC(O)CH3), 20.4 (OC(O)CH3), 20.4 (OC(O)CH3). HRMS (ESI) m/z: [M +
H+] calcd for C33H37N3O13H 684.23991, found 684.23920.
Nγ-[2,3,4,6-tetra-O-acetyl-β-d-galactopyranoside-(1→4)-6,3-di-O-
acetyl-2-deoxy-2-acetamido-β-d-glucopyranosyl]-Nα-
fluorenylmethoxycarbonyl-l-asparagine (3) Azidosugar 14 (0.74 mmol,
488 mg) was dissolved in THF (7.4 mL) and
a 1M solution of
trimethylphosphine in THF (1.5 eq, 1.1 mL, 1.1 mmol) was added and the
reaction was stirred at room temperature. H2O (50 eq, 0.67 mL, 37 mmol)
was added and the reaction was further stirred for 60 minutes. The
volatiles were removed in vacuo and the crude glycosyl amine was
redissolved in DMSO (7.4 mL). Fmoc-aspartic anhydride (1 eq, 0.74 mmol,
249 mg) was added and the reaction was stirred for 75 minutes. The
solvent was removed in vacuo and the crude was subjected to silica gel
column chromatography (0 → 8% MeOH in DCM, Δ = 1%) to yield the title
product (455 mg, 0.47 mmol, 63%). [α]D20 = +0,2 (c 1.00 in MeOH) 1H NMR
(400 MHz, DMSO-d6) δ 8.58 (d, J = 9.1 Hz, 1H, NγH), 7.89 (d, J = 7.7 Hz,
2H, Fmoc-Ar), 7.86 (d, J = 9.5 Hz, 1H, NHC(O)CH3), 7.71 (d, J = 7.5 Hz,
2H, Fmoc-Ar), 7.42 (t, J = 7.3 Hz, 3H, Fmoc-Ar, NαH), 7.33 (t, J = 7.4 Hz,
2H, Fmoc-Ar), 5.23 (d, J = 3.7 Hz, 1H, H4’), 5.16 (dd, J = 10.3, 3.6 Hz, 1H,
H3’), 5.10 (t, J = 9.5 Hz, 1H, H1), 4.97 (t, J = 9.5 Hz, 1H, H3), 4.84 (dd, J
= 10.3, 8.0 Hz, 1H, H2’), 4.70 (d, J = 8.0 Hz, 1H, H1’), 4.36 – 4.15 (m, 6H,
Asn-CH, Fmoc-CH2, H6a, H5’, Fmoc-CH), 4.09 – 3.95 (m, 3H, H6b, H6’),
3.81 (q, J = 9.5 Hz, 1H, H2), 3.73 – 3.55 (m, 2H, H4, H5), 2.63 (dd, J =
16.3, 5.3 Hz, 1H, Asn-CH2), 2.11 (s, 3H, C(O)CH3), 2.07 (s, 3H, C(O)CH3),
2.01 (s, 3H, C(O)CH3), 2.01 (s, 3H, C(O)CH3), 1.94 (s, 3H, C(O)CH3), 1.90
(s, 3H, C(O)CH3), 1.71 (s, 3H, NH(CO)CH3). 13C NMR (101 MHz, DMSO-
d6) δ = 173.1 (C=O), 170.4 (C=O), 170.0 (C=O), 169.9 (C=O), 169.6 (C=O),
169.5 (C=O), 169.3 (C=O), 169.2 (C=O), 155.8 (C=O), 143.8 (Fmoc-Ar),
140.7 (Fmoc-Ar), 127.7 (Fmoc-Ar), 127.1 (Fmoc-Ar), 125.3 (Fmoc-Ar),
120.2 (Fmoc-Ar), 99.9 (C1’), 77.9 (C1’), 76.2 (C4), 73.8 (C3), 73.5 (C5),
70.4 (C3’), 69.7 (C5’), 68.9 (C2’), 67.1 (C4’), 65.7 (Fmoc-CH2), 62.5 (C6),
60.9 (C6’), 52.3 (C2), 50.3 (Asn-CH), 46.6 (Fmoc-CH), 37.1 (Asn-CH2),
22.7 (NHC(O)CH3), 20.7 (C(O)CH3), 20.6 (C(O)CH3), 20.5 (C(O)CH3),
20.4 (C(O)CH3), 20.4 (C(O)CH3).HRMS (ESI) m/z: [M + H+] calcd for
C45H53N3O21H 972.32443, found 972.32357.
7
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10.1002/chem.202004076
Chemistry - A European Journal
FULL PAPER
Nγ-{2,3,4,6-tetra-O-acetyl-β-d-galactopyranoside-(1→4)-[3,4-di-O-
benzoyl-2-O-(4-methoxybenzyl)-α-l-fucopyranoside-(1→3)]-6-O-(4-
methoxybenzyl)-2-deoxy-2-acetamido-β-d-glucopyranosyl}-Nα-
fluorenylmethoxycarbonyl-l-asparagine (4) Glycosyl azide 11 (53 mg,
45 µmol) was dissolved in dry THF (0.45 mL) and cooled to 0°C in an
icebath. 75 µL of a 1 M trimethylphosphine solution in THF was added
dropwise. The reaction was stirred for 5 minutes at 0°C and for 5 minutes
at room temperature. H2O (50 eq, 40 µL, 2.25 mmol) was added and the
reaction was stirred for 2 hours at room temperature. The volatiles were
removed in vacuo and the crude glycosyl amine was redissolved in DMA
(450 µL). The reaction mixture was again cooled in an icebath and aspartic
anhydride[60] (1 eq, 15 mg, 45µmol) was added. The reaction was stirred
and allowed to warm to room temperature overnight. The solvent was
removed by evaporation and the crude glycoaminoacid was subjected to
silicagel column chromatography (0 → 25% acetone in DCM + 0.5% acetic
acid, Δacetone = 5%) to yield the title compound (49 mg, 33 µmol, 73%).
Traces of acetic acid were removed by sequential co-evaporation with
dioxane (3 x 2 mL), toluene (3 x 2 mL) and CHCl3 (3 x 2 mL). [α]D25 = -94.2
(c 1.00 in CHCl3). 1H NMR (400 MHz, CDCl3) δ 7.98 – 7.89 (m, 2H, CHarom),
7.78 – 7.64 (m, 5H, NγH, CHarom), 7.64 – 7.51 (m, 3H, CHarom), 7.51 – 7.40
(m, 3H, CHarom), 7.40 – 7.19 (m, 9H, NHC(O)CH3, CHarom), 7.07 (d, J = 8.6
Hz, 2H, CHarom), 6.91 (d, J = 8.6 Hz, 2H, CHarom), 6.66 (d, J = 8.3 Hz, 2H,
CHarom), 6.41 (d, J = 8.4 Hz, 1H, NαH), 5.63 – 5.54 (m, 2H, H4’, H3’), 5.47
(d, J = 3.4 Hz, 1H, H1’), 5.33 (d, J = 3.7 Hz, 1H, H4’), 5.09 (dd, J = 10.4,
8.0 Hz, 1H, H2’’), 4.99 (t, J = 7.8 Hz, 1H, H1), 4.85 (dd, J = 10.4, 3.6 Hz,
1H, H3’’), 4.81 – 4.70 (m, 1H, H5’), 4.69 – 4.43 (m, 5H, PMB-CH2, Asn-CH,
Fmoc-CH, H1’’), 4.39 – 4.22 (m, 5H, Fmoc-CH2, PMB-CHH, H6’’), 4.22 –
4.03 (m, 4H, PMB-CHH, H2, H2’, H4), 3.95 (t, J = 8.4 Hz, 1H, H3), 3.82 –
3.63 (m, 8H, OCH3, 6H, OCH3), 3.57 – 3.44 (m, 2H, H5, H5’’), 2.90 – 2.72
(m, 2H, Asn-CH2), 2.17 (s, 3H, C(O)CH3), 2.07 – 1.91 (m, 12H, 4 x
C(O)CH3), 1.24 (d, J = 6.5 Hz, 3H, H6’). 13C NMR (101 MHz, CDCl3) δ =
173.5 (C=O), 173.2 (C=O), 171.8 (C=O), 170.5 (C=O), 170.4 (C=O), 170.0
(C=O), 169.8 (C=O), 165.9 (C=O), 165.3 (C=O), 159.6 (Cq), 159.5 (Cq),
156.4 (C=O), 143.9 (Fmoc-Ar), 143.7 (Fmoc-Ar), 141.2 (Fmoc-Ar), 141.2
(Fmoc-Ar), 133.3 (CHarom), 133.1 (CHarom), 130.3 (CHarom), 129.8 (CHarom),
129.7 (CHarom), 129.6 (CHarom), 129.6 (Cq), 129.5 (Cq), 128.9 (Cq), 128.5
(CHarom), 128.3 (CHarom), 127.7 (CHarom), 127.1 (CHarom), 125.3 (CHarom),
125.2 (CHarom), 119.9 (CHarom), 114.1 (CHarom), 114.0 (CHarom), 99.4 (C1’’),
97.4 (C1’), 79.7 (C1), 76.0 (C5, C3) , 73.3 (C4), 73.3 (C2’), 73.3 (PMB-
CH2), 72.7 (PMB-CH2), 72.5 (C4’), 71.0 (C5’’), 70.8 (C3’’), 70.1 (C3’), 69.3
(C2’’), 67.8 (C6), 67.2 (Fmoc-CH2), 66.9 (C4’’), 65.8 (C5’), 61.0 (C6’’), 55.3
(OCH3), 55.2 (OCH3), 53.6 (C2), 50.5 (Asn-CH), 47.1 (Fmoc-CH), 37.9
(Asn-CH2), 22.8 (NHC(O)CH3), 20.8 (C(O)CH3), 20.8 (C(O)CH3), 20.7
(C(O)CH3), 20.6 (C(O)CH3), 16.1 (C6’). HRMS (ESI) m/z: [M + Na+] calcd
for C77H83N3O27Na 1504.51061, found 1504.51004.
CHH), 4.51 – 4.42 (m, 2H, H3, H5’), 4.38 (dd, J = 10.5, 4.2 Hz, 1H, H5),
4.13 (dd, J = 10.5, 3.4 Hz, 1H, H2’), 3.84 – 3.70 (m, 7H, PMB-OCH3,PMP-
OCH3, H6a), 3.70 – 3.57 (m, 2H, H6b, H4), 3.25 (td, J = 9.3, 6.9 Hz, 1H,
H2), 1.81 (s, 3H, NHC(O)CH3), 0.73 (d, J = 6.5 Hz, 3H, H6’). 13C NMR (101
MHz, CDCl3) δ 171.4 (C=O), 166.0 (C=O), 165.7 (C=O), 160.4 (Cq), 159.6
(Cq), 133.4 (CHarom), 133.2 (CHarom), 129.8 (CHarom), 129.7 (CHarom), 129.7
(CHarom), 129.6 (Cq), 129.6 (Cq), 128.6 (CHarom), 128.4 (CHarom), 127.8
(CHarom), 114.1 (CHarom), 113.7 (CHarom), 102.1 (PMP-CH), 98.6 (C1’), 87.9
(C1), 80.7 (C4), 75.5 (C3), 74.2 (C2’), 73.4 (PMB-CH2), 72.6 (C4’), 70.9
(C3’), 68.6 (C6), 68.5 (C5), 65.6 (C5’), 58.4 (C2), 55.4 (OCH3), 55.3
(OCH3), 23.4 (NHC(O)CH3), 15.6 (C6’). HRMS (ESI) m/z: [M + H+] calcd
for C44H46N4O13H 839.31341, found 839.31311.
Azido 3,4-di-O-benzoyl-2-O-(4-methoxybenzyl)-α-l-fucopyranoside-
(1→3)-6-O-(4-methoxybenzyl)-2-deoxy-2-(N-acetylacetamido)-β-d-
glucopyranoside (8) Dissacharide 7 (436 mg, 0.52 mmol) was dissolved
in anhydrous DCM and DiPEA (10 eq., 870 µL, 5 mmol) and acetyl chloride
(50 eq., 1.8 mL, 25 mmol) were added. The reaction was stirred for 2 hours
at room temperature, after which TLC (10% EtOAc in DCM) indicated full
conversion. The reaction mixture was diluted with DCM and the organic
layer was washed with saturated aqueous NaHCO3. The organic layer was
dried over MgSO4, filtered and concentrated. Silica gel column
chromatography (30% → 40% → 50% Et2O in pentane) yielded the
25
diacetylated intermediate (406 mg, 0.46 mmol, 89%). [α]D = -105.2 (c
0.50 in CHCl3). νmax/cm-1 2119.23 (N3), 1727.15 (CO) 1H NMR (400 MHz,
CDCl3) δ 7.94 – 7.87 (m, 2H, CHarom), 7.78 – 7.71 (m, 2H, CHarom), 7.63 –
7.55 (m, 1H, CHarom), 7.52 – 7.39 (m, 5H, CHarom), 7.32 – 7.26 (m, 2H,
CHarom), 7.12 – 7.04 (m, 2H, CHarom), 6.91 – 6.83 (m, 2H, CHarom), 6.68 (d,
J = 8.7 Hz, 2H, CHarom), 5.75 – 5.66 (m, 2H, H1, H3’), 5.51 (s, 1H, PMP-
CHacetal), 5.42 (dd, J = 3.3, 1.4 Hz, 1H, H4’), 4.79 (dd, J = 9.6, 8.6 Hz, 1H,
H3), 4.74 (d, J = 3.5 Hz, 1H, H1’), 4.52 – 4.37 (m, 4H, PMB-CH2, H5’, H5),
4.06 (dd, J = 10.6, 3.5 Hz, 1H, H2’), 3.85 – 3.70 (m, 8H, PMB-OCH3, PMP-
OCH3, H6), 3.70 – 3.61 (m, 2H, H4, H2), 2.50 (s, 3H, N(C(O)CH3)C(O)CH3),
2.30 (s, 3H, N(C(O)CH3)C(O)CH3), 0.52 (d, J = 6.4 Hz, 3H, H6’). 13C NMR
(101 MHz, CDCl3) δ 175.2 (C=O), 174.6 (C=O), 165.9 (C=O), 165.8 (C=O),
160.5 (Cq), 159.5 (Cq), 133.3 (CHarom), 133.2 (CHarom), 130.5 (CHarom),
129.8 (CHarom), 129.7 (CHarom), 129.4 (Cq), 129.2 (Cq), 128.5 (CHarom),
128.4 (CHarom), 128.0 (CHarom), 113.8 (CHarom), 113.7 (CHarom), 102.5
(PMP-CH), 98.8 (C1’), 87.5 (C1), 80.9 (C4), 73.6 (C3), 73.4 (PMB-CH2),
72.6 (C4’), 71.8 (C2’), 71.3 (C3’), 68.6 (C6), 68.0 (C5), 65.4 (C5’), 64.1
(C2), 55.4 (OCH3), 55.2 (OCH3), 28.6 (N(C(O)CH3)C(O)CH3), 25.6
(N(C(O)CH3)C(O)CH3), 15.2 (C6’). HRMS (ESI) m/z: [M + Na+] calcd for
C46H48N4O14Na
903.30592,
found
903.30478.
The
4-
methoxybenzylidene protected disaccharide (461 mg, 0.52 mmol) was
dissolved in dry THF and cooled to -70°C. BH3·THF was added as a 1.0
M solution in THF (5 eq, 2.6 mmol, 2.6 mL) and the reaction was stirred
for 15 minutes at this temperature. Then Bn2BOTf was added as a 1.0 M
solution in DCM (2 eq, 1 mmol, 1 mL) and the reaction was stirred for an
additional 15 minutes at -70°C. The reaction was then heated to -50°C and
stirred overnight. The reaction was quenched by careful addition of 0.5 mL
of Et3N followed by 15 mL MeOH and was stirred at room temperature for
30 minutes. The reaction mixture was concentrated in vacuo and subjected
to silica gel column chromatography (40% → 50% →60 % Et2O in pentane).
This yielded the title compound (370 mg, 0.42 mmol, 81%). [α]D25 = -93.2
(c 1.00 in CHCl3). νmax/cm-1 2117.80 (N3), 1724.29 (CO). 1H NMR (400
MHz, CDCl3) δ 7.95 – 7.88 (m, 2H, CHarom), 7.80 – 7.73 (m, 2H, CHarom),
7.67 – 7.59 (m, 1H, CHarom), 7.54 – 7.42 (m, 3H, CHarom), 7.35 – 7.26 (m,
4H, CHarom), 7.11 – 7.04 (m, 2H, CHarom), 6.90 (d, J = 8.6 Hz, 2H, CHarom),
6.74 (d, J = 8.6 Hz, 2H, CHarom), 5.67 – 5.59 (m, 3H, H1, H3’, H4’), 4.93 (d,
J = 3.6 Hz, 1H, H1’), 4.66 – 4.39 (m, 6H, PMB-CH2, PMB-CH2, H3, H5’),
4.10 (dd, J = 10.3, 3.6 Hz, 1H, H2’), 4.01 (s, 1H, 4-OH), 3.85 – 3.79 (m,
4H, H6a, OCH3), 3.78 – 3.72 (m, 4H, H6b, OCH3), 3.71 – 3.62 (m, 3H, H2,
H4, H5), 2.41 (s, 3H, N(C(O)CH3)C(O)CH3), 2.37 (s, 3H,
N(C(O)CH3)C(O)CH3)), 1.21 (d, J = 6.5 Hz, 3H, H6’). 13C NMR (101 MHz,
CDCl3) δ 175.4 (C=O), 174.3 (C=O), 165.8 (C=O), 165.4 (C=O), 159.5 (Cq),
159.4 (Cq), 133.5 (CHarom), 133.3 (CHarom), 130.0 (CHarom), 129.9 (CHarom),
129.7 (CHarom), 129.5 (CHarom), 129.2 (Cq), 128.6 (CHarom), 128.4 (CHarom),
113.9 (CHarom), 99.7 (C1’), 86.8 (C1), 82.6 (C3), 76.7 (C4), 73.4 (PMB-
CH2), 72.7 (PMB-CH2), 72.1 (C4’), 71.4 (C5), 71.3 (C2’), 70.3 (C3’), 68.7
Azido 3,4-di-O-benzoyl-2-O-(4-methoxybenzyl)-α-l-fucopyranoside-
(1→3)-4,6-O-(4-methoxybenzylidene)-2-deoxy-2-acetamido-β-d-
glucopyranoside (7) Donor 6 (1.5 eq., 1.76 mg, 3.0 mmol) and acceptor
5 (728 mg, 2.0 mmol) were co-evaporated 3 times with toluene, backfilling
the flask with N2 after every co-evaporation round, and placed under a N2
atmosphere. The sugars were dissolved in dry DCM (36 mL) with dry DMF
(4 mL). Activated 4Å molecular sieves (1 g) were added and the solution
was stirred for 90 minutes. The reaction mixture was then cooled in an
icebath and NIS (2.0 eq., 900 mg, 4.0 mmol) and TMSOTf (0.1 eq., 37 µL)
were added. The reaction was stirred and allowed to warm to room
temperature overnight. The reaction was filtered, diluted with DCM and
washed with a 1:1 mixture of 10% Na2S2O3 (aq) and saturated NaHCO3
(aq). The organic layer was dried over MgSO4, filtered and concentrated.
Silica gel column chromatography (30% → 40% → 50% → 60% EtOAc in
pentane) yielded the title compound (1.19 g, 1.42 mmol, 71%). [α]D25 = -
1
144.0 (c 1.00 in CHCl3). νmax/cm-1 2117.80 (N3), 1724.29 (CO) H NMR
(400 MHz, CDCl3) δ 8.00 – 7.89 (m, 2H, CHarom), 7.82 – 7.75 (m, 2H,
CHarom), 7.64 – 7.56 (m, 1H, CHarom), 7.54 – 7.40 (m, 5H, CHarom), 7.33 –
7.27 (m, 2H, CHarom), 7.15 – 7.08 (m, 2H, CHarom), 6.88 (d, J = 8.8 Hz, 2H,
CHarom), 6.74 (d, J = 8.6 Hz, 2H, CHarom), 6.05 (d, J = 6.9 Hz, 1H, NH), 5.73
(dd, J = 10.5, 3.3 Hz, 1H, H3’), 5.53 (s, 1H, PMP-CHacetal), 5.50 (dd, J =
3.4, 1.4 Hz, 1H, H4’), 5.28 (d, J = 9.3 Hz, 1H, H1), 5.15 (d, J = 3.5 Hz, 1H,
H1’), 4.61 (d, J = 11.4 Hz, 1H, PMB-CHH), 4.54 (d, J = 11.4 Hz, 1H, PMB-
8
This article is protected by copyright. All rights reserved.

10.1002/chem.202004076
Chemistry - A European Journal
FULL PAPER
(C6), 66.7 (C5’), 62.3 (C2), 55.4 (OCH3), 55.3 (OCH3), 28.4
(N(C(O)CH3)C(O)CH3), 25.6 (N(C(O)CH3)C(O)CH3), 16.2 (C6’). HRMS
(ESI) m/z: [M + NH4+] calcd for C46H50N4O14NH4 900.36618, found
900.36581.
8.3 Hz, 1H, H4), 3.85 – 3.78 (m, 5H, OCH3, H6), 3.75 (s, 3H, OCH3), 3.64
– 3.55 (m, 2H, H5’’, H5), 3.33 (q, J = 8.1 Hz, 1H, H2), 2.21 (s, 3H, C(O)CH3),
2.07 (s, 3H, C(O)CH3), 2.02 (s, 3H, C(O)CH3), 1.98 (s, 3H, C(O)CH3), 1.89
(s, 3H, NHC(O)CH3), 1.25 (d, J = 6.6 Hz, 3H, H6’). 13C NMR (101 MHz,
CDCl3) δ = 171.0 (C=O), 170.6 (C=O), 170.5 (C=O), 170.2 (C=O), 169.4
(C=O), 166.1 (C=O), 165.3 (C=O), 159.6 (Cq), 159.6 (Cq), 133.3 (CHarom),
133.0 (CHarom), 129.9 (CHarom), 129.8 (Cq), 129.8 (CHarom), 129.7 (CHarom),
129.7 (CHarom), 128.6 (CHarom), 128.3 (CHarom), 114.1 (CHarom), 114.0
(CHarom), 99.6 (C1’’), 97.3 (C1’), 87.2 (C1), 76.7 (C5), 73.6 (C2’, C4), 73.5
(C3), 73.4 (PMB-CH2), 73.1 (PMB-CH2), 72.8 (C4’), 71.1 (C5’’), 71.0 (C3’’),
71.0 (C3’), 69.2 (C2’’), 67.4 (C6), 67.0 (C4’’), 65.2 (C5’), 61.1 (C6’’), 57.0
(C2), 55.4 (OCH3), 55.3 (OCH3), 23.5 (NHC(O)CH3), 20.9 (C(O)CH3), 20.9
(C(O)CH3), 20.7 (C(O)CH3), 16.1 (C6’). HRMS (ESI) m/z: [M + Na+] calcd
for C58H66N4O22Na 1193.40609, found 1193.40573.
Azido 2,3,4,6-tetra-O-acetyl-β-d -galactopyranoside(1→4)-[3,4-di-O-
benzoyl-2-O-(4-methoxybenzyl)-α-l-fucopyranoside-(1→3)]-6-O-(4-
methoxybenzyl)-2-deoxy-2-(N-acetylacetamido)-β-d
-
glucopyranoside (10) Donor 9 (5 eq., 737 mg, 1.5 mmol) and acceptor 8
(266 mg, 0,3 mmol) were co-evaporated 3 times with toluene, backfilling
the flask with N2 after every co-evaporation round, and placed under a N2
atmosphere. The sugars were dissolved in dry DCM and activated 4Å
molecular sieves (300 mg) were added. The mixture was stirred 30
minutes at room temperature and subsequently cooled to -10°C. TMS
triflate (0.1 eq, 5.6 µl, 0.03 mmol) was added and the reaction was stirred
over night at -10°C. The reaction was quenched by addition of TEA (0.1
mL) and allowed to warm to room temperature. The reaction mixture was
diluted with DCM, filtered, further diluted with toluene and concentrated in
vacuo. Silica gel column chromatography (40 → 70% Et2O in pentane,
Azido
2,3,4,6-tetra-O-acetyl-β-d-galactopyranoside-(1→4)-6-(t-
butyldimethylsilyl)-2-deoxy-2-acetamido-β-d -glucopyranoside (13)
Donor 9 (1.5 eq, 368 mg, 0.75 mmol) and acceptor 12 (180 mg, 0.5 mmol)
were co-evaporated 3 times with toluene and put under N2. The sugars
were dissolved in dry DCM (5 mL) and stirred with activated 4 Å molecular
sieves (0.5 g) for 2 hours at room temperature. The reaction was cooled
to -40°C and BF3·Et2O (1.6 eq, 100 µL, 0.8 mmol) was added. The reaction
was stirred at -40°C overnight and formation of disaccharide product was
confirmed by TLC (70% EtOAc in pentane). The reaction was quenched
with Et3N (0.5 mL), diluted with DCM, filtered, diluted with toluene and
concentrated. Silica gel column chromatography (60% → 70% → 80%
EtOAc in pentane) yielded the title compound (193 mg, 0.28 mmol, 56%).
[α]D20 = +5,8 (c 1.00 in CHCl3) νmax/cm-1 2115.65 (N3), 1752.19 (CO). 1H
NMR (400 MHz, CDCl3) δ 6.17 (d, J = 8.5 Hz, 1H, NH), 5.40 (dd, J = 3.4,
1.0 Hz, 1H, H4’), 5.22 (dd, J = 10.5, 8.0 Hz, 1H, H2’), 4.99 (dd, J = 10.5,
3.4 Hz, 1H, H3’), 4.69 – 4.61 (m, 2H, H1, H1’), 4.15 (d, J = 6.5 Hz, 2H, H6’),
4.06 (bs, 1H, 3-OH), 4.01 (t, J = 6.5 Hz, 1H, H5’), 3.90 – 3.72 (m, 3H, H6,
H3), 3.69 – 3.57 (m, 2H, H4, H2), 3.43 (ddd, J = 9.6, 3.4, 1.5 Hz, 1H, H5),
2.17 (s, 3H, C(O)CH3), 2.08 (s, 3H, C(O)CH3), 2.07 (s, 3H, C(O)CH3), 2.03
(s, 3H, NHC(O)CH3), 1.99 (s, 3H, C(O)CH3), 0.92 (s, 9H, tBu), 0.11 (s, 3H,
SiCH3), 0.10 (s, 3H, SiCH3). 13C NMR (101 MHz, CDCl3) δ = 171.0 (C=O),
170.6 (C=O), 170.2 (C=O), 170.1 (C=O), 169.4 (C=O), 101.6 (C1’), 87.9
(C1), 80.5 (C4), 76.7 (C5), 71.9 (C3), 71.4 (C5’), 70.9 (C3’), 68.7 (C2’),
66.8 (C4’), 61.4 (C6’), 61.2 (C6), 55.6 (C2), 25.9 (tBu), 23.4 (NHC(O)CH3),
20.7 (C(O)CH3), 20.6 (C(O)CH3), 20.6 (C(O)CH3), 20.6 (C(O)CH3), 18.3
(Si-C), -5.0 (Si-CH3), -5.2 (Si-CH3). HRMS (ESI) m/z: [M + Na+] calcd for
C28H46N4O14SiNa 713.2672, found 713.2695.
25
Δ=5%) yielded the title compound (283 mg, 0.23 mmol, 77%). [α]D = -
104.4 (c 1.00 in CHCl3). 1H NMR (400 MHz, CDCl3) δ 7.99 – 7.92 (m, 2H,
CHarom), 7.78 – 7.71 (m, 2H, CHarom), 7.65 – 7.58 (m, 1H, CHarom), 7.50 –
7.42 (m, 3H, CHarom), 7.36 – 7.30 (m, 2H, CHarom), 7.26 (dd, J = 8.3, 7.4
Hz, 3H, CHarom), 7.13 (d, J = 8.6 Hz, 2H, CHarom), 6.97 (d, J = 8.7 Hz, 2H,
CHarom), 6.70 (d, J = 8.6 Hz, 2H, CHarom), 5.66 (dd, J = 3.3, 1.4 Hz, 1H,
H4’), 5.64 – 5.54 (m, 2H, H3’, H1), 5.38 (dd, J = 3.6, 1.0 Hz, 1H, H4’’), 5.14
(q, J = 6.5 Hz, 1H, H5’), 5.04 (dd, J = 10.3, 8.3 Hz, 1H, H2’’), 4.86 – 4.67
(m, 5H, H3’’, PMB-CHH, H1’, H1’’, H3), 4.60 (dd, J = 11.5, 6.1 Hz, 1H,
H6’’a), 4.50 (s, 2H, PMB-CH2), 4.46 – 4.38 (m, 2H, PMB-CHH, H6’’b), 4.11
(dd, J = 10.6, 3.7 Hz, 1H, H2’), 4.05 (dd, J = 10.0, 8.9 Hz, 1H, H4), 3.88 –
3.68 (m, 8H, OCH3, H6, OCH3), 3.59 (t, J = 9.4 Hz, 1H, H2), 3.56 – 3.49
(m, 2H, H5, H5’’), 2.51 (s, 3H, N(C(O)CH3)C(O)CH3), 2.25 (s, 3H,
N(C(O)CH3)C(O)CH3), 2.24 (s, 3H, C(O)CH3), 2.10 (s, 3H, C(O)CH3), 2.00
(s, 3H, C(O)CH3), 1.98 (s, 3H, C(O)CH3), 1.24 (d, J = 6.6 Hz, 3H, H6’). 13
C
NMR (101 MHz, CDCl3) δ = 175.5 (C=O), 174.8 (C=O), 170.9 (C=O), 170.5
(C=O), 170.2 (C=O), 168.9 (C=O), 166.1 (C=O), 165.4 (C=O), 159.8 (Cq),
159.5 (Cq), 133.3 (CHarom), 133.0 (CHarom), 130.8 (CHarom), 130.0 (Cq),
130.0 (CHarom), 129.9 (CHarom), 129.8 (Cq), 129.6 (CHarom), 129.4 (Cq),
128.5 (CHarom), 128.3 (CHarom), 114.3 (CHarom), 113.7 (CHarom), 99.7 (C1’’),
97.8 (C1’), 86.9 (C1), 76.6 (C5’’), 74.3 (C4), 73.7 (PMB-CH2), 73.5 (PMB-
CH2), 72.9 (C4’), 71.8 (C3’, C3, C2’), 71.3 (C3’’), 71.1 (C5), 69.2 (C2’’),
67.0 (C4’’), 66.9 (C6), 64.9 (C5’), 64.3 (C2), 61.1 (C6’’), 55.4 (OCH3), 55.3
(OCH3), 28.8 (N(C(O)CH3)C(O)CH3), 25.8 (N(C(O)CH3)C(O)CH3), 21.0
(C(O)CH3), 20.9 (C(O)CH3), 20.8 (C(O)CH3), 20.7 (C(O)CH3), 16.0 (C6’).
HRMS (ESI) m/z: [M + Na+] calcd for C60H68N4O23Na 1235.41666, found
1235.41654.
Azido 2,3,4,6-tetra-O-acetyl-β-d-galactopyranoside-(1→4)-6,3-di-O-
acetyl-2-deoxy-2-acetamido-β-d-glucopyranoside (14) Silyl protected
disaccharide 13 (517 mg, 0.75 mmol) was dissolved in dry THF (7.5 mL)
in a plastic tube. HF·pyridine complex (16 eq, 310 µL, 12 mmol) was added
and the reaction was stirred overnight. Completion of the reaction was
assessed by TLC (100% EtOAc) and the reaction mixture was diluted with
DCM. The organic layer was washed with aqueous saturated NaHCO3 (3:1
ratio of DCM:H2O) and the aqueous layer was back extracted with DCM.
The combined organic layers were dried over MgSO4, filtered and
concentrated, yielding 380 mg (0.66 mmol) of crude intermediate. The
crude desilylated disaccharide was dissolved in dry pyridine (6.6 mL) and
cooled to 0°C in an ice bath. Acetic anhydride (10 eq, 620 µL, 6.6 mmol)
and DMAP (0.1 eq, 9 mg, 0.07 mmol) were added. The reaction was stirred
overnight at room temperature and reaction completion was confirmed by
TLC (100% EtOAc). The reaction was quenched with methanol and
concentrated. Pyridine traces were removed with toluene co-evaporation.
Silica gel column chromatography (70% → 80% → 90% EtOAc in pentane)
Azido 2,3,4,6-tetra-O-acetyl-β-d-galactopyranoside-(1→4)-[3,4-di-O-
benzoyl-2-O-(4-methoxybenzyl)-α-l-fucopyranoside-(1→3)]-6-O-(4-
methoxybenzyl)-2-deoxy-2-acetamido-β-d-glucopyranoside
(11)
Protected trisaccharide 10 (61 mg, 50 µmol) was dissolved in dry THF (1
mL) and N,N-dimethylaminopropylamine (10 eq, 63 µL, 0.5 mmol) was
added. The reaction was stirred for 30 minutes at room temperature and
another portion of N,N-dimethylaminopropylamine (10 eq, 63 µL, 0.5
mmol) was added. After further stirring for 1 hour, TLC (15% EtOAc in
DCM) indicated full conversion. The reaction mixture was diluted with DCM
and washed with 1 M HCl (aq). The organic layer was dried over MgSO4,
filtered and concentrated in vacuo. Silica gel column chromatography (0%
→ 10% → 15% → 20% EtOAc in DCM) yielded the title compound (51 mg,
42 µmol, 87%). [α]D25 = -76.0 (c 1.00 in CHCl3). 1H NMR (400 MHz, CDCl3)
δ 8.02 – 7.94 (m, 2H, CHarom), 7.79 – 7.73 (m, 2H, CHarom), 7.65 – 7.58 (m,
1H, CHarom), 7.51 – 7.44 (m, 3H, CHarom), 7.33 – 7.28 (m, 3H, CHarom), 7.17
(d, J = 8.6 Hz, 2H, , CHarom), 6.95 (d, J = 8.6 Hz, 2H, CHarom), 6.76 (d, J =
8.7 Hz, 2H, CHarom), 6.03 (d, J = 7.5 Hz, 1H, NH), 5.68 – 5.61 (m, 2H, H4’,
H3’), 5.38 (dd, J = 3.6, 1.1 Hz, 1H, H4’’), 5.26 (d, J = 8.2 Hz, 1H, H1), 5.21
(d, J = 3.6 Hz, 1H, H1’), 5.08 (dd, J = 10.4, 8.1 Hz, 1H, H2’’), 4.99 – 4.86
(m, 2H, H5’, H3’’), 4.73 – 4.67 (m, 2H, PMB-CHH, H1’’), 4.64 (d, J = 11.6
Hz, 1H, PMB-CHH), 4.57 (d, J = 11.7 Hz, 1H, PMB-CHH), 4.46 – 4.31 (m,
4H, PMB-CHH, H6’’, H3), 4.18 (dd, J = 9.7, 3.5 Hz, 1H, H1’), 4.06 (t, J =
20
yielded the title compound (421 mg, 0.64 mmol, 85%). [α]D = -26,4 (c
1.00 in CHCl3) νmax/cm-1 2116.37 (N3), 1744.32 (CO). 1H NMR (400 MHz,
CDCl3) δ 6.53 (d, J = 9.6 Hz, 1H, NH), 5.37 (dd, J = 3.4, 1.2 Hz, 1H, H4’),
5.19 – 5.03 (m, 2H, H3, H2’), 4.99 (dd, J = 10.5, 3.4 Hz, 1H, H3’), 4.64 –
4.50 (m, 3H, H1, H1’, H6a), 4.21 – 4.01 (m, 4H, H6’, H6b, H2), 3.93 (t, J =
7.1 Hz, 1H, H5’), 3.84 (t, J = 9.1 Hz, 1H, H4), 3.73 (ddd, J = 9.1, 5.0, 2.2
Hz, 1H, H5), 2.17 (s, 3H, C(O)CH3), 2.14 (s, 3H, C(O)CH3), 2.11 (s, 3H,
C(O)CH3), 2.07 (s, 3H, C(O)CH3), 2.07 (s, 3H, C(O)CH3), 1.99 (s, 3H,
NHC(O)CH3), 1.97 (s, 3H, C(O)CH3). 13C NMR (101 MHz, CDCl3) δ =
9
This article is protected by copyright. All rights reserved.

10.1002/chem.202004076
Chemistry - A European Journal
FULL PAPER
171.0 (C=O), 170.5 (C=O), 170.4 (C=O), 170.3 (C=O), 170.1 (C=O), 170.0
(C=O), 169.3 (C=O), 101.3 (C1’), 88.3 (C1), 76.1 (C4), 74.5 (C5), 73.1 (C3),
70.8 (C3’), 70.7 (C5’), 69.0 (C2’), 66.6 (C4’), 61.9 (C6), 60.6 (C6’), 53.0
(C2), 23.0 (NHC(O)CH3), 20.9 (C(O)CH3), 20.8 (C(O)CH3), 20.6
(C(O)CH3), 20.6 (C(O)CH3), 20.5 (C(O)CH3), 20.5 (C(O)CH3). HRMS (ESI)
m/z: [M + Na+] calcd for C26H36N4O16Na 683.2019, found 683.2029.
[18]
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Acknowledgements
W.D. was financially supported by a NWO BBoL grant. M.H.S.M.
was supported by a Lundbeck Foundation Postdoc Abroad grant.
F.C. was financially supported by the Amsterdam Infection and
Immunity Institute Fellowship and by the NWO Spinoza award of
Y.K. C.A. would like to thank NWO for financial support in the form
of an ECHO grant. Furthermore, the authors would like to thank
A. Kros for granting us the access to the JASCO J-815 CD
spectrometer and F. van der Heijden for assistance with
compound characterization.
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Entry for the Table of Contents
A novel asparagine SPPS building block harboring a Lewis X type DC-SIGN ligand was synthesized and incorporated into the
immunodominant portion of Myelin Oligodendrocyte Glycoprotein (MOG31-55). This glycopeptide was evaluated for its ability to inhibit
citrullination induced aggregation of MOG35-55, to bind to DC-SIGN in vitro and for its immunomodulatory effects on human dendritic
cells, with the Lewis X yielding a tolerogenic response.
Institute and/or researcher Twitter usernames: @svankasteren, @araman_can
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