Heteromultivalent Glycooligomers as Mimetics of Blood Group Antigens

Article abstract of DOI:10.1002/chem.201804505

Precision glycomacromolecules have proven to be important tools for the investigation of multivalent carbohydrate–lectin interactions by presenting multiple glycan epitopes on a highly-defined synthetic scaffold. Herein, we present a new strategy for the versatile assembly of heteromultivalent glycomacromolecules that contain different carbohydrate motifs in proximity within the side chains. A new building block suitable for the solid-phase polymer synthesis of precision glycomacromolecules was developed with a branching point in the side chain that bears a free alkyne and a TIPS-protected alkyne moiety, which enables the subsequent attachment of different carbohydrate motifs by on-resin copper-mediated azide–alkyne cycloaddition reactions. Applying this synthetic strategy, heteromultivalent glycooligomers presenting fragments of histo-blood group antigens and human milk oligosaccharides were synthesized and tested for their binding behavior towards bacterial lectin LecB.

Full text of DOI:10.1002/chem.201804505

A Journal of
Accepted Article
Title: Heteromultivalent glycooligomers as mimetics of blood group
antigens
Authors: Katharina S. Bücher, Patrick B. Konietzny, Nicole L. Snyder,
and Laura Hartmann
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: Chem. Eur. J. 10.1002/chem.201804505
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Heteromultivalent glycooligomers as mimetics of blood group
antigens
Katharina Susanne Bücher[a], Patrick Benjamin Konietzny[a], Nicole L. Snyder[b], Laura Hartmann*[a]
Abstract: Precision glycomacromolecules have proven to be
important tools for the investigation of multivalent carbohydrate-lectin
interactions by presenting multiple glycan epitopes on a highly-
defined synthetic scaffold. Here we present a new strategy for the
versatile assembly of heteromultivalent glycomacromolecules
presenting different carbohydrate motifs in close proximity within the
side chains. A new building block suitable for the solid phase polymer
synthesis (SPPoS) of precision glycomacromolecules was developed
exhibiting a branching point in the side chain that bears a free alkyne
and a TIPS-protected alkyne moiety enabling the subsequent
attachment of different carbohydrate motifs by on-resin copper
mediated azide alkyne cycloaddition reactions (CuAAC). Applying this
synthetic strategy, heteromultivalent glycooligomers presenting
fragments of histo-blood group antigens (HBGA) and human milk
oligosaccharides (HMO) were synthesized and tested for their binding
behavior towards bacterial lectin LecB.
suitable model compounds to study the role of HBGAs, which in
turn may be used to develop new inhibitors e.g. in antibacterial or
antiviral treatment.[9]
In principle, glycomimetic structures of HBGAs are built from a
specific epitope fragment of the HBGA, in the simplest case a
fucose unit is presented in a multivalent fashion on a synthetic
scaffold.[10] There are numerous reports of glycosylated
macromolecular scaffolds and their binding interactions with
pathogen-related lectins such as LecB from Pseudomonas
aeruginosa.[11,12] However, the question remains as to how the
affinity and selectivity of HBGAs mimetics might be altered by
including not only one but different glycan fragments imitating
more closely the natural ligand’s heterofunctional structure.[13,14,15]
Indeed, it has been shown that the combination of different
carbohydrate moieties in heterofunctional glycoconjugates and
glycomimetic structures strongly affects their recognition
process.[16] Therefore, here we introduce a new strategy towards
obtaining glycomimetic ligands using an oligo(amidoamine)
scaffold with sequence-controlled divalent heterofunctional
glycan side chains based on different fragments of HBGA and
human milk oligosaccharides HMO ligands.
Introduction
Glycoconjugates are ubiquitous in nature and are important
components of the extracellular matrix and glycocalyx, a dense
layer of carbohydrate-based molecules on the cell surface. Their
specific interactions with carbohydrate recognition receptors such
as lectins, play important roles in many biochemical processes,[1]
including cell-cell communication, immune response, fertilization,
cell migration,[2,3] or cancer metastasis.[3,4] Furthermore they are
known to mediate interactions with pathogens[5,6] such as viruses
and bacteria that engage specific carbohydrates within the
glycocalyx to attach, enter and infect these cells. In particular,
histo-blood group antigens (HBGAs) including AB0 blood groups
and Lewis antigens, represent important target structures for
many pathogenic lectins.[6,7] While there are still many open
questions concerning the biological function and role of HBGAs
e.g. their specificity towards selected pathogens, it has already
been shown that glycomimetic structures[8] can be used as
Previously we reported the synthesis of sequence-controlled
glycooligo(amidoamines),
so-called
precision
glycomacromolecules, and their use as multivalent glycomimetic
ligands.[17] In short, tailor-made building blocks are assembled in
a stepwise fashion on solid support introducing functional
moieties in the side chains at defined positions within the scaffold
that provide for the attachment of different carbohydrate ligands.
Through the straightforward exchange of building blocks during
synthesis, we have been able to access
a library of
glycomacromolecules varying for example the number and
position of carbohydrates, the overall length or architecture of the
scaffold, as well as different linker or main chain motifs.[18,19,20]
Furthermore, we have developed different methods to obtain
heteromultivalent glycomacromolecules e.g. by using orthogonal
coupling strategies or sequential introduction of ligands during
scaffold assembly.[21] CuAAC has proven to be an important tool
for the introduction of carbohydrate ligands both for homo- and
heteromultivalent glycomacromolecules yielding highly efficient
coupling directly on solid support. The required alkyne and azido
functionalities can be either placed on the building block, e.g. TDS
(triple bond diethylenetriamine succinyl)[17] or BADS
(p-(azidomethyl)benzoyl diethylene-triamine succinyl)[19], or on
the carbohydrate ligand. In this study we extend this approach by
introducing a new building block carrying two alkyne groups (one
free and one protected) that allows for the controlled introduction
of different carbohydrates using CuAAC. After coupling of a first
carbohydrate ligand using the free alkyne, the second, protected
alkyne moiety is deprotected and can be conjugated with a
[a]
[b]
K. S. Bücher, P. B. Konietzny, Prof. Dr. L. Hartmann
Institute of Organic and Macromolecular Chemistry
Heinrich Heine University Düsseldorf
Düsseldorf, Germany
E-mail: laura.hartmann@hhu.de
Prof. Dr. N. L. Snyder
Department of Chemistry
Davidson College
Davidson, NC, USA
Supporting information for this article is given via a link at the end of
the document
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second carbohydrate ligand using the same reaction conditions
(Scheme 1).
Scheme 1. Introduction of new building block during SPPoS provides for the asymmetrical conjugation of ligands by first coupling a carbohydrate ligand to the free
alkyne using CuAAC (e.g. fucose in red), followed by deprotection of the second alkyne moiety and coupling of a second carbohydrate ligand (e.g. GalNAc in blue).
protected primary amine groups (Scheme 2) that allows for
introduction of functional side chains at the central secondary
Results and Discussion
amine
position.
For
iso-DTDS, the functional side chain exhibits a rigid phenylene-
linker as a branching point with two acetylene units, one of which
is protected with a TIPS (triisopropylsilyl) group (Scheme 1). TIPS
was selected because it is a well-established alkyne protecting
group that can be selectively cleaved on solid support[22] to
provide for subsequent CuAAC-based functionalization in the
presence of another alkyne, in this case with azido functionalized
carbohydrates.
Building Block Synthesis – isoDTDS (11)
In order to mimic the heteromultivalent presentation of
neighboring monosaccharide motifs in branched oligosaccharide
structures more closely, a new building block, iso-DTDS (iso-Di-
Triple-bond Diethylenetriamine Succinic acid) (11) was developed.
iso-DTDS is based on
a
previously established key
intermediate,[17,20]
a
diethylenetriamine with asymmetrically
Scheme 2. Synthetic route for new building block iso-DTDS 11, combining precursor 5[23] and key intermediate 6[17,20]
.
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Figure 1. 1H NMR spectra of iso-DTDS: (A) excerpt of spectrum in DMSO-d6; (B) full spectrum in methanol-d4.
Scheme 2 depicts the synthesis of iso-DTDS. First, compound 5
was synthesized according to literature protocols[23] starting from
asymmetrically meta-halogenized benzoic acid 1. After methyl
protection of the acid group (2), thermo-selective double
Sonogashira reactions were conducted using a Pd[P(Ph3)]4/CuI
catalytic system, first substituting TIPS-acetylene at C3 on the
benzene ring (3) followed by trimethylsilyl(TMS)-acetylene
substitution at the C5 position. Compound 4 was then treated with
KOH in THF to remove the TMS and the methyl-protecting groups
simultaneously to give 5 in 71% overall yield (see SI).
The synthesis of iso-DTDS is based on previous reports for key
intermediate 6 (trityl- and TFA-protected diethylenetriamine).[17,20]
Compound 5 was coupled to the free secondary amine of 6 using
PyBOP and DIPEA. The resulting intermediate 7 was treated with
TFA and triethylsilane (TES) as a scavenger to cleave the trityl
protecting group giving a mixture of 8 and triphenylmethane.
Crude 8 was used in the next reaction step without further
purification. The C-terminal carboxylic acid group was introduced
using succinic anhydride. After precipitation into aqueous citric
acid solution, compound 9 was isolated. In the next step, the TFA-
starting from 6 and 98% purity as determined by integration of UV
signals in RP-HPLC (see SI).
1H NMR spectra of iso-DTDS 11 were recorded in DMSO-d6 and
methanol-d4 (see Figure 1 and SI). In DMSO-d6, the characteristic
amide protons of isomerized building block 11 at 8.77 ppm and
7.90 ppm can be observed as well as the carboxylic acid proton
at 12.01 ppm as shown in figure 1A. Unfortunately, the broad
solvent peak at 3.38 ppm complicates the analysis of the
methylene protons of iso-DTDS (see SI). Therefore, additional
analysis was performed in methanol-d4 (Figure 1, B). Signals of
TIPS protecting group at 1.12 ppm and Fmoc protecting group
between 7.0-8.0 ppm for aromatic protons as well as 4.39 ppm
1
and 4.16 ppm for aliphatic protons are clearly visible in H NMR
(for detailed analysis see SI).
Synthesis of glycooligomers
Syntheses of precision glycooligo(amidoamines) were performed
by applying previously reported procedures of Fmoc-based
SPPoS.[17] iso-DTDS was combined with the previously
established ethylenedioxy-bis(ethylamine) succinyl building block
(EDS)[17] using PyBOP as coupling reagent (Scheme 3). Coupling
efficiency of iso-DTDS was evaluated by Fmoc quantification
based on UV/Vis measurements of the cleavage solution for test
sequence (EDS-DTDS-EDS) for single coupling with 5 eq. as well
as double coupling with 3 eq. of building block, respectively.
Coupling efficiencies for the introduction of iso-DTDS were about
86% for the single coupling and 95% for the double coupling. The
use of alternative coupling reagents did not improve the coupling
protecting group was removed. As already described,
a
rearrangement can occur during this step.[19] The formation of
primary amine and rearrangement product was monitored by
LC/MS (see SI). Running the reaction at 60 °C for 8 h followed by
stirring at room temperature for 16 h afforded only isomerized
product 10 with minor impurities from the TIPS-cleaved side
product. After removal of solvent, crude product 10 was subjected
to Fmoc-chloride yielding the final iso-DTDS building block 11
after purification. Iso-DTDS was obtained in 40% overall yield
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efficiency, with DIC showing 21% and HATU 80% product
formation for a single coupling (compared to PyBOP: 86%). EDS
building block coupling onto the iso-DTDS chain end yielded 96%
efficiency under standard coupling conditions (see SI) showing
that chain elongation is successful after introduction of iso-DTDS.
Based on these results, further glycooligo(amidoamines) were
synthesized using standard coupling conditions for EDS (see SI)
and double coupling for iso-DTDS (3 eq. of building block and
PyBOP with 30 eq. of DIPEA in DMF for 1.5 h).
Scheme 3. Synthetic strategy for the synthesis of heteromultivalent glycooligomers applying iso-DTDS; introduction of different carbohydrate units by consecutive
CuAAC on solid support.
common monosaccharide motif of different HBGAs. This
Scheme 3 shows the solid phase synthesis of heteromultivalent
glycooligomers using iso-DTDS. After assembly of the oligomer
backbone and acetylation of the final amine group, carbohydrate
ligands are conjugated to the iso-DTDS side chain. As a first step
for all glycooligomers, acetylated α-L-fucopyranosylazide 12c
(Fuc) was coupled using previously established conditions for
CuAAC reaction. After removing excess reagents through
successive washing, the TIPS-protecting group was cleaved
using TBAF in DMF.[24] The corresponding glycooligomer could
then be subjected to a second CuAAC reaction with another azido
functionalized carbohydrate derivative (e.g. GalNAc (13c), Gal
(14c), Lac (15) or Sia (16)). In the final step, carbohydrate side
chains were deacetylated on resin and the crude final
glycooligomers were cleaved off the resin under acidic conditions
and isolated by precipitation and lyophilization.
glycooligomer was then split into four batches and further
functionalized with either a GalNAc, Gal, Lac or Sia generating
glycooligomers 17-20 (Figure 2). In addition, a homomultivalent
all-Fuc glycooligomer 21 and an all-Gal glycooligomer 22 were
synthesized for comparison in later binding studies. The aromatic
unit in the glycomimetic structures was installed with the goal of
mimicking the branching sugar unit in the natural trisaccharide,
with an additional ethyl-linker introduced via the functionalized
monosaccharides in an effort to balance the rigidity of the
aromatic branching unit.
Nomenclature of glycooligomers follows previously introduced
systematics: the carbohydrates attached to a single branching
unit (iso-DTDS) are written in brackets (e.g. (Fuc,Gal)) followed
by the position of iso-DTDS within the oligomer chain and the
overall chain length as given by the total number of building blocks,
e.g. (Fuc,Gal)[2,5]-6 for glycooligomer 18.
Following this protocol,
a first generation of homo- and
heteromultivalent glycooligomers introducing two iso-DTDS
building blocks and thereby four carbohydrate ligands were
synthesized (Figure 2). In total, six glycooligomers were
synthesized using a scaffold with the sequence EDS-DTDS-EDS-
EDS-DTDS-EDS. As first carbohydrate we introduced Fuc, a
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glycooligomers were measured as the reduction of LecB
adhesion to the fucosylated sensor chip surface with increasing
amount
of
glycooligomer.
α-L-Methylfucose (MeFuc) was measured as reference
compound. Galactosylated oligomer 22 was used as negative
control whereas homomultivalent fucosylated oligomer 21 served
as
a positive control as comparison for the effect of
heteromultivalency in the HBGA mimetics 17-20. Negative control
glycooligomer 22 showed no binding to LecB (see SI). Table 2
shows the results of the inhibition-competition assay of
glycooligomers 17-21 and α-L-methylfucose.
Table 1. Analytical data of glycooligomers 17-22.
Type of
sugar
MW
[g/mol]
Yield[a]
[%]
Entry
17
Natural model fragment
HBGA A
Fuc/GalNAc
2701.88
40
Figure 2. Hetero- and homomultivalent glycooligomer structures as HBGA and
18
19
Fuc/Gal
Fuc/Lac
Lewisa
2619.78
2855.95
49
46
HMO mimicry.
Crude glycooligomers were directly analyzed by RP-HPLC/MS
(see SI) after cleavage off the resin. The results demonstrate that
corresponding glycooligomers are synthesized in good purity (72-
85%). Nevertheless, all structures were further purified by using
an ion exchange resin and semi-preparative RP-HPLC giving the
final structures in high purities (> 99%) (see SI). Glycooligomer 20
carrying two Sia ligands was isolated carrying a methyl protecting
group (20-Me). Only after purification, glycooligomer 20-Me was
subjected to cleavage of the methyl group giving glycooligomer
20 (see Table 1). Final analysis of glycooligomer structures was
2`Fucosyllactose (2`FL)
20-Me
20
Fuc/Sia(Me)
Fuc/Sia
-
2906.06
2878.01
40
36
Sialyl Lewisa
21
22
Fuc/Fuc
Control
2587.78
2651.77
26
36
1
performed by H-NMR, RP-HPLC/MS, HR-ESI-MS and MALDI-
TOF-MS (see SI).
Gal/Gal[b]
Negative control
Lectin binding studies of heteromultivalent glycooligomers
towards LecB
[a] Overall yield after purification by ion exchange resin and semi-preparative
HPLC with
galactose.
a gradient of water/acetonitrile. [b] Alpha/beta mixture of
After the successful synthesis of the first generation of
heteromultivalent glycooligomers imitating fragments of HBGAs,
we investigated their potential to act as HBGA mimetics by
studying their binding behavior towards LecB from Pseudomonas
aeruginosa. It is well known that LecB binds to different HBGAs
and HMOs of which Lea shows the highest affinity (KD = 210 nM)
compared to monovalent ligand L-fucose (KD = 2.9 µM).[25,12] We
have previously reported a surface plasmon resonance (SPR)
inhibition competition assay that allows for the measurement of
half maximum inhibitory concentration (IC50) values of
glycooligomers binding to LecB.[26] In short, a commercially
available streptavidin-coated sensor chip was functionalized with
commercially available biotinylated polyacrylamide bearing either
fucose (PAA-Fuc) as positive control or galactose (PAA-Gal) as
negative control at the reference cell. Glycooligomers were
preincubated for 1 h at different concentrations with LecB
(200 nM) in TRIS-buffer and the ligand-LecB-complex was
injected to the SPR sensor chip. Inhibitory potencies of
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DTDS building block e.g. by means of molecular modelling and
light scattering.
Table 2. Results from SPR inhibition competition assay of glycooligomers
17-21 and LecB (200 nM).
Ligand
MeFuc
n[a]
1
IC50 [nM][b]
300 ± 28
61 ± 16
RIP[c]
RIP/n[c,d]
Conclusions
1 ± 0.1
4.9 ± 0.3
1
2
2.5
A new building block, iso-DTDS, suitable for solid phase polymer
synthesis was introduced providing for the introduction of closely
neighboring carbohydrate ligands in the side chains of precision
glycomacromolecules. iso-DTDS can be used to create
heteromultivalent glycooligomeric constructs combining different
carbohydrate motifs and thereby more closely mimicking complex
oligosaccharide ligands. In this report, iso-DTDS was applied to
the synthesis of glycooligomers presenting fragments of HBGAs.
Inhibitory potencies of these glycomimetic oligomers towards
LecB were investigated. Interestingly, a glycooligomer presenting
only Fuc-ligands showed similar inhibitory effects as
glycooligomers combining Fuc with GalNAc, Lac or Sia. Only the
glycooligomer combining Fuc and Gal showed a slight increase in
the inhibitory potential indicating additional binding of the Gal
ligands within the structure based on fragments of the natural Lea
ligand. Overall this strategy gives straightforward access to a
variety of heteromultivalent glycooligomers and extends our
platform of precision glycomacromolecules. Following the
presented concept, macromolecular mimetics of other
oligosaccharides or combinations of carbohydrates with
additional non-carbohydrate binding motifs are now accessible.
2
2
35 ± 9
8.6 ± 0.3
5.0 ± 0.2
4.3
2.5
60 ± 11
2
4
76 ± 8
54 ± 8
3.9 ± 0.1
5.6 ± 0.2
2.0
1.4
[a] Number of fucose units within the ligand. [b] IC50 values determined by
two independent measurements with standard error of the mean (SEM). [c]
Relative inhibitory potencies (RIP) based on α-L-methylfucose (MeFuc), RIP
=
IC50(MeFuc) / IC50(glycooligomer). [d] Relative inhibitory potency
normalized on fucose units per oligomer (RIP/n).
Compared to α-L-methylfucose, all glycooligomers showed
increased inhibition of LecB (Table 2). Previously we could show
that binding to LecB increases with an increasing number of
fucose units on the oligomeric backbone.[26] Surprisingly, positive
control 21 exhibiting a total of four fucose side chains does not
show an increase in binding in comparison to the
heteromultivalent glycooligomers presenting only two fucose
ligands. For further comparison we normalized the IC50 values on
the IC50 value of α-L-methylfucose to provide relative inhibitory
potencies (RIP) that can be further normalized to the number of
fucose ligands (RIP/n) (Table 2). Indeed, RIP values fit well with
previously investigated homomultivalent glycooligomers binding
to LecB, where an RIP/n of about 2-3 was observed.[26] Similar
values for heteromultivalent glycooligomers 17, 19 and 20
indicate that the second carbohydrate motif seems to play no role
in improving the overall binding to LecB. Only glycooligomer 18
with an additional galactose ligand in close proximity to the fucose
ligand shows a lower IC50 value and thereby increased binding.
One possible explanation could be a partial mimicking of the Lea
ligand. Interestingly, sLea mimetic 20 did not show increased
binding although sLea is also known as a potent binder of LecB.[12]
Thus further studies are required to investigate participation of the
different carbohydrate motifs in LecB binding in more detail e.g.
by STD-NMR or crystallography. Ongoing studies also include the
analysis of the glycooligomer conformation and thereby
distancing between carbohydrate side chains attached via the
Experimental Section
Synthesis of iso-DTDS
Key intermediate 6 was synthesized according literature.[17,20] Synthesis of
precursor intermediate 5 was performed according to literature.[23]
3-Ethynyl-5-((triisopropylsilyl)ethynyl)benzoic acid (5): To a solution of
methyl-3-((triisopropylsilyl)ethynyl)-5-((trimethylsilyl)ethynyl)benzoate (4)
(5.98 g, 14.5 mmol, 1 eq.) in THF (15 mL) was added 15 mL aq. KOH
solution (0.2 g/mL, 3 g, 53 mmol, 3.7 eq.) and the reaction mixture was
stirred for 24 h at room temperature. Upon completion, 15 mL of water
were added and a solid precipitated. The THF was evaporated, and the
remaining aqueous suspension was cooled with an ice bath. Aqueous HCl
(58 mL, 174 mmol, 3 M, 12 eq.) was added, and the mixture was stirred
for at least one hour. The obtained solid precipitate was filtered in vacuum
and dried under high vacuum overnight to afford 3-ethynyl-5-
((triisopropylsilyl)ethynyl)benzoic acid (5) as colorless solid (4.35 g, 13.3
mmol, 92 %). 1H NMR (600 MHz, CDCl3): δ [ppm] = 8.15 (p, 4J = 1.6 Hz,
2H, HAr-2, HAr-6), 7.8 (t, 4J = 1.6 Hz, 1H, HAr-4), 3.15 (s, 1H, CΞC-H), 1.15-
1.13 (m, 21H, -CH-(CH3)2). 13C NMR (75.5 MHz, CDCl3): δ [ppm] = 170.7
(-COOH), 140.3 (CAr-4), 133.8 (CAr-2), 133.4 (CAr-6), 129.9 (CAr-1), 124.8
(CAr-5), 123.2 (CAr-3), 104.7 (CΞC-TIPS), 93.6 (CΞC-TIPS), 81.7 (CΞC-H),
79.1 (CΞC-H), 18.8 (CH3), 11.4 (CH(CH3)2). Rf = 0.46 (DCM/MeOH (10:1)).
ESI-MS: m/z calcd. for C20H26O2Si [M+H]+ 327.2, found 327.2. HR-ESI-
MS: m/z calcd. for C20H26O2Si [M+H]+ 327.1775, found 327.1772. RP-
HPLC: (gradient from 80 % to 100 % eluent B in 10 min, proceeding at
100 % B, 17 min, 25 °C) tR = 5.8 min, determined purity 98 %.
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3-Ethynyl-N-(2-(2,2,2-trifluoroacetamido)ethyl)-5-((triisopropylsilyl)-
excess citric acid followed by washing with cold DCM. The product was
obtained as white solid (13.30 g, 22 mmol, 91%). 1H NMR (300 MHz,
CD3OD, CDCl3): δ [ppm] = 7.57 (t, 4J = 1.5 Hz, 1H, p-HBz), 7.37 (s, 2H, o-
HBz), 3.72-3.67 (m, 1H, NCH2CH2), 3.63-3.56 (m, 2H, NCH2CH2), 3.52-
3.44 (m, 2H, NCH2CH2), 3.40-3.33 (m, 3H, NCH2CH2, CΞCH, overlapping
with signal from CD2HOD signal), 3.25-3.20 (m, 1H, N-CH2CH2), 2.63-2.53
(m, 2H, (CO)CH2), 2.48-2.37 (m, 2H, (CO)CH2), 1.09 (s, 21H, CH(CH3)2).
ESI-MS: m/z calcd. for C30H40F3N3O5Si [M+H]+ 608.3, found 608.3;
[M+Na]+ 630.3, found 630.2. HR-ESI-MS: m/z calcd. for C30H40F3N3O5Si
[M+H]+ 608.2762, found 608.2753. RP-HPLC: (gradient from 40% to 100%
eluent B in 30 min, 25 °C) tR = 15.6 min, determined purity 87%. Rf = 0.52
(DCM/MeOH (9:1) + one droplet AcOH).
ethynyl)-N-(2-(tritylamino)ethyl)benzamide (7): To
2,2,2-trifluoro-N-(2-((2-(tritylamino)ethyl)amino)ethyl)acetamide
a
solution of
(key
intermediate, 6) (2.58 g, 5.84 mmol, 1 eq.) in DMF (40 mL) were added
dialkyne acid 5 (2 g, 6.13 mmol, 1.05 eq.), PyBOP (3.19 g, 6.13 mmol,
1.05 eq.), HOBt (0.89 g, 5.84 mmol, 1 eq.) and DIPEA (1.66 mL,
17.52 mmol, 3 eq.). The mixture was stirred for 16 h at room temperature.
The resulting yellowish solution was poured into 400 mL of water and left
overnight. The suspension was centrifuged and the water was decanted.
The solid product was redissolved in ethyl acetate and extracted three
times with water, dried over MgSO4, filtered and concentrated in vacuum.
Column chromatography (gradient of hexane/ethyl acetate (3:1-2:1))
afforded the product as colorless crystals (3.68 g, 4.9 mmol, 84%). 1H NMR
(600 MHz, CDCl3): δ [ppm] = 7.85 (s, 1H, NH), 7.66 (s, 1H, HBz), 7.53 (s,
1H, HBz), 7.46 (s, 1H, HBz), 7.37 (d, 3J = 7.8 Hz, 6H, C(C6H5)3), 7.28 (t, 3J
= 7.7 Hz, 6H, C(C6H5)3), 7.19 (t, 3J = 7.3 Hz, 3H, C(C6H5)3), 3.63-3.61 (m,
2H, (C=O)NH-CH2-CH2), 3.51-3.48 (m, 2H, (C=O)NH-CH2-CH2), 3.43 (t, 3J
= 5.9 Hz, 2H, C(Ph)3-NH-CH2-CH2), 3.13 (s, 1H, CΞC-H), 2.25 (t, 3J = 5.9
Hz, 2H, C(Ph)3-NH-CH2), 1.13-1.12 (m, 21H, -CH-(CH3)2). 13C NMR (151
MHz, CDCl3): δ [ppm] = 172.6 ((C=O)Ph), 157.8 ((C=O)CF3), 157.6, 145.3
4-((2-((2-(3-ethynyl-5-((triisopropylsilyl)ethynyl)benzamido)ethyl)amino)-
ethyl)amino)-4-oxobutanoic acid (10): Compound 9 (10.0 g, 16.5 mmol,
1 eq.) was suspended in a mixture of MeOH (250 mL) and EtOH (180 mL)
and stirred at 40 °C until the starting material was dissolved completely.
K2CO3 (16 g, 116 mmol, 7 eq.) in water (100 mL) was added and the
mixture was heated to 60 °C and allowed to stir at 60 °C for 8 h, and then
at room temperature overnight. Remaining solvents were removed under
reduced pressure at 40 °C. The crude product was analyzed by RP-HPLC
and used without further purification. ESI-MS: m/z calcd. for C28H41N3O4Si
[M+H]+ 512.29, found 512.04. RP-HPLC: (gradient from 100% to 50%
eluent A in 5 min, decreasing to 0% A until 12 min, proceeding at 0% A,
17 min, 25 °C) tR = 8.8 min, determined purity 92%.
(C-1Phenyl), 136.8 (C-4Bz), 136.0 (C-1Bz), 130.4 (C-2Bz, C-6Bz), 129.8 (C-2Bz
,
C-6Bz), 128.4 (o-CPhenyl, m-CPhenyl), 128.1 (o-CPhenyl, m-CPhenyl), 126.6 (p-
CPhenyl), 124.8 (C-5Bz), 123.0 (C-3Bz), 116.7 (CF3), 104.7 (C-TIPS), 93.5
(CΞC-TIPS), 81.8 (CΞC-H), 79.2 (CΞC-H), 71.0 (C-Ph3), 60.4 (residual
ethyl acetate), 50.4 (N(CH2)2), 44.2 (N(CH2)2), 42.2 (NH-CH2), 39.5 (NH-
CH2), 21.1 (residual ethyl acetate), 18.6 (CH3), 14.2 (residual ethyl
acetate), 11.3 (CH(CH3)2). HR-ESI-MS: m/z calcd. for C45H50F3N3O2Si
(monoisotopic mass 749.3624) [M+H]+ 750.3697, found 750.3699. RP-
HPLC: (gradient from 80% to 100% eluent B in 10 min, proceeding at
100% B, 17 min, 25 °C) tR = 11.2 min, determined purity 99%. Rf = 0.79
(Hex/EE (1:1)).
4-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(3-ethynyl-5-((triisopropyl-
silyl)ethynyl)benzamido)ethyl)amino)ethyl)amino)-4-oxobutanoic
acid
(11): The slurry of 10 was redissolved in THF (200 mL) and water (200 mL).
Fmoc‐Cl (4.67 g, 18 mmol, 1.1 eq.) was added and the reaction mixture
was stirred for 18 h at room temperature. The emulsion was evaporated
under reduced pressure. The pH was tested and needs to be
pH > 7. The gel-like residue was redissolved in ethyl acetate (50 mL), water
(300 mL) and brine (200 mL). The aqueous layer was washed two times
with ethyl acetate to remove remaining Fmoc-based byproducts. Citric acid
(1L, 10% solution in water) was added to the aqueous layer to adjust the
pH < 4. The product was then extracted three times extensively from the
aqueous solution with ethyl acetate. The collected organic layers were
dried over MgSO4, filtered and concentrated under reduced pressure.
Crude product (11.69 g, 15.9 mmol, 96%) was further purified by silica gel
column chromatography with DCM/MeOH (20:1) to afford 6.3 g (8.5 mmol,
52%) of pure product 11. 1H NMR (600 MHz, CD3OD): δ [ppm] = 7.84 (dd,
4,4J = 21.4, 8.0 Hz, 2H, o-HBz), 7.76 (d, 3J = 7.6 Hz, 2H, HAr (Fmoc)), 7.64-
7.49 (m, 3H, p-HBz, HAr (Fmoc)), 7.35 (m, 2H, HAr (Fmoc)), 7.27 (dt, 3,4J =
21.5, 7.3 Hz, 2H, HAr (Fmoc)), 4.44 (d, 3J = 5.8 Hz, 1H, OCH2CH), 4.34 (d,
3J = 6.1 Hz, 1H, OCH2CH), 4.16 (dt, 3,4J = 36.4, 5.7 Hz, 1H, OCH2CH),
3.63 (d, 5J = 23.9 Hz, 1H, CΞC-H), 3.49 (s, br, 2H, NHCH2CH2), 3.45-3.40
(m, 1H, NHCH2CH2), 3.39-3.34 (m, 2H, NHCH2CH2), 3.34-3.30 (m,
overlapping with methanol-d4, NHCH2CH2), 3.21 (t, 3J = 5.9 Hz, 1H,
NHCH2CH2), 3.06 (t, 3J = 5.8 Hz, 1H, NHCH2CH2), 2.56 (t, 3J = 6.9 Hz, 2H,
(CO)CH2), 2.42 (t, 3J = 7.0 Hz, 2H, (CO)CH2), 1.15-1.08 (m, 21H,
CH(CH3)2). 13C NMR (151 MHz, DMSO-d6): δ [ppm] = 173.8 (CO2H), 172.0
(CH2(CO)NH), 171.1 (CH2(CO)NH), 164.5 (PhCO), 155.6 (O(CO)NH),
155.5 (O(CO)NH), 143.8 (CAr (Fmoc)), 143.8 (CAr (Fmoc)), 140.7 (CAr
(Fmoc)), 136.7, 136.6 (o-CBz), 135.5, 135.4 (o-CBz), 130.7, 130.5 (C-1Bz),
127.6 (CAr (Fmoc)), 127.6 (CAr (Fmoc)), 127.1 (CAr (Fmoc)), 125.1 (CAr
(Fmoc)), 125.0 (CAr (Fmoc)), 123.1 (C-5Bz), 123.0 (C-5Bz), 122.6 (C-3Bz),
122.5 (C-3Bz), 120.1 (CAr (Fmoc)), 120.1 (CAr (Fmoc)), 105.1 (CΞC-TIPS),
92.0 (CΞC-TIPS), 91.9 (CΞC-TIPS), 82.5 (CΞC-H), 82.3 (CΞC-H), 81.7
(CΞC-H), 67.9 (Fmoc-CH-CH2), 66.9 (Fmoc-CH-CH2), 46.9, 46.7, 46.6,
46.5, 38.1, 37.5, 36.8 (all HNCH2CH2), 30.1 (succinyl-CH2), 30.0 (succinyl-
CH2), 29.1 (succinyl-CH2), 21.1 (CH(CH3)2), 18.5 (CH3), 10.6. ESI-MS: m/z
calcd. for C43H51N3O6Si [M+H]+ 734.4, found 734.3; [M+Na]+ 756.3, found
756.2. HR-ESI-MS: m/z calcd. for C43H51N3O6Si (monoisotopic mass
2,2,2-trifluoroacetaldehyde,2-(3-ethynyl-N-(2-(2,2,2-trifluoroacetamido)-
ethyl)-5-((triisopropylsilyl)ethynyl)benzamido)ethan-1-aminium salt (8): To
a solution of 7 (18.09 g, 24.1 mmol, 1 eq.) in DCM (240 mL) were added
TES (10.8 mL, 68 mmol, 2.8 eq.) and TFA (26 mL, 337 mmol, 10% v/v).
The colorless solution was stirred 1 h at room temperature and the reaction
progress was determined by TLC (hexane/ethyl acetate (1:1)). Then TFA
was co-evaporated with toluene. The crude product containing
triphenylmethane as a side product (1:1 mixture) was obtained as white
solid (20.86 g crude mixture: calc. containing product: 14.9 g, 24 mmol,
quant.). Remaining triphenylmethane was not successfully separated but
could be removed in the next reaction step. The obtained crude product
was used without further purification. 1H NMR (600 MHz, CDCl3): δ [ppm]
= 8.18 (s, 3H, NH), 7.96 (s, 1H, (CO)NH), 7.63 (s, 1H, HBz), 7.43 (s, 1H,
HBz), 7.38 (s, 1H, HBz), 7.30-7.27 (m, 6H, C5H6), 7.25 (residual toluene),
7.23-7.20 (m, 3H, C5H6), 7.17 (residual toluene), 7.13-7.11 (m, 6H, C5H6),
5.56 (s, 1H, CH3-Ph3), 3.81 (s, 2H, CH2) 3.72 (s, 2H, NH), 3.55 (s, 2H,
CH2), 3.36-3.31 (m, 4H, CH2), 3.15 (s, 1H, CΞC-H), 2.36 (residual toluene),
1.14-1.09 (m, 21H, CH(CH3)2). ESI-MS: m/z calcd. for C26H36F3N3O2Si
[M+H]+ 508.3, found 508.2. HR-ESI-MS: m/z calcd. for C26H36F3N3O2Si
[M+H]+ 508.2602, found 508.2607. Rf = 0.72 (DCM/MeOH (5:1)).
4-((2-(3-ethynyl-N-(2-(2,2,2-trifluoroacetamido)ethyl)-5-((triisopropylsilyl)-
ethynyl)benzamido) ethyl)amino)-4-oxobutanoic acid (9): To a solution of
20.86 g of crude product 8 (about 14.97 g, 24 mmol, 1 eq. of product 8
respectively) in 240 mL DCM (0.1 M) were added succinic anhydride
(2.411 g, 24 mmol, 1 eq.) and NEt3 (10 mL, 72 mmol, 3 eq.). The reaction
mixture was stirred for 2 h at room temperature. The reaction progress was
determined by TLC (DCM/MeOH (10:1) v/v, acetic acid 1 droplet). After
complete consumption the reaction mixture was concentrated to a total
volume of 100 mL under reduced pressure. The mixture was precipitated
in 2 L of aq. citric acid (10%) and stirred for 1 h. The precipitate was filtered
in vacuum and the solid was washed intensively with water to remove
This article is protected by copyright. All rights reserved.

10.1002/chem.201804505
Chemistry - A European Journal
FULL PAPER
733.3547) [M+H]+ 734.3620, found 734.3623. RP-HPLC: (gradient from
80% to 100% eluent B in 10 min, proceeding at 100% B, 17 min, 25 °C) tR
= 6.1 min, determined purity 98%. Rf = 0.71 (DCM/MeOH (10:1) + droplet
AcOH).
Hz, 2H, Sia-H8), 3.76-3.43 (m, 73H, CH2-O-(CH2)2-O-CH2, N-CH2-CH2-NH,
Fuc-H2, Fuc-H3, Fuc-H4, Sia-H4-H7, Sia-H9), 3.41-3.25 (m, 17H, O=C-
NH-CH2-CH2-O), 3.20 (dt, 3,3J = 18.7, 5.2 Hz, 2H, Fuc-5), 3.16-3.11 (m, 2H,
Sia-H), 2.76-2.70 (m, 4H, O=C-CH2-CH2-C=O), 2.62 (dd, 2,3J = 12.9, 4.6
Hz, 2H, Sia-He3), 2.55-2.47 (m, 21H, O=C-CH2-CH2-C=O), 2.00 (s, 6H,
(NH)CO-CH3 (Sia)), 1.95, 1.95 (s, s, 3H, O=C-CH3), 1.79 (t, 2,3J = 12.4 Hz,
2H, Sia-Ha3), 0.94-0.93 (m, 6H, Fuc-H6). MALDI-TOF-MS: m/z calcd for
C124H193N29O51 [M+Na]+ 2927.34, found 2927.47. HR-ESI-MS: m/z calcd
for C124H193N29O51 (monoisotopic mass 2904.3400): [M+3H]3+ 969.1206,
found 969.1191. RP-HPLC: (gradient from 100% to 50% eluent A in 30 min,
25 °C): tR = 14.6 min, determined purity 99%.
Glycooligomers analysis
1
(Fuc,GalNAc)[2,5]-6 (17): H NMR (600 MHz, D2O): δ [ppm] = 8.50-8.48
(m, 2H, N=N-N-CH), 8.46-8.44 (m, 2H, N=N-N-CH), 8.12-8.09 (m, 2H,
p-HPh), 7.94-7.90 (m, 4H, o-HPh), 4.88 (d, 3J = 3.6 Hz, 2H, Fuc-H1α), 4.84-
4.82 (m, 2H, GalNAc-H1α), 4.75-4.69 (m, 8H, N=N-N-CH2), 4.16 (dt, 2,3J =
10.1, 4.5 Hz, 2H, N=N-N-CH2-CH2), 4.10-4.00 (m, 6H, N=N-N-CH2-CH2
GalNAc-H3), 3.95 (dt, 2,3J = 10.5, 4.8 Hz, 2H, N=N-N-CH2-CH2), 3.87 (d,
3J = 2.9 Hz, 2H, GalNAc-H2), 3.82-3.79 (m, 2H, GalNAc-H4), 3.74-3.44 (m,
62H, CH2-O-(CH2)2-O-CH2, N-CH2-CH2-NH, Fuc-H2, GalNAc-H6, Fuc-H3,
Fuc-H4), 3.40-3.26 (m, 16H, O=C-NH-CH2-CH2-O), 3.22 (dt, 3,3J = 17.7,
5.4 Hz, 2H, Fuc-H5), 3.14-3.09 (m, 2H, GalNAc-H5), 2.76-2.69 (m, 4H,
O=C-CH2-CH2-C=O), 2.55-2.47 (m, 20H, O=C-CH2-CH2-C=O), 1.96, 1.95
(s, s, 3H, (CH2-NH)-(O=)C-CH3), 1.78-1.77 (m, 6H, NH-(O=)C-CH3)
(GalNAc)), 0.93-0.91 (m, 6H, Fuc-H6). MALDI-TOF-MS: m/z calcd. for
C116H181N29O45 [M+Na]+ 2723.28, found 2723.34. HR-ESI-MS: m/z calcd.
for C116H181N29O45 (monoisotopic mass 2700.2766): [M+3H]3+ 901.0995,
found 901.0988. RP-HPLC: (gradient from 100% to 50% eluent A in
30 min, 25 °C): tR = 14.0 min, determined purity 99%.
(Fuc,Sia)[2,5]-6 (20): 1H NMR (300 MHz, D2O): δ [ppm] = 8.46-8.43 (m,
2H, N=N-N-CH), 8.38-8.34 (m, 2H, N=N-N-CH), 8.05-8.01 (m, 2H, p-HPh),
7.89-7.82 (m, 4H, o-HPh), 4.88 (d, 3J = 3.0 Hz, 2H, Fuc-H1α), 4.74-4.60 (m,
8H, N=N-N-CH2), 4.25-4.20 (m, 2H, N=N-N-CH2-CH2), 4.13-3.95 (m, 8H,
N=N-N-CH2-CH2, Sia-H8), 3.85-3.11 (m, 94H, CH2-O-(CH2)2-O-CH2, N-
CH2-CH2-NH, Fuc-H2, Fuc-H3, Fuc-H4, Sia-H4-H7, Sia-H9, O=C-NH-
CH2-CH2-O, Fuc-H-5), 2.77-2.49 (m, 28H, O=C-CH2-CH2-C=O, Sia-H3eq),
2.00 (s, 6H, NC(=O)CH3 (Sia)), 1.95, 1.94 (s, s, 3H, O=C-CH3), 1.75 (t, 2,3
J
= 12.1 Hz, 2H, Sia-H3ax), 0.93 (d, 3J = 6.6 Hz, 6H, Fuc-H6). HR-ESI-MS:
m/z calcd. for C122H189N29O51 (monoisotopic mass 2878.3087): [M+3H]3+
959.7769, found 959.7761. RP-HPLC: (gradient from 100% to 50% eluent
A in 30 min, 25 °C): tR = 13.6 min, 13.8 min, determined purity 96%.
(Fuc,Fuc)[2,5]-6 (21): 1H NMR (600 MHz, D2O): δ [ppm] = 8.39 (s, 4H,
N=N-N-CH), 7.99-7.97 (m, 2H, p-HPh), 7.82-7.79 (m, 4H, o-HPh), 4.78 (d,
3J = 3.1 Hz, 4H, Fuc-H1α), 4.67-4.56 (m, 8H, N=N-N-CH2), 4.04-3.89 (m,
8H, N=N-N-CH2-CH2), 3.63-3.09 (m, 76H, Fuc-H2, Fuc-H3, Fuc-H4, CH2-
O-(CH2)2-O-CH2, N-CH2-CH2-NH, O=C-NH-CH2-CH2-O), 3.06-2.99 (m, 4H,
Fuc-H5), 2.68-2.59 (m, 4H, O=C-CH2-CH2-C=O), 2.47-2.36 (m, 20H, O=C-
CH2-CH2-C=O), 1.86, 1.86 (s, s, 3H, O=C-CH3), 0.82 (d, 3J = 6.5 Hz, 12H,
Fuc-H6). MALDI-TOF-MS: m/z calcd. for C112H175N27O43 [M+Na]+ 2609.2,
found 2609.3. HR-ESI-MS: m/z calcd. for C112H175N27O43 (monoisotopic
mass 2586.2337): [M+3H]3+ 863.0852, found 863.0851. RP-HPLC:
(gradient from 100% to 50% eluent A in 30 min, 25 °C): tR = 14.3 min,
determined purity 99%.
(Fuc,Gal)[2,5]-6 (18): 1H NMR (600 MHz, D2O): δ [ppm] = 8.47-8.44 (m,
4H, N=N-N-CH), 8.05-8.03 (m, 2H, p-HPh), 7.89-7.86 (m, 4H, o-HPh), 4.98
3
(d, J = 3.7 Hz, 2H, Gal-H1α), 4.88 (d, 3J = 3.5 Hz, 2H, Fuc-H1α), 4.75-
4.68 (m, 8H, N=N-N-CH2), 4.17-4.15 (m, 2H, N=N-N-CH2-CH2), 4.10-4.07
(m, 2H, N=N-N-CH2-CH2), 4.02-3.98 (m, 4H, N=N-N-CH2-CH2), 3.83-3.79
(m, 4H, Gal-H2, Gal-H4), 3.76-3.69 (m, 6H, Fuc-H2, Gal-H3, Gal-H6),
3.67-3.43 (m, 55H, CH2-O-(CH2)2-O-CH2, N-CH2-CH2-NH, Fuc-H3, Fuc-
H4), 3.41-3.24 (m, 18H, O=C-NH-CH2-CH2-O, Gal-H6), 3.20 (dt, 3,3J = 19.6,
5.1 Hz, 2H, Fuc-H5), 3.16-3.13 (m, 2H, Gal-H5), 2.75-2.69 (m, 4H, O=C-
CH2-CH2-C=O), 2.54-2.48 (m, 20H, O=C-CH2-CH2-C=O), 1.95, 1.94 (s, s,
3H, O=C-CH3), 0.94-0.92 (m, 6H, Fuc-H6). MALDI-TOF-MS: m/z calcd. for
C
112H175N27O45 [M+Na]+ 2641.2, found 2641.3. HR-ESI-MS: m/z calcd. for
C112H175N27O45 (monoisotopic mass 2618.2235): [M+3H]3+ 873.7485,
found 873.7479. RP-HPLC: (gradient from 100% to 50% eluent A in 30 min,
25 °C): tR = 13.7 min, determined purity 99%.
(Gal,Gal)[2,5]-6 (22): Negative control 22 exhibits galactose units of an
α/β-mixture (3:1). 1H NMR (600 MHz, D2O): δ = 8.38-8.34 (m, 4H, N=N-N-
CH), 7.97-7.91 (m, 2H, p-HPh), 7.81-7.75 (m, 4H, o-HPh), 5.00 (d, 3J =
1.4 Hz, 1H, Gal-H1α), 4.74-4.65 (m, 8H, N=N-N-CH2), 4.45 (d, 3J = 7.4 Hz,
Gal-H1β), 4.39-4.32 (m, 3H, N=N-N-CH2-CH2), 4.19-4.12 (m, 4H, N=N-N-
CH2-CH2), 4.08-4.01 (m, 3H, N=N-N-CH2-CH2, Gal-H2α), 3.92 (d, J = 3.2
Hz, 4H, Gal-H2β), 3.79-3.12 (m, 89H, Gal-H2, Gal-H4, Gal-H5, Gal-H3,
Gal-H6, CH2-O-(CH2)2-O-CH2, N-CH2-CH2-NH, O=C-NH-CH2-CH2-O),
2.76-2.68 (m, 4H, O=C-CH2-CH2-C=O), 2.52-2.47 (m, 20H, O=C-CH2-
CH2-C=O), 1.94-1.92 (m, 3H, O=C-CH3). MALDI-TOF-MS: m/z calcd. for
(Fuc,Lac)[2,5]-6 (19): 1H NMR (600 MHz, D2O): δ [ppm] = 8.58 (s, 2H,
N=N-N-CH), 8.40-8.39 (m, 2H, N=N-N-CH), 8.01-7.09 (m, 2H, p-HPh),
7.88-7.79 (m, 4H, o-HPh), 5.87 (dd, 3,4J = 9.1, 1.7 Hz, 2H, Glc-H1β), 4.87
(d, 3J = 3.4 Hz, 2H, Fuc-H1α), 4.73-4.64 (m, 4H, N=N-N-CH2), 4.57 (d, 3J
= 7.7 Hz, 2H, Gal-H1β), 4.15 (dt, 2,3J = 8.9, 2.0 Hz, 2H, N=N-N-CH2-CH2),
4.08-4.05 (m, 4H, N=N-N-CH2-CH2, Glc-H), 4.00-3.94 (m, 12H, Glc-H, Gal-
H), 3.88-3.84 (m, 2H, Glc-H), 3.82-3.79 (m, 4H, Gal-H2, Gal-H4), 3.74-
3.69 (m, 6H, Fuc-H2, Gal-H3, Gal-H6), 3.64-3.44 (m, 51H, CH2-O-(CH2)2-
O-CH2, N-CH2-CH2-NH, Fuc-H3, Fuc-H4), 3.42-3.27 (m, 17H, O=C-NH-
CH2-CH2-O), 3.24-3.12 (m, 6H, Gal-H6, Fuc-H5, Gal-H5), 2.73-2.68 (m,
4H, O=C-CH2-CH2-C=O), 2.55-2.45 (m, 20H, O=C-CH2-CH2-C=O), 1.94,
1.93 (s, s, 3H, O=C-CH3), 0.95-0.93 (m, 6H, Fuc-H6). MALDI-TOF-MS:
m/z calcd. for C120H187N27O53 [M+Na]+ 2877.3, found 2877.3. HR-ESI-MS:
m/z calcd. for C120H187N27O53 (monoisotopic mass 2854.2768): [M+3H]3+
952.4329, found 952.4325. RP-HPLC: (gradient from 100% to 50% eluent
A in 30 min, 25 °C): tR = 12.8 min, determined purity ≥99%.
C
112H175N27O47 [M+Na]+ 2673.2134, found 2673.294. HR-ESI-MS: m/z
calcd for C112H175N27O47 (monoisotopic mass 2650.2134): [M+3H]3+
884.4117, found 884.4114. RP-HPLC: (gradient from 100% to 50% eluent
A in 30 min, 25 °C): tR = 12.9 min, 13.1 min, 13.3 min, determined purity
99%.
Acknowledgements
The authors would like to thank Mirko Lindic for his help with
(Fuc,Sia(methyl-protected))[2,5]-6 (20-Me): 1H NMR (600 MHz, D2O):
δ [ppm] = 8.48-8.46 (m, 2H, N=N-N-CH), 8.37-8.36 (m, 2H, N=N-N-CH),
8.08-8.06 (m, 2H, p-HPh), 7.92-7.88 (m, 4H, o-HPh), 4.88 (d, J = 3.5 Hz,
synthesizing
azidoethyl-2,3,4,6-tetra-O-acetyl-α-D-
3
galactopyranoside and Mischa Baier for helpful discussions. LH
thanks the DFG for support through the FOR 2327 ViroCarb (HA
5950/5-1) and large equipment grant INST 208/735-1. NLS
2H, Fuc-H1α), 4.76-4.62 (m, 11H, COO-CH3, N=N-N-CH2, overlapping
with HDO-signal), 4.24-4.22 (m, 2H, N=N-N-CH2-CH2), 4.10-4.07 (m, 2H,
3
N=N-N-CH2-CH2), 4.02-3.98 (m, 4H, N=N-N-CH2-CH2), 3.83 (t, J = 10.2
This article is protected by copyright. All rights reserved.

10.1002/chem.201804505
Chemistry - A European Journal
FULL PAPER
J. Org. Chem. 2010, 6, 801; I. Deguise, D. Lagnoux, R. Roy, New J. Chem. 2007,
31, 1321. Review article: J. L. Jiménez Blanco, C. Ortiz Melleta, J. M. García
Fernández, Chem. Soc. Rev. 2013, 42, 4518.
thanks the Davidson College VPAA and the Dean Rusk
International Studies Program for sabbatical support.
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This article is protected by copyright. All rights reserved.

10.1002/chem.201804505
Chemistry - A European Journal
FULL PAPER
FULL PAPER
Katharina Susanne Bücher, Patrick
Benjamin Konietzny, Nicole L. Snyder,
Laura Hartmann*
TIPS
Heteromultivalent glycooligomers are
synthesized by solid phase assembly using
a building block with a free alkyne and a
TIPS-protected alkyne moiety enabling the
subsequent attachment of different
carbohydrate motifs by on-resin copper
mediated azide alkyne cycloaddition
reactions. Different fragments of blood
group antigens are thereby conjugated onto
a macromolecular scaffold and investigated
for their binding to bacterial lectin LecB.
HN
O
O
HO
N
Fmoc
N
Page No. – Page No.
H
O
Heteromultivalent glycooligomers as
mimics of blood group antigens
TIPS
TIPS
This article is protected by copyright. All rights reserved.