Synthesis of Azido-Glycans for Chemical Glycomodification of Proteins

Article abstract of DOI:10.1002/ejoc.201800602

Chemically produced, accurately linkable oligosaccharides are of importance for the synthesis of neo-glycoproteins. On the route to high-mannose type N-glycans, we present a convenient synthesis of several glycans bearing an azide moiety at the reducing end. An azido-glycan core structure as valuable precursor was modified into the protected N-glycan pentasaccharide core structure and the possibility of modular attachment of different antenna was demonstrated through synthesis of a pentamannose donor and glycosylation with the core structure. The azido function allows for chemical ligation with recombinantly modified proteins featuring noncanonical cyclooctyne amino acids, providing access to customized glycopatterns of glycoproteins, e.g., of antibodies that are of high interest for biopharmaceutical applications.

Full text of DOI:10.1002/ejoc.201800602

A Journal of
Accepted Article
Title: Synthesis of Azido-Glycans for Chemical Glycomodification of
Proteins
Authors: Mirella Wawryszyn, Paul F. Sauter, Martin Nieger, Martin R.
Koos, Christine Koehler, Burkhard Luy, Edward A. Lemke,
and Stefan Bräse
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201800602
Link to VoR: http://dx.doi.org/10.1002/ejoc.201800602
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10.1002/ejoc.201800602
European Journal of Organic Chemistry
FULL PAPER
Synthesis of Azido-Glycans for Chemical Glycomodification of
Proteins
M. Wawryszyn, † [a,b] P. F. Sauter, † [a] M. Nieger,[c] M. R. M. Koos,[d] C. Koehler,[e,f,g] B. Luy,[a,d]
E. A. Lemke,[e,f,g] and S. Bräse*[a,b,h]
Abstract: Chemically produced, accurately linkable oligosaccharides
are of importance for the synthesis of neo-glycoproteins. On the route
to high-mannose type N-glycans, we present a convenient synthesis
of several glycans bearing an azide moiety at the reducing end. An
azido-glycan core structure as valuable precursor was modified into
the protected N-glycan pentasaccharide core structure and the
possibility of modular attachment of different antenna was
demonstrated through synthesis of a pentamannose donor and
glycosylation with the core structure. The azido function allows for
chemical ligation with recombinantly modified proteins featuring non-
natural cyclooctyne amino acids, providing access to customized
glycopatterns of glycoproteins, e.g., of antibodies that are of high
interest for biopharmaceutical applications.
for energy storage and as structural components, they also occur
as a part of many important biomolecules where their role is still
not completely understood.[2] Oligosaccharides N-linked to
proteins via the carboxamide side chain of asparagine – the so
called N-glycans – are the most abundant post-translational
modification of eukaryotic proteins.[1b, 3] Naturally occurring
N-glycans are categorized into three structural classes: the high-
mannose, the complex and the hybrid type.[1b, 4] These N-glycans
share a common pentasaccharide core structure, which derives
from an evolutionary preserved biosynthetic pathway.[4b, 5]
Modifications beyond this core structure are carried out by a set
of glycan processing enzymes that overlap in their localization.
Thus, a large variety of different oligosaccharides is formed in a
combinatorial manner.[6] Therefore, glycoproteins exist as
heterogeneous mixtures of different glycoforms.[7] However,
glycosylation plays a central role in secretion, stability, function,
and immunogenicity of glycoproteins (GPs) and a detailed
understanding of their structure-activity relationship is of pivotal
interest.[8] Thus, an easy way to the access of homogeneous
glycoforms is required.[8a, 8c-e] The impaired isolation of
homogeneous glycoforms for biological studies creates an ever-
growing need for synthetic N-glycans. In contrast, protein design
is well understood and even site-specific incorporation of
unnatural amino acids (nAAs) is routinely possible since the
1980s and several proteins have been approved for cancer
therapy.[8a, 8b, 9]
Introduction
Carbohydrates are ubiquitously present in nature and a main
constituent of all biomass.[1] Besides their biological importance
[a]
[b]
Institute of Organic Chemistry, Karlsruhe Institute of Technology
(KIT), Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany.
Soft Matter Synthesis Laboratory, Institute for Biological
Interfaces 3, Karlsruhe Institute of Technology (KIT), Hermann-
von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen,
Germany.
Multiple syntheses of N-glycans have been established for
elucidation of their biological roles. Kerekgyarto et al. reported the
synthesis of a benzylated core pentasaccharide glycosyl azide.[10]
Several di-, and triantennary N-glycans and respective
glycoconjugates were successfully synthesized by Danishefsky et
al.[11] The synthesis of a complex type glycan containing a core
fucose was reported by Fukase et al. and high-mannose type by
Matsuo et al.[12] Unverzagt et al. established syntheses of various
N-glycans, including core-fucosylated multiantennary bisected
structures.[13] Chemoenzymatic approaches for synthesis of N-
glycans were investigated by Ito et al.,[14] Wong et al.,[15] and
Boons et al.[16] A fully automated synthesis of several glycans was
reported by Seeberger et al.[17]
[c]
[d]
Department of Chemistry, University of Helsinki, P. O. Box 55,
00014 University of Helsinki, Finland.
Institute for Biological Interfaces 4, Karlsruhe Institute of
Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344
Eggenstein-Leopoldshafen, Germany.
Departments of Biology and Chemistry, Pharmacy and
Geosciences, Johannes Gutenberg-University Mainz, Johannes-
von-Mullerweg 6, 55128 Mainz, Germany.
Institute of Molecular Biology (IMB), Ackermannweg 4, 55128
Mainz, Germany.
Structural and Computational Biology Unit & Cell Biology and
Biophysics Unit, European Molecular Biology Laboratory (EMBL),
Meyerhofstraße 1, 69117 Heidelberg, Germany.
Institute of Toxicology and Genetics, Karlsruhe Institute of
Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344
Eggenstein-Leopoldshafen, Germany.
[e]
[f]
[g]
[h]
Without enzymatic treatment, however, a native glycan with a
hydroxyl group at the reducing end is neither reactive without
harsh chemical conditions, nor does it allow site specific reactions
under physiological conditions.[18] To provide strictly
homogeneous glycoforms, we propose a biorthogonal approach:
Glycans synthesized bearing a suitable moiety at their reducing
end are combined with recombinantly modified proteins featuring
corresponding nAA. In this way, arbitrary glycans can be created
and directed to designated positions in a protein to achieve any
†
*
M. W. and P. F. S. contributed equally to this work.
Correspondence should be addressed to Stefan.Braese@kit.edu,
www.ioc.kit.edu/braese/
Electronic Supplementary Information (ESI) available: Supporting
Information (SI) see DOI: 10.1039/x0xx00000x. Crystallographic
data can be obtained free of charge from The Cambridge
Crystallographic
Data
Centre
via
www.ccdc.cam.ac.uk/data_request/cif: CCDC 1565150 (4a), CCDC
1565151 (6b), CCDC 1565152 (7b), CCDC 1565152 (11c), CCDC
1565154 (19' only shown in the SI).
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10.1002/ejoc.201800602
European Journal of Organic Chemistry
FULL PAPER
glycopattern that may be of interest. Azido-functionalized mono-, Results and Discussion
di-, and oligosaccharides are well known in the literature and can
be particularly helpful in a variety of glyco-conjugation reactions,
e.g. copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC)
To show a possible way towards neo-glycoproteins which are
manufacturable via biorthogonal reactions such as strain-
promoted 1,3-dipolar cycloaddition between azide and
cyclooctyne derivatives, we synthesized the azido-glycans 1, 2,
and 3 shown in Figure 2.
click-reaction.[19]
Using
strain-promoted
alkyne-azide
cycloaddition (SPAAC) ligation instead, as an example of copper-
free click-chemistry, glycoproteins with a defined glycopattern can
be obtained as shown in Figure 1 and Scheme 9.[9f, 20] The ligation
protein bears a cyclooctynyl group as an nAA, obtained by genetic
code expansion. We established a fast and easy access to three
biologically inspired azido-glycans, 1, 2, and 3, via several
strategies, outlined in the retrosynthetic approach shown in
Figure 2 and explained in the following.
The smallest target structure, 3, represents the unbranched core
structure of all N-glycans. The glycopattern of pentamer 2
represents the conserved pentasaccharide core structure, which
all naturally occuring N-glycans have in common. The octameric
structure 1 is close to a typical high-mannose glycopattern, even
though in living systems more complex patterns are encountered.
To give a brief overview of our strategy, we initially relied on a
linear glycosylation approach for the synthesis of chitobiosyl
azide 9 featuring parallel steps for the monomeric donor (4b–7b)
and acceptor (4a–7a) building blocks. Since the azide moieties of
the acceptor building blocks provide excellent protection of the
reducing end and modular approaches gave inferior yields, we
chose azide protection at the very beginning. The β-manno-
inversion of β-gluco-trisaccharide 14 to compound 15 on the way
to the trimeric core structure represented one of the major
synthetic challenges, which was overcome by inversion via a SN2-
substitution of the triflate moiety of compound 14.
Figure 1. Access to homogeneous glycoproteins: Illustration of an azido-glycan
on the left, exemplarily shown with a high-mannose glycopattern, as well as an
arbitrary recombinantly modified protein featuring a cyclooctyne nAA. The
attachment of glycan to a protein is achieved via strain-promoted azide-alkyne
cyclo-addition (SPAAC).
Figure 2. Retrosynthetic approach for azido-glycans 1, 2, and 3: Man6(GlcNPhth)2N3 (1) was obtained by convergent block glycosylation of key acceptor 17 and
pentamannoside donor 28; pentasaccharide structure Man3(GlcNPhth)2N3 (2) was obtained by tandem glycosylation of key acceptor 17 and an arbitrary mannose
based donor; the native trimeric key structure Man(GlcNPhth)2N3 (2) was achieved by glycosylation of chitobiosyl azide 10 and thio-tolyl donor 11 followed by
subsequent deprotection steps.
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Scheme 1. Synthesis of chitobiosyl azide 10; a) NaOMe, MeOH, rt, 30 min (R = SEt, N3, quant.); b) p-methoxybenzaldehyde dimethyl acetal, p-TsOH, MeCN, rt,
4 h, (R = SEt, 84%; R = N3, 72%); c) Ac2O, Pyridine, rt, 16 h, (R = SEt, 97%; R = N3, 92%); d) BH3∙THF, TMSOTf, 0 °C, 1.5 h, (R = SEt, 69%; R = N3, 85%); e)
Ac2O, pyridine, rt, 16 h, (R = SEt, 99%; R = N3, 92%); f) DDQ, CH2Cl2/H2O, rt, 4 h, 79%; g) NIS, AgOTf, CH2Cl2, 30 °C, 2.5 h; h) DDQ, CH2Cl2/H2O, rt, 4 h, 86% (2
steps).
The key acceptor compound 17 was obtained after deprotection
steps proceeding from 15. Further, the pentamannoside 28 was
obtained by glycosylation of the trichloroacetimidate (TCA)
disaccharide donor 22 and 3,6-diol acceptor 27 in a good yield.
For the synthesis of the conserved pentasaccharide core
structure 2, we performed a tandem glycosylation of key
structure 17 as an acceptor and 29 as donor as shown in
Scheme 6. The protected octameric glycan 1 was obtained by the
assembly of branched pentamannose antenna 28 and
trisaccharide azide 17 by block glycosylation. Global deprotection
of key structure 17 yielded the native trisaccharide 3, which was
successfully clicked to the Fab fragment of Herceptin121→BCN via
SPAAC ligation, leading to previously published 3-BCN-Her.[20]
(PMB) deprotection of 7a resulted in the monomeric C-3
acceptor 8. The desired azide-bearing acceptor and
8
thioglycosilated donor 7b were prepared in excellent overall yields
of 24% over eleven steps and 33% over ten steps, respectively.
Subsequent glycosylation of donor 7b and acceptor 8 yielded the
chitobiosyl azide 9.[23] Finally, repeating PMB deprotection of
compound 9 furnished chitobiosyl azide 10, which served as an
acceptor for glycosylation with β-thio-tolyl donor 11. This highly
reproducible two step protocol opened access to disaccharide
acceptor 10 on a multigram scale with a yield of 86% over two
steps.
Synthesis of the core trimer: N-glycan Man3(GlcNAc)2N3 (17)
The following glycosylation reaction for β-gluco trisaccharide 12c
was explored using the three prepared β-thio-tolyl donors 11a–c
(see Table 1). For activation of the thioglycosides two different
promoter systems were tested. On the one hand the NIS/AgOTf-
system and on the other the DMDS/Tf2O-system was
attempted.[24] As shown in Table 1, the DMDS/Tf2O activation
method was found to be superior for acetal protected donors 11a
and 11b, but still providing low yields of 16% and 31%,
respectively. Fortunately, utilizing thio-tolyl donor 11c by
activation with NIS/AgOTf promoter system at −40 °C, proved to
be the most efficient method and gave a satisfying yield of 50%.
Next, one of the major synthetic challenges was the β-manno-
inversion of β-gluco-trisaccharide 14 described in Scheme 2 (b-d).
Mild hydrolysis of the mono-chloroacetic group of
trisaccharide 12c using thiourea unblocked the C-2 position.
Introducing a trifluoromethylsulfonyloxy group (triflate) at the C-2
position of compound 13 by treatment with Tf2O in
pyridine/dichloromethane at −15 °C led to compound 14, on which
inversion from the gluco- to manno-form was carried out: The
triflate group of trimeric building block 14 was displaced by an
acetyl group at C-2 position to obtain the β-manno-trimer 15,
presented in Scheme 2.[25]
Synthesis of chitobiosyl azide (GlcNAc)2N3 (10)
As outlined in Scheme 1, starting from monosaccharide
precursors 4a and 4b, which were accessible in a five-step
sequence commencing from glucosamine hydrochloride on multi
gram scale (see supplementary data), the synthesis of chitobiosyl
azide 9 was performed in a modular way.[21] Early introduction of
the azido moiety at the reducing end for precursor 4a turned out
to be advantageous for chemical stability against later
glycosylation steps. Likewise, implementation of the thioethyl
moiety for monomer 4b as a prevalent donor for specific activation
with N-iodosuccinimide (NIS) proved to be suitable for the
following protecting and deprotecting steps.[22] Deacetylation
under basic conditions was followed by acid-catalyzed 4,6-p-
methoxybenzylidene-acetal protection to form 4,6-di-O-
benzylidene acetals 5b and 5a. The remaining 2-OH group of
compounds 5b and 5a underwent esterification by treatment with
acetic anhydride in pyridine, delivering acetylated monomers 6b
and 6a.[21] The fully protected compounds 6b and 6a were then
regioselectively deprotected at the 4-position by reductive ring
opening in the presence of BH3∙THF complex and trimethylsilyl
triflate, then acetylated at the 6-position to give access to 7b and
7a. The configuration of 7b was confirmed by X-ray
crystallography (see CCDC). Afterwards, para-methoxy benzyl
Reaction conditions regarding the SN2-inversion protocol to obtain
the β-manno-configuration of core trisaccharide 15 at the C-2
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10.1002/ejoc.201800602
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position were optimized. Using 18-crown-6 and CsOAc as a base
in DMF at 60 °C, a yield of 70% of trimeric structure 13 could be
achieved and full inversion was gained; no other isomer was
detected. The β-manno configuration of compound 13 was
dimethylsilyl (TBS) group at the anomeric position the
dimannoside 20 was removed in the presence of
tetrabutylammonium fluoride (TBAF) yielding 62% of the
29b]
disaccharide 21.[28c,
Subsequent conversion using
1
confirmed by NMR-experiments, measuring characteristic JCH
trichloroacetonitril and 1,8-diazabicycloundec-7-ene (DBU)
provided the Schmidt donor, trichloroacetimidate compound 22,
with a yield of 72% (Scheme 3).[28e]
coupling constant of 157.5 Hz for the C-2 inverted position.[26]
To achieve the 3,6-diol acceptor 17, which was needed for later
azido-N-glycan synthesis towards octameric and pentameric key
structures (1 and 2), first, the p-methoxy benzyl (PMB) moiety at
C-6 position of trimer 15 was cleaved oxidatively with DDQ in a
mixture of water and dichloromethane. Subsequently, oxidative
debenzylation of C-3 position of compound 16 was performed by
using NaBrO3 and Na2S2O4 in a biphasic system of water and
ethyl acetate at room temperature, resulting in 3,6-diol acceptor
15.[27]
Table 1. Exploration of the glycosylation reaction to trisaccharide
compounds with chitobiosyl acceptor 10. Three different thio-tolyl donors
11a–c were examined; promotor systems and temperature were varied to
obtain compounds 12a–c at different yields. The NIS/AgOTf promoter
system in combination with compound 11c proved to be the most efficient
and additionally simplified the protecting group strategy for the following
steps.
Scheme 2: Synthesis of trimeric key structure 17; a) NIS, AgOTf, CH2Cl2,
−40 °C, 2 h, 50%; b) thiourea, NaHCO3, EtOH, reflux, 6 h, 99%; c) Tf2O,
pyridine, CH2Cl2, −15 °C to rt, 2.5 h, quant.; d) CsOAc, 18-crown-6, DMF, 60 °C,
16 h; 70%; e) DDQ, CH2Cl2/H2O, rt, 2.5 h, 93%; f) NaBrO3, Na2S2O4, EtOAc/H2O,
rt, 9 h, 58%.
Entry
12a
Promotor-System
DMDS/Tf2O
Temp.
0 °C
Yield
16%
31%
50%
12b
12c
DMDS/Tf2O
0 °C
NIS/AgOTf
−40 °C
Synthesis of the branching point: 3,6-diol acceptor (27)
Synthesis of an N-glycan arm: dimannoside-donor Man2SEt
(22)
In Scheme 4 we present a brief reaction sequence for 3,6-diol-
functionalized acceptor 27, which at the same time serves as a
donor for further glycosylation purposes due to its thioethyl moiety.
The trifunctional donor/acceptor monomer 27 was obtained
starting from commercially available D-mannose 23. After
peracetylation, mannoside 24 was coupled with ethanthiol in the
presence of BF3∙OEt2 to obtain thioethyl compound 18, whose
structure could be confirmed by X-ray cristallography.[28a] After
deprotection of the acylester, the 2,3:4,6-di-O-benzylidene
protection of compound 26 was achieved by using benzaldehyde
dimethyl acetal and camphor-10-sulfonic acid.[30] Subsequent
Cu(OTf)2-catalyzed regioselective reductive O-3,6-ring opening
as a late-stage introduction of the acceptor’s 1,3- and 1,6-arms
was conducted by adding boron hydride complex BH3∙THF at 0 °C
to the reaction mixture.[31] The regioselective 3,6-diol formation to
compound 27 was verified by the HMBC NMR experiment.
We proceeded with the sequence of dimannoside-donor 22
(Scheme 3), which was needed as a precursor building block for
branched pentamannoside antenna 28 (Scheme 5). Thus, the
monomeric thioethyl donor 18 and 2-OH acceptor 19 were
synthesized by standard protection and deprotection procedures,
described in detail in the supplementary data.[22, 28] Afterwards,
the disaccharide building block 20 was obtained via glycosylation
with the compounds 18 and 19 with NIS/TMSOTf as a promotor
system at −40 °C (Scheme 3). Since strongly electron-
withdrawing groups like the 2,3,4,6-tetra-O-acetyl groups of
mannoside 16 are expected to disfavour oxacarbenium ion
formation, glycosylation occurs by SN2-like displacement of the
β-anomeric sulfonium ion, leading to α-glycosidic linkage, as
observed for compound 20.[29] Afterwards, the tert-butyl
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10.1002/ejoc.201800602
European Journal of Organic Chemistry
FULL PAPER
position of protection groups could be confirmed. Unfortunately,
for the larger compounds, the amount of sample was too low for
similar NMR analysis.
Scheme 5: Synthesis of branched pentamannose antenna 28; a) TMSOTf,
CH2Cl2, MS 4 Å, −20 °C to 0 °C, 50%.
Scheme 3: Synthesis of disaccharide trichloroacetimidate donor 22; a) NIS,
TMSOTf, CH2Cl2, MS 4 Å, −40 °C, 2 h, 40%; b) TBAF, THF, 0 °C, 1 h, 62%; c)
CCl3CN, DBU, CH2Cl2, 0 °C, 1 h, 72%.
Synthesis of Azido-N-glycan Man3(GlcNAc)2N3 (2)
The simple mannose-based donor 29 was obtained in eight steps
from commercially available D-mannose (see supporting
information) and used in tandem glycosylation with trimeric 3,6-
acceptor 17 in the presence of NIS/AgOTf at −40 °C to yield the
pentameric glycan Man3Glc(GlcNAc)2 2 (Scheme 6).[24a, 34] The
doubly-glycosylated product could be isolated in a satisfying yield
of 38%. Only minor quantities of singly-glycosylated side product
were isolated alongside. The glycopattern of pentamer 2 is of
interest as it represents the common pentasaccharide core
structure of naturally occuring N-glycans.
Scheme 4: Synthesis 3,6-diol acceptor 27; a) Ac2O, I2, rt, 2 h, 91%; b) EtSH,
BF3∙OEt2, CH2Cl2, 0 °C to rt, 16 h; c) NaOMe, MeOH, rt, 6 h, 64% (2 steps); d)
benzaldehyde dimethyl acetal, CSA, DMF, rt, 16 h, 84%; e) BH3∙THF, Cu(OTf)2,
CH2Cl2, 0 °C to rt, 2 h, 31%.
Synthesis of branched core pentamannoside Man5SEt (28)
As shown in Scheme 5, Schmidt tandem glycosylation was
carried out with trichloroacetimidate donor 22 and 3,6-diol-
functionalized acceptor 27 under TMSOTf activation conditions at
−20 °C, leading to branched pentamannoside 28 with a yield of
50%.[32] The thioethyl moiety of compound 27 remains unaffected
under the reaction conditions, leaving 28 as a donor for the
following reactions.
Scheme 6: Synthesis of key pentasaccharide Man3NAc2 2; a) NIS, AgOTf,
CH2Cl2, MS 4 Å, −40 °C, 2 h, 38%; b) TBAF, THF, 0 °C to rt.
To confirm the structure of pentamer 28, full NMR assignment
was desired. The high grade of signal overlap especially in the 1H
frequency necessitated acquiring spectra beyond the common set
of 1H-1H and 1H-13C 2D experiments. Therefore,
a 1,1-
ADEQUATE experiment was recorded, providing highly resolved
information on 13C-13C pairs at the price of long measurement
duration.[33] Full assignment of all signals as shown in Figure 3
could be achieved, and subsequently inter-ring linkage and the
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Global deprotection of Azido-N-glycan Man(GlcNAc)2N3 (3)
In Scheme 8 the global deprotection of trimeric structure 16 to
provide native ManNAc2 3 is shown. Removal of phthalimide
moieties and saponification of acetyl esters were carried out
simultaneously with an excess of ethylenediamine in n-butanol at
90 °C.[27] Afterwards, the crude product bearing free amine
positions underwent direct N-acetylation in a mixture of methanol
and water in the presence of Ac2O to obtain the native
trisaccharide ManGlcNAc2 3 [27]
byproducts, two consecutive rounds of purification via HPLC were
.
Due to many unidentified
necessary to isolate the pure core trisaccharide
over the two steps.
3 in 55% yield
Figure 3. 1H-13C HSQC spectrum (heteronuclear 2D NMR) of pentameric
structure 28 in CD2Cl2 – The ring CH-group region is shown with the full
assignment of the ring CH-groups achieved by evaluating 1,1-ADEQUATE,
HSQC, HMBC, and homonuclear spectra (data not shown).
Synthesis of Azido-N-glycan Man6(GlcNPhth)2N3 (1)
As the assembly of pentamer 2 proved to be successful, we
proceeded with the convergent block glycosylation of core
trisaccharide 16 and an excess amount of branched
pentamannose antenna 28 via activation with NIS/TMSOTf at
−40 °C (Scheme 7). Surprisingly, just one of the acceptor
positions formed a glycosidic bond, resulting in azido-glycan 1,
confirmed with MALDI-TOF experiments (see supplementary
data). As the C-6-position is known to be more reactive than the
C-3 position we assume structure 1 as shown in Scheme 7 was
obtained. After subsequent deprotection of the remaining acetyl-
groups under Zemplén conditions with NaOMe in methanol,
however, only fragments of the product could be detected my
mass spectrometry and the deprotection of the azido-glycan could
not be verified.
Scheme 8: Deprotection strategies of compound 17 to obtain native
trisaccharide 3; a) Ethylenediamine, 1-Butanol, 90 °C, 16 h; b) Ac2O/MeOH/H2O
(1:10:1.5), rt, 16 h, 55%.
The general applicability of azido-glycans for chemical
glycomodification of proteins via SPAAC was already
demonstrated by Koehler et al. using native azido-trisaccharide 3.
In Scheme 9, the corresponding SPAAC of the azido-glycan and
the Fab fragment of Herceptin (Her121→BCN) containing the
cyclooctyne nAA endo-bicyclo[6.1.0]non-4-yne-lysin (BCN), is
shown.[20]
Scheme 9: Chemical ligation via SPAAC of native azido-N-glycan 3 and
recombinantly modified Fab fragment of Herceptin BCN-Her (Her121→BCN) to
create neo-glycoprotein 3-BCN-Her.
Scheme 7: Assembly of building blocks 17 and 28 to octameric azido-glycan
Man(GlcNPhth)2N3 1; a) NIS, TMSOTf, CH2Cl2, MS 4 Å, −45 °C, 2.5 h, 70%; b)
NaOMe, MeOH, rt, 4 h.
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10.1002/ejoc.201800602
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HSQC (heteronuclear single quantum coherence), and HMBC
(heteronuclear multiple bond correlation). The HSQC .and 1H-13C-
multiplicity-edited HSQC were recorded with 2048 × 512 points in 142 and
8.5 ms acquisition time, respectively. The coupling delay was matched to
145 Hz. Two scans were added with an overall measurement time of
29 min. The HMBC was recorded with 8192 × 512 points in 570 and
8.5 ms acquisition time, respectively. The transfer delay was matched to
Conclusions
We demonstrated a facile large scale chemical synthesis of the
azide-functionalized N-glycans 1, 2, and 3 via several strategies
mentioned above. The key step for the common precursor
structure was the assembly of the β-mannosidic linkage via
inversion of a β-glucoside trisaccharide. From this precursor, an
octameric glycan was created via block glycosylation and a
deprotected core trisaccharide was successfully conjugated to the
Fab-fragment of a recombinantly modified human antibody
containing a nAA to modify its glycostructure.
We will go on to attach different antenna to create a small library
of glycans for biological evaluation and production of single
glycoforms of proteins of therapeutical interest. As the
carbohydrate motif has a significant effect on several properties
of glycoproteins such as stability, function and immunogenicity,
glycosylation needs to be considered but also provides new
possibilities for pharmaceutical development.
1
8 Hz, a threefold lowpass filter was utilized to remove JCH-correlations.
Eight scans were added with an overall measurement time of 1 h 53 min.
For the pentameric structure 28, also an 1,1-ADEQUATE (adequate
double quantum transfer experiment) was recorded on the 600 MHz
spectrometer using
a sample of 10 mg substance dissolved in
dichloromethane-d2. The 1,1-ADEQUATE was recorded with 4096 × 1024
points in 285 and 10.6 ms acquisition time, respectively. The
heteronuclear coupling delay was matched to 145 Hz, the homonuclear
coupling delay was matched to 45 Hz; 64 scans were added with an overall
measurement time of 28 hours.
ESI-MS (electrospray ionization mass spectrometry) and FAB-MS (fast
atom bombardment mass spectrometry) measurements were performed
by the use of an AGILENT TECHNOLOGIES 6230 TOF LC/MS and a FINNAGAN
MAT 95 device. The molecule ion peak [M]+ or the protonated [M + H]+ and
deprotonated [M − H]+, [M + Na]+ or [M + K]+ form as well as characteristic
fragments were indicated as the ratio of mass to charge with respect to
their relative intensity to the base signal (100%). High resolution mass was
indicated by the calculated and measured value of the relating compound.
MALDI-TOF (matrix assisted laser desorption/ionization˗time of flight)
mass spectra were recorded on an AB SCIEX 4800 Proteomics analyzer
and a BRUKER Autoflex II Smartbeam device, using a nitrogen laser (λ =
337 nm) and software FlexControl version1.1 as well as XMASS˗XTOF
version 5.1.1. On a standard aluminum target plate (386 spots) from
BRUKER CHCA and DHB (1:1) in H2O+0.1% THF/ACN+0.1% THF (1:1)
were utilized as matrix. Mass spectra were acquired in the reflector positive
ion mode, default calibration.
Experimental Section
General information: Unless otherwise indicated, utilized solvents and
chemicals were purchased from commercial suppliers (ABCR, ACROS,
ALFA AESAR, CARL ROTH; FLUKA, FISHER SCIENTIFIC, MERCK, SIGMA ALDRICH,
VWR) and starting materials were used without further purification.
Analytical thin-layer chromatography (TLC) was performed on silica gel 60
F254 precoated aluminium plates from MERCK with a layer thickness of
0.25 mm and with detection by UV (λ = 254 nm, 365 nm). SEEBACH-
reagent (2.5% phosphomolybdic acid, 1.0% cerium-(IV) sulfate
tetrahydrate, 6.0% concentrated sulfuric acid, 90.5% destilled water) as
dipping solution with subsequent heating by warm airflow. Purification was
performed with preparative chromatography using normal-phase silica gel
(Silica gel 60, 0.040–0.063 mm, from MERCK).
IR (infrared spectroscopy) data were recorded on a BRUKER IFS 88. The
measurements were performed either in between KBr plates or via ATR
(attenuated total reflection) technique. The basis intensities were
characterized in the following way: frequency of absorption (ṽ in cm–1) and
intensity of absorption as s = strong (10–40% transmission), m = medium
(41–70% transmission), w = weak (71–90% transmission) and vw = very
weak (91–100% transmission).
NMR samples were prepared as solutions and measured at room
temperature. Spectra were recorded on a BRUKER 300 MHz AVANCE
spectrometer with room temperature Dual 13C/1H probe head (1H-NMR
spectra: 300 MHz, 13C-NMR spectra: 63 MHz), a BRUKER 400 MHz
AVANCE spectrometer with room temperature BBI probe head (1H-NMR
spectra: 400 MHz, 13C-NMR spectra: 100 MHz), a BRUKER 500 MHz
AVANCE III HD spectrometer with cryogenically cooled BBO probe head
(1H-NMR spectra: 500 MHz, 13C-NMR spectra: 125 MHz), and a BRUKER
600 MHz AVANCE III spectrometer with cryogenically cooled TCI probe
head (1H-NMR spectra: 600 MHz, 13C-NMR spectra: 150 MHz). For 1H-1D
spectra, 65536 points were acquired in 5.3 s acquisition time with 16 scans,
for 13C-1D, 65536 points were acquired in 0.9 s acquisition time with 128
scans. Chemical shifts (δ) are expressed in parts per million (ppm) and
referenced to the deuterated solvents, either chloroform (1H: δ = 7.26 ppm;
13C: δ = 77.16 ppm), methanol (1H: δ = 2.50 ppm; 13C: δ = 49.00 ppm),
DMSO (1H: δ = 2.50 ppm; 13C: δ = 39.43 ppm) or dichloromethane (1H: δ
= 5.32 ppm; 13C: δ = 53.5 ppm) as internal standards. Coupling constants
(J) are expressed in Hertz (Hz) as absolute values. Splitting patterns are
designated as s (singlet), d (doublet), t (triplet), q (quartet), quin (quintet),
m (multiplet), dd (doublet of doublets). Further, diastereotopic methylene
protons are marked as Ha and Hb. When obtained from DEPT
(distortionless enhancement by polarization transfer) experiments, the
number of protons attached to the respective carbon signals is indicated
as ‘+’ for CH- and CH3- groups and ‘–’ for CH2-groups. For structural
determination of proton and carbon ring positions the following 2D
experiments were recorded: COSY (correlation spectroscopy), 1H-13C-
O-(2,4-Di-O-acetyl-3-O-benzyl-6-O-p-methoxybenzyl-β-D-
mannopyranosyl)-(1→4)-O-(3,6-di-O-acetyl-2-deoxy-2-phthalimido-β-
D-glucopyranosyl)-(1→4)-3,6-di-O-acetyl-2-deoxy-2-phthalimido-β-D-
glucopyranosyl azide (15): To a solution of 14 (280 mg, 0.209 mmol,
1.00 eq.) in DMF (5.4 mL), dry CsOAc (64.8 mg, 0.230 mmol, 1.10 eq.)
and 18-crown-6 (64.8 mg, 0.230 mmol, 1.10 eq.) were added and stirred
at 60 °C overnight. The reaction mixture was diluted with dichloromethane,
evaporated to dryness followed by column chromatographic purification
using the eluent mixture ratio stated below to give compound 15 as a white
solid (58%).
Rf = 0.37 (toluene/acetone = 5:1). – 1H-NMR (500 MHz, CDCl3): δ = 7.89–
7.82 (m, 4 H, HAr), 7.79–7.72 (m, 4 H, HAr), 7.35–7.17 (m, 7 H, HAr), 6.88
(d, 3J = 8.6 Hz, 2 H, CHAr), 5.76 (dd, 3J = 10.2 Hz, 3J = 9.0 Hz, 1 H, 3-H),
5.70 (dd, 3J = 10.7 Hz, 3J = 9.1 Hz, 1 H, 3'-H), 5.57 (d, 3J = 9.4 Hz, 1 H, 1-
H), 5.44 (d, 3J = 8.4 Hz, 1 H, 1'-H), 5.48 (d, 3J = 2.8 Hz, 1 H, 2''-H), 5.05 (t,
3J = 9.7 Hz, 1 H, 4''-H), 4.64 (d, 2J = 12.2 Hz, 1 H, PhCHaHb), 4.44 (s, 1 H,
1''-H), 4.42–4.33 (m, 5 H, 6-Hb, 6'-Hb, PhCHaHb, MeOPhCH2), 4.17 (dd, 3J
3
= 10.8 Hz, J = 8.4 Hz, 1 H, 2'-H), 4.27–4.23 (m, 1 H, 6'-Ha), 4.07 (t, 3J =
9.9 Hz, 1 H, 2-H), 3.96 (t, 3J = 9.4 Hz, 1 H, 4-H), 3.90 (t, 3J = 9.5 Hz, 1 H,
4’-H), 3.80 (s, 3 H, OCH3), 3.83–3.69 (m, 3 H, 5-H, 6-Ha, 5'-H), 3.52–3.45
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800602
European Journal of Organic Chemistry
FULL PAPER
(m, 3 H, 3''-H, 6''-H2), 3.41–3.36 (m, 1 H, 5''-H), 2.10 (s, 3 H, CH3CO), 2.09
(s, 3 H, CH3CO), 1.95 (s, 3 H, CH3CO), 1.90 (s, 3 H, CH3CO), 1.88 (s, 3 H,
CH3CO), 1.82 (s, 3 H, CH3CO) ppm. – 13C-NMR (125 MHz, CDCl3): δ =
170.8 (Cq, CO), 170.7 (Cq, CO), 170.3 (Cq, CO), 170.2 (Cq, CO), 169.9 (Cq,
CO), 169.8 (Cq, CO), 168.0 (Cq, CO), 167.4 (Cq, CO), 159.4 (Cq, CAr), 137.5
(Cq, CAr), 134.6 (+, CHAr), 131.3 (Cq, CAr), 129.8 (Cq, CAr), 129.6 (+, CHAr),
128.5 (+, CHAr), 128.0 (+, CHAr), 128.5 (+, CHAr), 127.9 (+, CHAr), 124.0 (+,
CHAr), 113.9 (+, CHAr), 98.0 (+, C-1''), 97.6 (+, C-1’), 85.5 (+, C-1), 76.5 (+,
C-3’’), 75.3 (+, C-4), 74.1 (+, C-5'’), 74.5 (+, C-4'), 74.6 (+, C-5), 73.3 (–,
PhCH2), 72.8 (+, C-5'), 71.0 (–, PhCH2), 70.0 (+, C-3'), 70.7 (+, C-3), 69.4
(–, C-6''), 68.3 (+, C-4''), 67.1 (+, C-2'’), 62.4 (–, C-6'), 61.8 (–, C-6), 55.0
(+, C-2'), 55.4 (+, OCH3), 54.6 (+, C-2), 21.0 (+, CH3CO), 20.6 (+, 2 ×
CH3CO), 20.8 (+, CH3CO), 20.9 (+, CH3CO) ppm. – IR (ATR): ṽ = 2929
(vw), 2117 (vw), 1777 (vw), 1741 (w), 1714 (w), 1612 (vw), 1513 (vw), 1467
(vw), 1366 (w), 1217 (w), 1172 (w), 1028 (w), 972 (w), 873 (vw), 819 (vw),
740 (vw), 720 (w), 643 (vw), 599 (vw), 566 (vw), 530 (vw), 477 (vw), 455
(vw), 388 (vw) cm–1. – MS (3-NBA, FAB): m/z(%): 1273 (84) [M + Na]+,
1251 (5) [M + H]+, 662 (69), 647 (82), 365 (100).
layer was evaporated to dryness followed by column chromatographic
purification with the eluent mixture ratio stated below to give compound 17
as a white solid (58%).
Rf = 0.19 (toluene/acetone = 3:1). – 1H-NMR (500 MHz, CDCl3): δ = 7.88–
7.82 (m, 4 H, HAr), 7.79–7.72 (m, 4 H, HAr), 5.79–5.72 (m, 2 H, 3-H, 3'-H),
5.56 (d, 3J = 9.4 Hz, 1 H, 1-H), 5.41 (d, 3J = 8.3 Hz, 1 H, 1'-H), 5.33 (d, 3J
3
= 3.1 Hz, 1 H, 2''-H), 4.90 (t, J = 9.7 Hz, 1 H, 4''-H), 4.59 (s, 1 H, 1''-H),
3
3
4.39–4.32 (m, 3 H, 6-Hb, 6'-H2), 4.17 (dd, J = 10.6 Hz, J = 8.4 Hz, 1 H,
2'-H), 4.06 (dd, 3J = 10.2 Hz, 3J = 9.6 Hz, 1 H, 2-H), 3.93–3.97 (m, 1 H, 4-
2
3
H), 3.86–3.90 (m, 1 H, 4’-H), 3.60 (dd, J = 12.0 Hz, J = 4.9Hz, 1 H, 6''-
Ha), 3.843.68 (m, 5 H, 5-H, 6-Ha, 5'-H, 3''-H, 6''-Hb), 3.36–3.32 (m, 1 H, 5''-
H), 2.46 (bs, 1 H, OH), 2.41 (bs, 1 H, OH), 2.14 (s, 3 H, CH3CO), 2.13 (s,
3 H, CH3CO), 2.11 (s, 3 H, CH3CO), 1.98 (s, 3 H, CH3CO), 1.91 (s, 3 H,
CH3CO), 1.88 (s, 3 H, CH3CO) ppm. – 13C-NMR (125 MHz, CDCl3): δ =
171.6 (Cq, CO), 170.9 (Cq, CO), 170.8 (Cq, CO), 170.5 (Cq, CO), 170.3 (Cq,
CO), 169.9 (Cq, CO), 167.9 (Cq, CO), 167.4 (Cq, CO), 134.7 (+, CHAr),
131.3 (Cq, CAr), 124.0 (+, CHAr), 97.2 (+, C-1’), 97.9 (+, C-1''), 85.5 (+, C-
1), 75.0 (+, C-5''), 75.2 (+, C-4), 75.4 (+, C-4'), 74.6 (+, CH), 72.8 (+, CH),
70.2 (+, C-3'), 70.4 (+, C-3), 70.8 (+, C-4''), 70.9 (+, CH), 69.6 (+, C-4'’),
62.6 (–, C-6'), 61.9 (–, C-6), 61.7 (–, C-6''), 55.0 (+, C-2'), 54.6 (+, C-2),
21.0 (+, CH3CO), 21.0 (+, CH3CO), 20.6 (+, CH3CO), 20.7 (+, CH3CO),
20.8 (+, CH3CO), 20.9 (+, CH3CO) ppm. – IR (ATR): ṽ = 2912 (w), 2852
(vw), 2118 (w), 1740 (w), 1713 (m), 1368 (w), 1218 (m), 1033 (m), 972 (w),
873 (w), 795 (vw), 720 (m), 644 (vw), 600 (vw), 566 (vw), 530 (w), 454
(vw) cm–1. – MS (ESI): m/z(%): 1062 (100) [M + Na]+.
O-(2,4-Di-O-acetyl-3-O-benzyl-β-D-mannopyranosyl)-(1→4)-O-(3,6-di-
O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→4)-3,6-di-O-
acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl azide (16): To a
solution of 15 (66.1 mg, 52.9 µmol, 1.00 eq.) in dichloromethane (1 mL)
and water (0.05 mL), DDQ (18.0 mg, 79.3 µmol 1.50 eq.) was added and
stirred for 2.5 h. Then, NaHCO3 solution was added and the crude product
extracted with ethyl acetate. Afterwards the organic layer was washed with
water and brine and dried over Na2SO4. The organic layer was evaporated
to dryness followed by column chromatographic purification with the eluent
mixture shown below to give compound 16 as a white solid (93%).
Ethyl 2,4-di-O-benzyl-3,6-di-O-(2,3,4,6-tetra-O-acetyl-D-
mannopyranosyl-α-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-1-
thio-β-D-mannopyranoside (28): A solution mixture of acceptor 27
(54.9 mg, 0.140 mmol, 1.00 eq.) and donor 22 (314 mg, 0.340 mmol,
2.50 eq.) in dichloromethane (15 mL) together with molecular sieve (20 mg
MS 4 Å) were stirred for 15 min at room temperature. Afterwards the
solution mixture was cooled down to 0 °C and TMSOTf (6.00 µL,
0.0300 mmol, 0.100 eq.) was added. The mixture was stirred at 0 °C for
3.5 h and afterwards neutralized with a drop of NEt3 and filtered through a
plug of Celite. The solution was evaporated to dryness followed by column
chromatographic purification with the eluent mixing ratio shown below to
obtain 28 as colorless oil (49%).
Rf = 0.13 (toluene/acetone = 5:1). – 1H-NMR (500 MHz, CDCl3): δ = 7.93–
7.80 (m, 4 H, HAr), 7.80–7.71 (m, 4 H, HAr), 7.38–7.21 (m, 5 H, HAr), 5.80–
5.72 (m, 2 H, 3-H, 3'-H), 5.56 (d, 3J = 9.4 Hz, 1 H, 1-H), 5.52 (d, 3J = 3.1 Hz,
1 H, 2''-H), 5.43 (d, 3J = 8.4 Hz, 1 H, 1'-H), 4.98 (t, 3J = 9.7 Hz, 1 H, 4''-H),
2
4.65 (d, J = 12.2 Hz, 1 H, PhCHaHb), 4.51 (s, 1 H, 1''-H), 4.41–4.30 (m,
4 H, 6-Hb, 6'-H2, PhCHaHb), 4.17 (dd, 3J = 10.5 Hz, 3J = 8.5 Hz, 1 H, 2'-H),
4.06 (t, 3J = 9.9 Hz, 1 H, 2-H), 3.98–3.93 (m, 1 H, 4-H), 3.90–3.85 (m, 1 H,
4’-H), 3.82–3.70 (m, 3 H, 5-H, 6-Ha, 5'-H), 3.54 (dd, 3J = 9.7 Hz, 3J = 3.3 Hz,
1 H, 3''-H), 3.67–3.58 (m, 2 H, 6''-H2), 3.28–3.23 (m, 1 H, 5''-H), 2.50 (bs,
1 H, OH), 2.14 (s, 3 H, CH3CO), 2.11 (s, 3 H, CH3CO), 2.03 (s, 3 H,
CH3CO), 1.96 (s, 3 H, CH3CO), 1.92 (s, 3 H, CH3CO), 1.89 (s, 3 H,
CH3CO) ppm. – 13C-NMR (125 MHz, CDCl3): δ = 170.7 (Cq, 3 × CO), 170.6
(Cq, CO), 170.3 (Cq, CO), 169.9 (Cq, CO), 167.9 (Cq, CO), 137.4 (Cq, CAr),
134.7 (+, CHAr), 131.3 (Cq, CAr), 128.0 (+, CHAr), 128.1 (+, CHAr), 128.6 (+,
CHAr), 124.0 (+, CHAr), 98.0 (+, C-1''), 97.3 (+, C-1’), 85.5 (+, C-1), 76.3 (+,
C-3''), 75.0 (+, C-4'), 75.3 (+, C-4), 75.4 (+, C-5''), 74.6 (+, C-5), 72.8 (+,
C-5'), 71.2 (–, PhCH2), 70.2 (+, C-3'), 70.5 (+, C-3), 67.2 (+, C-2'’), 67.8 (+,
C-4''), 62.5 (–, C-6'), 61.9 (–, C-6), 61.8 (–, C-6''), 55.0 (+, C-2'), 54.6 (+,
C-2), 21.0 (+, CH3CO), 21.0 (+, CH3CO), 20.6 (+, CH3CO), 20.7 (+,
CH3CO), 20.8 (+, CH3CO), 20.9 (+, CH3CO) ppm. – IR (ATR): ṽ = 2923
(vw), 2118 (w), 1741 (m), 1714 (m), 1367 (m), 1218 (m), 1030 (m), 972 (w),
874 (w), 795 (vw), 720 (m), 644 (w), 601 (w), 530 (w), 480 (vw) cm–1. – MS
(ESI): m/z(%): 1152 (100) [M + Na]+, 1168 (75) [M + K]+.
Rf = 0.40 (cyclohexane/ethyl acetate 1:1). – 1H-NMR (600 MHz, CD2Cl2):
δ = 7.38–7.22 (m, 40 H, Harom), 5.42–5.35 (m, 4 H, 1I-H, 2IV-H, 3IV-H, 3V-
H), 5.28–5.19 (m, 3 H, 1II-H, 4IV-H, 4V-H), 5.02 (s, 1 H, 1III-H), 4.99 (s, 1 H,
1V-H), 4.90–4.87 (m, 2 H, CH2Ph), 4.76 (s, 1 H, 1IV-H), 4.73–4.47 (m, 14 H,
CH2Ph), 4.23–3.85 (m, 18 H, 2I-H, 2II-H, 2III-H, 3I-H, 3II-H, 3III-H, 4I-H, 4II-H,
4III-H, 5I-H, 5II-H, 5IV-H, 5V-H, 6I-H, 6IV-H, 6V-H), 3.75–3.61 (m, 6 H, 5III-H,
6I-H, 6II-H, 6III-H,), 2.64–2.52 (m, 2 H, SCH2CH3), 2.16–1.94 (m, 24 H, 8x
OCH3), 1.59 (s, 3 H, SCH2CH3) ppm. – 13C-NMR (150 MHz, DMSO): δ =
170.4–169.5 (Cq, COCH3), 138.5 (Cq OBn), 128.3–127.6 (CH, OBn), 101.1
(CH, C-1II), 99.2 (CH, C-1IV), 99.2 (CH, C-1V), 98.8 (CH, C-1III), 81.1 (CH,
C-3I), 80.8 (CH, C-1I), 79.6 (CH, C-2I), 79.3 (CH, C-3II), 79.1 (CH, C-3III),
77.1 (CH, C-2II), 75.8 (CH, C-2III), 74.9 (CH, C-4II, C-4I), 74.6 (CH, C-4III),
74.9 (CH2, CH2Ph), 73.2 (CH2, CH2Ph), 73.1 (CH2, CH2Ph), 72.9 (CH2,
CH2Ph), 72.6 (CH, C-5I), 72.3 (CH2, 2 × CH2Ph), 71.6 (CH2, 2 × CH2Ph),
71.7 (CH, C-5III), 71.5 (CH, C-5II), 69.7 (CH2, C-6II), 69.5 (CH, C-2IV, C-2V),
69.1 (CH, C-3IV, C-3V), 69.0 (CH, C-5IV, C-5V), 68.8 (CH2, C-6III), 66.5 (CH2,
C-6I), 66.0 (CH, C-4IV, C-4V), 62.8 (CH2, C-6IV), 62.5 (CH2, C-6V), 26.9 (CH,
SCH2CH3 ), 25.3 (CH, SCH2CH3), 20.5 (8 × CH3, OAc) ppm. – MS (MALDI-
TOF, Matrix: DHB:CHCA 1:1), m/z = 1951 [M + Na]+.
O-(2,4-Di-O-acetyl-β-D-mannopyranosyl)-(1→4)-O-(3,6-di-O-acetyl-2-
deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→4)-3,6-di-O-acetyl-2-
deoxy-2-phthalimido-β-D-glucopyranosyl azide (17): To a solution of 16
(239 mg, 0.211 mmol, 1.00 eq.) in ethyl acetate (4.2 mL), a solution of
NaBrO3 (106 mg, 0.634 mmol, 3.00 eq.) in water (2.1 mL) was added.
Afterwards a solution of Na2S2O5 (130 mg, 0.634 mmol, 3.00 eq.) in water
(4.2 mL) was added under vigorous stirring and was stirred for another 9 h
at room temperature after the addition was completed. The reaction
mixture was diluted with ethyl acetate, separated, washed with Na2S2O3
solution and the organic layer was dried over Na2SO4. Finally, the organic
2,4-Di-O-benzyl-3,6-di-O-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl-
(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-mannopyranosyl-
(1→6)-(2,4-di-O-acetyl-β-D-mannopyranosyl)-(1→4)-(3,6-di-O-acetyl-
2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→4)-3,6-di-O-acetyl-2-
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201800602
European Journal of Organic Chemistry
FULL PAPER
deoxy-2-phthalimido-β-D-glucopyranosyl azide (1): A solution of 28
(106 mg, 0.0549 mmol, 2.40 eq.) and 17 (23.8 mg, 0.0229 mmol, 1.00 eq.)
in dichloromethane (5.00 mL , MS 4 Å) was stirred for 1 h at room
temperature. Then, the mixture was cooled down to −40 °C and NIS
(29.6 mg, 0.132 mmol, 2.40 eq.) and afterwards TMSOTf (0.500 µL,
0.610 µg, 0.003 mmol, 0.0500 eq.) were added. The reaction mixture was
stirred for 3 h, then it was diluted with ethyl acetate and filtered through a
plug of Celite. Afterwards, the organic layers were washed with NaHSO3-
solution and brine, dried over Na2SO4 and evaporated to dryness followed
by column chromatographic purification using the eluent mixture ratio
stated below to give the octameric glycan 1 (40%).
Gupta, G. R. D. B. Thimiri, A. Aubert, P. Drncova, F. Garzoni, I. Berger,
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Rf = 0.42 (cyclohexane/ethyl acetate 2:1). – MS (MALDI-TOF, Matrix:
DHB:CHCA 1:1), m/z = 2908 [M + H]+.
[10] L. Kalmar, K. Agoston, Z. Szurmai, B. Doenczo, J. Kerekgyarto, J.
Carbohydr. Chem. 2012, 31, 203-219.
For more information concerning the characterization and working
procedures please refer to the supporting information.
[11] a) M. A. Walczak, S. J. Danishefsky, J. Am. Chem. Soc. 2012, 134,
16430-16433; b) M. A. Walczak, J. Hayashida, S. J. Danishefsky, J. Am.
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Acknowledgements
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We thank the Karlsruhe School of Optics & Photonics (KSOP),
the BW Stiftung and the GRK2039 for financial support. B.L. and
M.R.M.K. thank the Fonds der Chemischen Industrie, the
Deutsche Forschungsgemeinschaft (instrumentation facility
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Keywords: Azido-Glycan • N-Glycan • High-Mannose Motif •
Heterogeneous/ Homogeneous Glycoforms • Oligosaccharide
Synthesis Strategies • Glycoproteins • Strain-Promoted Alkyne-
Azide Cycloaddition (SPAAC) Ligation • Glycomodification of
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