A Single Route to Mammalian N-Glycans Substituted with Core Fucose and Bisecting GlcNAc

Article abstract of DOI:10.1002/anie.201807742

The occurrence of α1,6-linked core fucose on the N-glycans of mammalian glycoproteins is involved in tumor progression and reduces the bioactivity of antibodies in antibody-dependent cell-mediated cytotoxicity (ADCC). Since core-fucosylated N-glycans are difficult to isolate from natural sources, only chemical or enzymatic synthesis can provide the desired compounds for biological studies. A general drawback of chemical α-fucosylation is that the chemical assembly of α1,6-linked fucosides is not stereospecific. A robust and general method for the α-selective fucosylation of acceptors with primary hydroxy groups in α/β ratios exceeding 99:1 was developed. The high selectivities result from the interplay of an optimized protecting group pattern of the fucosyl donors in combination with the activation principle and the reaction conditions. Selective deprotection yielded versatile azides of all mammalian complex-type core-fucosylated N-glycans with 2-4 antennae and optional bisecting GlcNAc.

Full text of DOI:10.1002/anie.201807742

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: A Single Route to Mammalian N-Glycans substituted with Core
Fucose and Bisecting GlcNAc
Authors: Carlo Unverzagt, Thomas Luber, Mathäus Niemietz,
Theodoros Karagiannis, Manuel Mönnich, Dimiti Ott, Lukas
Perkams, Janika Walcher, Lukas Berger, Matthias Pischl,
Markus Weishaupt, Steffen Eller, and Joanna Siebenhaar
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: Angew. Chem. Int. Ed. 10.1002/anie.201807742
Angew. Chem. 10.1002/ange.201807742
Link to VoR: http://dx.doi.org/10.1002/anie.201807742
http://dx.doi.org/10.1002/ange.201807742

10.1002/anie.201807742
Angewandte Chemie International Edition
A Single Route to Mammalian N-Glycans substituted with Core Fucose and Bisecting
GlcNAc
Thomas Luber, Mathäus Niemietz, Theodoros Karagiannis, Manuel Mönnich, Dimitri Ott,
Lukas Perkams, Janika Walcher, Lukas Berger, Matthias Pischl, Markus Weishaupt, Steffen
Eller, Joanna Hoffman and Carlo Unverzagt*
[*] Prof. C. Unverzagt, Bioorganische Chemie, Gebäude NW1, Universität Bayreuth, 95440 Bayreuth (Germany),
Fax: (+49) 921-555365, E-mail: carlo.unverzagt@uni-bayreuth.de
This work is dedicated to the 150th anniversary of TUM
Abstract:
The occurrence of a1,6-linked core-fucose on the N-glycans of mammalian glycoproteins is
involved in tumor progression and reduces the bioactivity of antibodies in ADCC. Since core-
fucosylated N-glycans are difficult to isolate from natural sources only chemical or enzymatic
synthesis can provide the desired compounds for biological studies. A general drawback of
chemical a-fucosylation is that the chemical assembly of a1,6-linked fucosides is not
stereospecific. We have developed a robust and general method for the a-selective fucosylation
of acceptors with primary hydroxyl groups in a/b ratios exceeding 99:1. The high selectivities
result from the interplay of an optimized protecting group pattern of the fucosyl donors in
combination with the activation principle and the reaction conditions. Selective deprotection
yielded versatile azides of all mammalian complex-type core-fucosylated N-glycans with 2-4
antennae and optional bisecting GlcNAc.
Mammalian N-glycoproteins frequently display core fucose or bisecting GlcNAc in the core
region of N-glycans.[1] These ubiquitous modifications are influencing the conformational
behavior of the N-glycan[2] and the biological properties of the parent N-glycoprotein. Of high
relevance is the lowered antibody-dependent cellular cytotoxicity (ADCC) of core fucosylated
antibodies.[3] Furthermore core fucose and bisecting GlcNAc are frequently associated with
cancer progression and serve as a tumor marker.[4]
The chemical synthesis of complex-type N-glycans is challenging and compounds with
substitutions in the core require additional efforts. Thus, only a limited number of examples are
reported with core fucose[5] or a bisecting GlcNAc[6] or even both modifications.[7]
Chemoenzymatic
approaches
using
recombinant
a1,6-fucosyltransferases[8]
or
1
This article is protected by copyright. All rights reserved.

10.1002/anie.201807742
Angewandte Chemie International Edition
endoglycosidases[9] are simplifying the introduction of core fucose only for appropriate
substrates. Recently, a mutated fucosidase was developed capable of adding a core fucose onto
glycopeptides and glycoproteins.[10] Here we show that chemical a1,6-fucosylation of complex
N-glycans can reliably be carried out with nearly exclusive a-selectivity using suitably
protected fucosyl donors. The new approach is compatible with the modular one-pot
introduction of a bisecting GlcNAc moiety and can furnish anomeric azides of all mammalian
complex-type core-fucosylated N-glycans with an optional bisecting GlcNAc.
AcO
O
AcO
AcO
O
NHTFAc
AcO
O
O
O
O
O
AcO
AcO
N3
N3
N3
N3
AcO
AcO
NHTFAc
G1
G2
G3
G4
O
O
CCl3
D
B
NH
NH
OMPM
OMPM
AcO
AcO
AcO
O
AcO
OMPM
SEt
AcO
AcO
H3
C
O
NHTFAc
CCl3
α1,6-arm
F
core fucose
N3
N3
N3
N3
G5
G6
G7
G8
1. modular
synthesis
AcO
O
OMe
bisecting
GlcNAc
O
O
OH
NPhth
AcO
Ph
O
CF3
NPh
AcO
O
O
O
BnO
O
O
HO
BnO
N3
NHTFAc
O
BnO
NPhth
E
2. selective
deprotection
A
N3
N3
N3
N3
α1,3-arm
G9
G10
G11
G12
AcO
AcO
O
O
O
O
AcO
AcO
AcO
AcO
NHTFAc
O
O
CCl3
NH
B
C
AcO
AcO
O
O
AcO
O
AcO
AcO
N3
N3
N3
N3
NHTFAc
AcO
G13
G14
G15
G16
CCl3
NH
O
AcO
AcO
NHTFAc
GlcNAc
Man
Fuc
Scheme 1: Modular set of building blocks designed for the envisioned all-in-one-route to
mammalian complex N-glycans G1-G16 with optional bisecting GlcNAc and core fucose.
Bn=benzyl, NPhth=phthalimido, Ac=acetyl, TFAc=trifluoracetyl, MPM=p-methoxybenzyl,
MP=p-methoxyphenyl.
The N-TFAc-protected building blocks B-E efficiently provide N-glycans with an optional
bisecting modification (G1-G8).[6k] We thus intended to combine the latter approach with the
core trisaccharide A[5i] functionalized for the late introduction of a1,6-linked fucose.[7c] This
should consequently allow the synthesis of all the core variations of mammalian N-glycans
(G1-G16) via a single pathway (Scheme 1). However, the low steroselectivity (a/b ratios
around 10:1) observed for chemical a1,6-fucosylation of N-glycan acceptors posed an
unresolved obstacle.[5g, 5i] The formation of b-fucoside side products can be determined by
analytical HLPC-MS of the crude reaction mixture but is easily overlooked when relying only
on NMR. Typically, the separation of a- and b-fucosylated N-glycans by flash chromatography
is not possible. When purifying these mixtures by preparative RP-HPLC we experienced
2
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10.1002/anie.201807742
Angewandte Chemie International Edition
coelution of the anomers leading to significant loss of the valuable compounds after repetitive
separations.[5i] The key for a general approach to core fucosylated N-glycans was thus to find a
highly stereoselective and reliable method for chemical a1,6-fucosylation.
We initially pursued the modular approach outlined in Scheme 1 by using the analogous
thioglycoside donors instead of the imidate donors B-E. Although the syntheses were successful
we observed side reactions on the electron-rich p-methoxyphenyl (MP) protecting group of A.
When activating aromatic thioglycosides with NIS/TfOH we found halogenations or aglycon
transfer on the MP group affecting the purity of the reaction products (data not shown). In
contrast, glycosylation of A with the imidates B or C and catalytic activation with BF3-OEt2
proceeded smoothly, in high yield and exclusive a1,3 regio- and stereoselectivity. Only minor
amounts of double glycosylation products were formed, which could be removed by flash
chromatography. The regioselectivity generally observed for A results from a higher reactivity
of the equatorial OH-group and is assisted by the surrounding protecting groups.
Pentasaccharide 1 (89 % yield) and hexasaccharide 2 (88 % yield) were routinely[6k] converted
to the diols 3 and 4 followed by introduction of the 1,6-arm using the imidates B or D and
activation with TMSOTf. Again a1,6 regio- and stereoselectivity was observed for compounds
5-8 (78-85 % yield) and confirmed by 2D-NMR analysis including 13C-1H coupling constants
for the anomeric carbons.[11] The corresponding bisected N-glycans were synthesized in a one-
pot procedure[6k] by first reacting the primary OH-group of the diols 3 or 4 with the donors B
or D and subsequent addition of GlcNAc donor E (10 equiv.) and TfOH (4 equiv.) at -45 °C.
Following a single protocol with two nearly quantitative conversions the four bisected N-
glycans 9-12 were obtained in high yields (72-80 %). Even the pentaantennary bisected N-
glycan H was obtained in 86 % yield by the one-pot approach whereas the early introduction
of a bisecting GlcNAc gave the analogous pentaantennary intermediate in a far lower yield (19
%).[7c] The glycosylations proceeded without side reactions facilitating purification of the N-
TFAc protected glycans by flash chromatography. Subsequently, the N-glycans 5-8 were
acetylated and selectively deprotected with CAN giving the acceptors 13-16. The bisected
compounds 9-12 were directly oxidized to the acceptors 17-20.
3
This article is protected by copyright. All rights reserved.

10.1002/anie.201807742
Angewandte Chemie International Edition
MPO
NPhth
1)
OH
O
Ph
O
O
O
O
BnO
O
N3
HO
BnO
O
BnO
NPhth
A
a) B or C, BF3xOEt2, CH2Cl2, -45 °C
AcO
R1O
O
AcO
AcO
AcO
O
O
AcO
MPO
AcO
AcO
AcO
NPhth
OH
Ph
O
O
O
O
O
O
NHTFAc
O
BnO
O
N3
1 R1 = Ac (89 %)
2 R1 = Ac3GlcNHTFAc (88 %)
BnO
O
= Ac3GlcNHTFAc
NHTFAc
BnO
NPhth
b) 1. Ac2O/Pyr (5:1); 2. pTosOHxH2O, MeCN
AcO
O
O
R1O
MPO
NPhth
AcO
AcO
AcO
AcO
HO
OAc
O
O
O
O
O
HO
BnO
O
N3
3 R1 = Ac (83 %)
BnO
O
4 R1 = Ac3GlcNHTFAc (79 %)
NHTFAc
BnO
NPhth
R2O
AcO
AcO
AcO
O
O
O
O
O
AcO
AcO
c) B or D, TMSOTf, CH2Cl2, -45 °C
or
d) one-pot: 1. B or D, TMSOTf, CH2Cl2, -45 °C; 2. E, TfOH, CH2Cl2, -45 °C
NHTFAc
O
AcO
5 R1 = Ac, R2 = Ac, R3 = H (78 %)
c)
R1O
AcO
MPO
NPhth
6 R1 = Ac, R2
7 R1 = Ac3GlcNHTFAc, R2 = Ac, R3 = H (79 %)
8 R1 = Ac3GlcNHTFAc, R2 Ac3GlcNHTFAc, R3 = H (72 %)
=
Ac3GlcNHTFAc, R3 = H (83 %)
OAc
O
AcO
AcO
O
O
O
=
BnO
O
O
N3
BnO
AcO
O
R3O
9 R1 = Ac, R2 = Ac, R3 = Ac3GlcNHTFAc (85 %)
d)
BnO
NPhth
NHTFAc
10 R1 = Ac, R2 = Ac3GlcNHTFAc, R3 = Ac3GlcNHTFAc (82 %)
11 R1 = Ac3GlcNHTFAc, R2 = Ac, R3 = Ac3GlcNHTFAc (80 %)
12 R1 = Ac3GlcNHTFAc, R2 = Ac3GlcNHTFAc, R3 = Ac3GlcNHTFAc (81 %)
R2O
AcO
AcO
AcO
O
O
e) 1. Ac2O/Pyr (5:1); 2. CAN, toluene, MeCN, H2O
or
f) CAN, toluene, MeCN, H2O
O
O
O
AcO
AcO
NHTFAc
O
O
13 R1 = Ac, R2 = Ac, R3 = Ac (80 %)
e)
f)
AcO
14 R1 = Ac, R2
=
Ac3GlcNHTFAc, R3 = Ac (69 %)
OH
R1O
AcO
15 R1 = Ac3GlcNHTFAc, R2 = Ac, R3 = Ac (71 %)
16 R1 = Ac3GlcNHTFAc, R2 Ac3GlcNHTFAc, R3 = Ac (83 %)
NPhth
=
OAc
AcO
O
O
O
BnO
O
O
BnO
AcO
AcO
N3
17 R1 = Ac, R2 = Ac, R3 = Ac3GlcNHTFAc (93 %)
O
R3O
18 R1 = Ac, R2 = Ac3GlcNHTFAc, R3 = Ac3GlcNHTFAc (87 %)
19 R1 = Ac3GlcNHTFAc, R2 = Ac, R3 = Ac3GlcNHTFAc (87 %)
20 R1 = Ac3GlcNHTFAc, R2 = Ac3GlcNHTFAc, R3 = Ac3GlcNHTFAc (86 %)
BnO
NPhth
NHTFAc
2)
AcO
AcO
AcO
AcO
AcO
O
O
100
AcO
a)
b)
c)
NHTFAc
4G
O
AcO
AcO
O
4
4G + TMS
O
O
O
O
NHTFAc
AcO
AcO
AcO
NHTFAc
O
O
O
O
AcO
AcO
AcO
-40
100
O
O
CCl3
AcO
AcO
AcO
NHTFAc
O
AcO
G
H
NHTFAc
O
NH
4G
AcO
AcO
4
NHTFAc
one-pot: 1. G, TMSOTf, CH2Cl2, -40 °C;
2. E, TfOH, CH2Cl2, -45 °C
-40
100
AcO
AcO
O
O
AcO
AcO
O
H
O
AcO
MPO
NHTFAc
O
NPhth
OAc
O
AcO
O
O
BnO
O
O
AcO
AcO
N3
BnO
O
-40
BnO
NPhth
NHTFAc
O
2.0
4.0
6.0
time [min]
8.0
10.0
AcO
AcO
AcO
O
H (86 %)
NHTFAc
Scheme 2: 1.) Modular synthesis of eight N-glycan acceptors with (17-20) and without
bisecting GlcNAc (13-16) using acceptor A and imidate donors B-E. The introduction of the
1,6-arm and the bisecting GlcNAc moiety was carried out in a one-pot procedure (step d); 2.)
One-pot synthesis of pentaantennary bisected N-glycan H and HPLC-chromatograms: a)
crude reaction mixture of 4 with G and resulting decasaccharide 4G, b) crude reaction mixture
after addition of E, c) product H after flash chromatography.
4
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10.1002/anie.201807742
Angewandte Chemie International Edition
An exploratory a-fucosylation of acceptor 20 with donor F under the in-situ anomerization
conditions used previously[5a, 5i, 7b, 7c] gave only moderate a-selectivity. For undecasaccharide
21 an a/b ratio of 85:15 was determined by HPLC after flash chromatography (Scheme 3). At
this point we set out for a more selective a1,6-fucosylation method. There is a broad knowledge
on influencing the selectivity of 1,2-cis-glycosylations.[12] For a-fucosylations a non-
participating protecting group at O-2 is required.[13] Despite many examples of a-fucosylations,
there are only few reports where a-fucosylations of acceptors with primary alcohol groups[14]
were studied systematically. It was found that the a-selectivity with primary alcohols is
generally lower compared to secondary alcohols and susceptible mainly to the protecting
groups in the donor in conjunction with the particular reaction conditions.
We started the search for highly a-selective fucosyl donors with the bicyclic fucosyl-oxathianes
Fa and Fb[15] and the readily available chitobiose 22 as a model acceptor. LC-MS analysis
revealed low reactivity towards 22 and product a/b-ratios of 92:8 (Fa) and 86:14 (Fb). The use
of TMS-protected fucosyl iodide Fc[16] gave an a/b ratio of 95:5.
a)
AcO
AcO
20 + F
O
O
AcO
CuBr2, Bu4NBr,
CH2Cl2/DMF (2 : 1)
NHTFAc
AcO
O
O
O
O
O
AcO
AcO
AcO
AcO
HPLC of 21 after flash chromatography:
OMPM
OMPM
NHTFAc
100
O
O
21α
AcO
AcO
O
OMPM
AcO
AcO
H3C
O
O
AcO
21β
NHTFAc
NPhth
O
OAc
O
AcO
0
O
O
BnO
O
O
AcO
AcO
22.00
26.00
30.00
34.00
38.00
N3
BnO
time [min]
O
AcO
AcO
AcO
O
BnO
NPhth
NHTFAc
O
21 (85 %) α/β = 85 : 15
NHTFAc
R1
R2
α/β ratio4
b)
c)
donordr productdr
OR2
OR2
23a[a]
23b[b]
23c[b]
23d[c]
23d[d]
23e[c]
23f[c]
H
Ac
Bn
H
92 : 8
Fa
Fb
Fc
F
OR1
H3C
O
H
86 : 14
95 : 5
OH
H
donors Fa-Fk
O
MPM
MPM
Bn
MPM
MPM
Bn
81 : 19
87 : 13
86 : 14
90 : 10
91 : 9
NPhth
O
NPhth
O
O
O
conditions
F
O
BnO
AcO
BnO
AcO
N3
N3
BnO
BnO
Fe
Ff
Fg
Fh
O
MPM
MPM
MPM
Ac
BnO
NPhth
BnO
NPhth
23g[c]
23h[c]
ClAc
Bz
22
23a-k
96 : 4
conditions:
[a] 1. Tf2O, DIPEA, ClCH2CH2Cl, PhOMe3 (-30 to -10 °C,
2. 22 (-10 to 50 °C), 3. MeOH, CH2Cl2, HCOOH
[b] 1. Bu4NI, DIPEA,CH2Cl2,, 2. MeOH, Amberlyst 15
[c] CuBr2, Bu4NBr, CH2Cl2/DMF (8:1)
OAc
OBn
OBn
OTMS
OTMS
OMe
O
OMe
O
OAc
O
O
S
OTMS
[d] CuBr2, Bu4NBr, DMF
Ph
Ph
H3C
O
S
H3C
O
H3C
O
I
R1
R2
solvent2 α/β ratio4
donordr productd
Fa
Fb
Fc
d)
MPM
MPM
MPM
MPM
Bn
Ac
Bz
Bz
Bz
Bz
DMF
83 : 17
93 : 7
99 : 1
99 : 1
99 : 1
99 : 1
Ff
23f
23h
23h
23h
23i
DMF
Fh
Fh
Fh
Fi
OMPM
OBn
OR
OR
OR
OR
DCM
CHCl3
CHCl3
OMPM
OBn
OMPM
SEt
OBn
SEt
OMPM
OBn
SEt
H3C
O
H3C
O
H3C
O
H3C
O
Bn
pMeO-Bz CHCl3
Fk
23k
SEt
F
Fe
Ff: R = Ac
Fg: R = ClAc
Fh: R = Bz
Fi: R = Bz
Fk: R = pMeO-Bz
conditions: CuBr2 (30 eq.), Bu4NBr (15 eq.), solvent
5
This article is protected by copyright. All rights reserved.

10.1002/anie.201807742
Angewandte Chemie International Edition
Scheme 3: a) fucosylation of N-glycan acceptor 20 with donor F was accompanied by b-linked
fucoside 21b, which can only be removed by HPLC; b) 1,6-fucosylations of chitobiosyl acceptor
22 using donors Fa-Fk. c) a/b-selectivities observed under established conditions. d) a/b-
selectivities observed in neat solvents.
Since we could not improve the a-selectivity with donors Fa-Fc any further we returned to
donor F. The selectivity under in-situ-anomerization conditions (CuBr2/Bu4NBr) increased
slightly by replacing the solvent DCM/DMF (a/b 81:19) by DMF (a/b 87:13). However,
varying the amounts of promotor reagents in neat DMF showed nearly no effect (data not
shown). We thus compared a series of fucosyl donors with different protecting groups (F, Fe-
Fh) under identical reaction conditions. The a/b ratio increased to 86:14 with the perbenzylated
donor Fe. The fucosyl donor (Ff) with two acetates improved the selectivity further (a/b 90:10).
Best results were obtained with the 3,4-benzoylated donor Fh (a/b 96:4). However, when using
neat DMF instead of DCM/DMF the selectivity for donors Ff and Fh dropped. Gratifyingly,
the a/b-ratio of all 3,4-benzoylated donors Fh-Fk was exceeding 99:1 when utilizing pure
DCM or CHCl3 as a solvent (Scheme 3d).
We then tested the donors Fi and Fk under the most promising conditions with N-glycan
acceptor 16. Due a highly viscous reaction mixture the amounts of CuBr2 and TBAB were
reduced to 10 equiv. each. Surprisingly, the a-selectivity of donors Fi and Fk decreased
(Scheme 4a) relative to the reaction with the disaccharide 22 (compounds 23i and 23k). By
doubling the amount of TBAB relative to CuBr2 the targeted a/b-ratio of 99:1 could be restored
for the coupling of the systematically varied donors Fi-Fn with acceptor 15. When omitting the
TBAB completely from the reaction mixtures the a/b-ratio was lowered depending on the p-
substituent (Scheme 4b,c). Notably, in the absence of TBAB the unsubstituted benzoate (Fi)
showed the strongest loss of a-selectivity whereas the p-fluoro derivative Fn was barely
affected. The p-fluorobenzoylated donor Fn was chosen for the further syntheses since the a/b-
ratio even under non-optimal conditions (no TBAB) was already high (97:3). Under optimal
starting conditions donor Fn should provide a/b-ratios of 99:1 even after solvent loss during
preparative fucosylations (2-4 d).
6
This article is protected by copyright. All rights reserved.

10.1002/anie.201807742
Angewandte Chemie International Edition
a)
16 + Fi or Fk
AcO
O
AcO
O
AcO
CuBr2 10eq.
Bu4NBr 10eq.
CHCl3
3d
NHTFAc
AcO
AcO
AcO
O
O
O
O
AcO
AcO
OR
OR
NHTFAc
X
O
O
AcO
AcO
AcO
X
AcO
O
O
OBn
H3C
O
O
AcO
NHTFAc
O
NPhth
O
OAc
O
AcO
O
O
AcO
BnO
O
O
O
AcO
AcO
N3
O
O
BnO
O
BnO
NPhth
NHTFAc
OBn
H3C
O
SEt
24a: R = Bz α/β = 95 : 5
24b: R = pMeO-Bz α/β = 97 : 3
Fi: X = H
Fk:X = OMe
Fl:X = Cl
Fm:X = Ph
Fn:X = F
15 + Fi - Fn
b)
X
X
AcO
AcO
CuBr2 10eq.
± Bu4NBr 20eq.
CHCl3
AcO
AcO
AcO
O
O
O
O
O
AcO
2-4d
10 eq. CuBr2
10 eq. CuBr2
20 eq. Bu4NBr
α/β ratio4
NHTFAc
O
O
O
O
no Bu4NBr
α/β ratio4
O
O
AcO
AcO
O
donordpr roductdrX1
OBn
AcO
AcO
H3C
O
O
AcO
81 : 19
91 : 9
91 : 9
94 : 6
97 : 3
Fi
25i
H
99 : 1
NHTFAc
O
NPhth
O
OAc
O
AcO
Fk
Fl
25k
25l
OMe 99 : 1
O
AcO
BnO
O
O
BnO
AcO
AcO
Cl
Ph
F
99 : 1
99 : 1
99 : 1
N3
O
Fm
Fn
25m
25n
BnO
NPhth
NHTFAc
25i-n
c)
100
100
25iα
25iα
CuBr + TBAB
2
CuBr
2
25iβ
25iβ
28.00
0
0
12.00
16.00
20.00
24.00
32.00
12.00
16.00
20.00
24.00
28.00
32.00
time [min]
time [min]
d)
19
a) Fo (3 eq.)
F
F
TMSOTf (0.2 eq.),
CHCl3 -45 °C, 3h
or
F
F
F
F
AcO
AcO
AcO
AcO
AcO
b) Fp (3 eq.),
TfOH (0.2 eq.),
CHCl3, , -45 °C, 1h
or
O
O
O
O
O
O
O
AcO
O
O
O
O
NHTFAc
O
O
O
O
OBn
OBn
O
AcO
c) Fn (3 eq.) NIS (3 eq.),
TfOH (0.2 eq.), CHCl3
-45 °C, 1h
H3C
O
H3C
O
CF3
NPh
O
AcO
O
O
OBn
AcO
AcO
O
H3C
O
O
AcO
CCl3
O
NHTFAc
O
Fo
Fp
NPhth
O
OAc
O
AcO
O
O
NH
BnO
O
O
BnO
AcO
AcO
N3
O
AcO
O
BnO
NPhth
NHTFAc
O
AcO
AcO
32
NHTFAc
a) α/β = 99 : 1 (85 % conversion of acceptor, HPLC)
b) α/β = 99 : 1 (80 % yield)
c) α/β = 97 : 3 (93 % conversion of acceptor, HPLC)
Scheme 4: a) Promotor ratio affects a-selectivity in fucosylations of N-glycan acceptor 16 with
donors Fi and Fk; b) a1,6-fucosylations of acceptor 15 using fucosyl donors Fi-Fn are affected
by the p-substituents X of the benzoyl groups and by omitting Bu4NBr; c) RP-HPLC
chromatograms of the synthesis of 25i with (left) and without TBAB (right) d) rapid a1,6-
fucosylation of 19 with imidates Fo, Fp and thioglycoside Fn.
7
This article is protected by copyright. All rights reserved.

10.1002/anie.201807742
Angewandte Chemie International Edition
The donors Fi-Fn can be regarded as superdisarmed[12] requiring large excess of donor and long
reaction times. Most likely the corresponding a-bromide generated after activation with
CuBr2/TBAB is not reactive enough to give a glycoside whereas the small amounts of more
reactive b-bromide generated via in-situ-anomerization[17] lead to the desired a-glycoside[18]
under the conditions found to be optimal for a1,6-selective fucosylation (halogenated solvent,
no DMF, excess TBAB). Under less optimal conditions (e.g. CuBr2 without TBAB) other
pathways may operate and compromise the otherwise nearly exclusive a-selectivity. In those
cases the p-substituted benzoyl groups[19] of the donors Fi-Fn may provide a valuable tool for
tuning the a-selectivity in glycosylations lacking in-situ-anomerization. This assumption was
tested with the trichloacetimidate[20] of the p-fluorobenzoylated fucose (Fo) and the acceptor
19 (Scheme 4d). Using 0.2 equiv of TMSOTf the decasaccharide 32 was obtained with an
excellent a/b-ratio (99:1) and a much shorter reaction time (3 h, -45 °C). Since the imidate Fo
gave only 85 % conversion we also tested the corresponding N-phenylimidate[21] Fp. Here full
conversion was observed with identical a/b-ratio (99:1). Thus in-situ anomerization conditions
are not essential for high a-selectivity in the fucosylation of primary acceptors. The
fucosylation of acceptor 19 with thioglycoside Fn in CHCl3 and activation with NIS/TfOH at -
45° C gave a slightly lower a/b-ratio (97:3).
The preparative fucosylations were carried out using donor Fn under the robust in-situ-
anomerization conditions established above. The N-glycan acceptors 13-20 were reacted with
10 equiv. of F, 10 equiv. of CuBr2 and 12 equiv. of TBAB in CHCl3 for 2-7 days. After
purification by flash chromatography the yields for the core fucosylated compounds 26-33 were
generally high (76-83 %) and the observed a/b-ratio was exceeding 99:1. For selective removal
of the benzyl groups in the presence of an azide an oxidative debenzylation was carried out by
[22]
using either Na2S2O4/NaBrO3
or the recently established NBS[23] as a more controllable
source for bromine radicals. After cleavage of the base-labile protecting groups and acetylation
the core fucosylated N-glycan azides G9-G16 were obtained in good yields. The final products
can be purified by RP-HPLC since the presence of 1,6-linked fucose leads to stronger retention
on the column. The NMR spectra of the N-glycans G9-G16 were fully assigned confirming the
regio- and stereoselectivity. The core fucosylated N-glycans showed the typical 13C signal for
C-61 at 67.8 ppm. By applying the deprotection sequence to the non-fucosylated precursors 13-
20 the synthesis of all mammalian type N-glycan cores G1-G16 through a single pathway can
be formally completed.
8
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10.1002/anie.201807742
Angewandte Chemie International Edition
13-20
Fn (10 eq.)
CuBr2,(10 eq.)
TBAB (12 eq.)
CHCl3
R2
O
AcO
AcO
O
O
O
O
O
AcO
OFBz
OFBz
AcO
NHTFAc
AcO
26 R1 = Ac, R2 = Ac, R3 = Ac (81 %)
27 R1 = Ac, R2 Ac3GlcNHTFAc, R3 = Ac (77 %)
28 R1 = Ac3GlcNHTFAc, R2 = Ac, R3 = Ac (80 %)
29 R1 = Ac3GlcNHTFAc, R2 Ac3GlcNHTFAc, R3 = Ac (76 %)
O
O
OBn
=
H3C
O
AcO
R1
O
=
O
AcO
NPhth
O
OAc
AcO
30 R1 = Ac, R2 = Ac, R3 = Ac3GlcNHTFAc (83 %)
O
O
O
BnO
O
O
BnO
AcO
AcO
31 R1 = Ac, R2 = Ac3GlcNHTFAc, R3 = Ac3GlcNHTFAc (79 %)
32 R1 = Ac3GlcNHTFAc, R2 = Ac, R3 = Ac3GlcNHTFAc (81 %)
33 R1 = Ac3GlcNHTFAc, R2 = Ac3GlcNHTFAc, R3 = Ac3GlcNHTFAc (79 %)
N3
R3
O
BnO
NPhth
NHTFAc
26-29:
30-33:
1. Na2S2O4, NaBrO3, EtOAc, H2O
2. ethylene diamine, n-BuOH 80 °C
3. Ac2O/pyridine
1. NBS, CH2Cl2, phosphate buffer (2M, pH 3) 21-24
2. ethylene diamine, n-BuOH 80 °C
3. Ac2O/pyridine
4. MeNH2 (40 % in H2O), MeOH
4. MeNH2 (40 % in H2O), MeOH
GlcNAc
Man
Fuc
N3
N3
N3
N3
N3
N3
N3
N3
G9 (71 %)
G10 (44 %)
G11 (79 %)
G12 (70 %)
G13 (56 %)
G14 (48 %)
G15 (43 %)
G16 (53 %)
Scheme 5: optimized a-selective synthesis of core-fucosylated and bisected N-glycans 27-34
using donor Fn followed by deprotection to N-glycan azides G9-G16.
In summary, a modular synthesis of all the 16 cores of mammalian complex-type N-glycans
with optional core fucose and bisecting GlcNAc was established following a single approach.
Key features for highly a-selective 1,6-fucosylations of N-glycans were superdisarmed donors
and mild activation in halogenated solvents. By integrating a one-pot protocol even the most
demanding core fucosylated and bisected N-glycans were synthesized with unprecedented
efficiency and purity. Enzymatic elongation of these glycans can provide the tools needed for
the investigation of the biological interplay of the ubiquitous bisecting GlcNAc and core fucose
modifications.
We are grateful for financial support by Deutsche Forschungsgemeinschaft (DFG, SPP 1623).
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