A unified strategy for the synthesis of mucin cores 1-4 saccharides and the assembled multivalent glycopeptides

Article abstract of DOI:10.1002/chem.201302921

By displaying different O-glycans in a multivalent mode, mucin and mucin-like glycoproteins are involved in a plethora of protein binding events. The understanding of the roles of the glycans and the identification of potential glycan binding proteins are major challenges. To enable future binding studies of mucin glycan and glycopeptide probes, a method that gives flexible and efficient access to all common mucin core-glycosylated amino acids was developed. Based on a convergent synthesis strategy starting from a shared early stage intermediate by differentiation in the glycoside acceptor reactivity, a common disaccharide building block allows for the creation of extended glycosylated amino acids carrying the mucin type-2 cores 1-4 saccharides. Formation of a phenyl-sulfenyl-N-Troc (Troc=trichloroethoxycarbonyl) byproduct during N-iodosuccinimide-promoted thioglycoside couplings was further characterized and a new methodology for the removal of the Troc group is described. The obtained glycosylated 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acid building blocks are incorporated into peptides for multivalent glycan display. One for all! In a convergent approach starting from a shared early-stage intermediate by differentiation in the glycoside acceptor reactivity, a common disaccharide building block allows for the creation of extended glycosylated amino acids carrying the mucin type-2 cores 1-4 saccharides (see scheme). The amino acids are incorporated into peptides for multivalent glycan display. Ac=acetyl, Troc=trichloroethoxycarbonyl, TBS=tert-butyl dimethylsilyl

Full text of DOI:10.1002/chem.201302921

FULL PAPER
DOI: 10.1002/chem.201302921
A Unified Strategy for the Synthesis of Mucin Cores 1–4 Saccharides and the
Assembled Multivalent Glycopeptides
Christian Pett, Manuel Schorlemer, and Ulrika Westerlind*[a]
Dedicated to Professor Thomas Norberg on the occasion of his 65th birthday
Abstract: By displaying different O-
glycans in a multivalent mode, mucin
and mucin-like glycoproteins are in-
volved in a plethora of protein binding
events. The understanding of the roles
of the glycans and the identification of
potential glycan binding proteins are
major challenges. To enable future
binding studies of mucin glycan and
glycopeptide probes, a method that
gives flexible and efficient access to all
a convergent synthesis strategy starting
from a shared early stage intermediate
by differentiation in the glycoside ac-
ceptor reactivity, a common disaccha-
a
phenyl-sulfenyl-N-Troc (Troc=tri-
chloroethoxycarbonyl) byproduct
during N-iodosuccinimide-promoted
thioglycoside couplings was further
characterized and a new methodology
for the removal of the Troc group is
described. The obtained glycosylated 9-
fluorenylmethoxycarbonyl (Fmoc)-pro-
tected amino acid building blocks are
incorporated into peptides for multiva-
lent glycan display.
AHCTUNGTRENNrGUN ide building block allows for the crea-
tion of extended glycosylated amino
acids carrying the mucin type-2
cores 1–4 saccharides. Formation of
Keywords: amino acids · carbohy-
drates · glycopeptides · glycosyla-
tion · reactivity
common
mucin
core-glycosylated
amino acids was developed. Based on
Introduction
often extended with Gal and GlcNAc residues and terminat-
ed with fucose, sialic acid, sulfation, Gal, and/or GalNAc.
Various bioactive glycoconjugates contain the type-2 disac-
charide unit N-acetyllactosamine [b-d-Gal-(1!4)-b-d-
GlcNAc], which serves as a common scaffold for the Lewis
antigens for example, SLex, Lex, and Ley. These epitopes are
responsible for generating diversity by extending or termi-
nating the mucin-type core glycan structures.[5]
Several syntheses of mucin-type core-glycosylated amino
acids and glycopeptides have been described,[6] which have
mainly focused on specific protein targets, in particular
tumor-associated mucin-1 epitopes and E-, P-, and L-selectin
ligands.[7] In contrast to previously prepared P-selectin gly-
copeptide ligands involving a single glycan binding site, the
multivalent mucin peptide tandem repeats are densely gly-
cosylated, thereby posing a synthesis problem that differs
fundamentally from the challenge posed by monovalent gly-
copeptide structures. In the diverse mucin glycopeptide
structures oligosaccharide diversity is generated by differing
the extensions of structurally common core-glycosylated
amino acids. Therefore, a synthesis strategy could be envis-
aged that gives flexible and efficient access to all mucin
core-glycosylated amino acids, and in extension also glyco-
peptides, based on a small set of common building blocks.
We here report a unified synthesis of N-acetyllactos-
Mucins are highly glycosylated proteins that populate the
cell surface of epithelial tissues in a membrane-bound or se-
creted form.[1] By displaying different O-glycans in a multiva-
lent mode, mucins and mucin-like glycoproteins are involved
in a plethora of protein binding events.[2] With the diversity
of glycan structures found in biological systems in mind, the
understanding of the roles of the glycan and the identifica-
tion of potential glycan binding proteins is a major chal-
lenge. Here, chemical synthesis can enable glycobiology by
supplying well-defined glycan and glycopeptide probes, thus
making systematic glycan binding studies feasible. In spite
of the structural diversity of mucin-type O-glycans, they all
have in common that a GalNAc (Ac=acetyl) residue is con-
nected to the protein backbone through an O-glycosidic
linkage to serine or threonine.[3] The branching of the
GalNAc residue in the 3- or 6-position with Gal, GlcNAc,
or GalNAc gives rise to different core structures (1–8), and
the core structures 1–4 are the most common structures
identified in humans (Figure 1A).[4] These structures are
[a] Dipl.-Chem. C. Pett, M. Sc. M. Schorlemer, Dr. U. Westerlind
Gesellschaft zur Fçrderung der Analytischen
Wissenschaften e.V. ISAS–Leibniz
Institute for Analytical Sciences
Otto-Hahn-Strasse 6b, 44227 Dortmund (Germany)
Fax : (+49)231-1392-4850
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
and mucin 5B (MUC5B) glycopeptides. The method em-
ploys a disaccharide key building block responsible for se-
lective extension of GalNAc- (Tn) or Gal-GalNAc-Thr (T-
E-mail: ulrika.westerlind@isas.de
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201302921.
Chem. Eur. J. 2013, 19, 17001 – 17010
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
17001

Figure 1. A) Core 1–4 structures of mucin-type glycosylation. B) A unified synthesis strategy for building up extended core 1–4 structures by using
common precursor building blocks. tBu=tert-butyl, Troc=trichloroethoxycarbonyl, TBS=tert-butyl dimethylsilyl, PMP=p-methoxyphenyl.
antigen) amino acids. By using orthogonal protecting groups
that direct the reactivity, the stereoselectivity, and are fur-
ther removable under mild conditions, the obtained core-
glycosylated 9-fluorenylmethoxycarbonyl solid-phase pep-
tide synthesis (Fmoc-SPPS) building blocks are readily
available for glycopeptide synthesis after just a few steps
from the common precursors.
lected, because these groups can be removed under mild
conditions like diluted sodium methoxide in methanol.[9]
The acetyl groups further stabilize the O-glycosidic bond
during the acidic trifluoroacetic acid (TFA) treatment, re-
quired for resin cleavage of the synthesized peptides.[10] For
the design of a straightforward route to mucin-type Fmoc-
protected amino acid building blocks, a retrosynthetic analy-
sis was carried out. We focused on a convergent coupling
approach starting from common precursor building blocks,
for example, an N-trichloroethoxycarbonyl (NHTroc)-pro-
tected thioglycoside disaccharide donor and Tn or T-antigen
acceptors. By using this strategy and further relying on or-
thogonal protecting groups, as well as internal reactivity dif-
ferences of the acceptors, the different type-2 N-acetyllac-
tosamine-extended core 1, core 2, core 3, and core 4 Fmoc-
SPPS amino acids were obtained in a few steps from the
precursor building blocks. We used the thioglycoside donor
4, containing a tert-butyldimethylsilyl group in the 6-position
and N-Troc protection in the 2-position of the GlcN residue
as a common building block for core extension. The choice
of the glycan protecting groups was considered to be critical
for the proceeding key glycosylation steps. The O-TBS
Results and Discussion
Synthetic strategy: We outline a strategy to synthesize
mucin glycopeptides by using glycosylated Fmoc-protected
amino acid building blocks incorporated by a stepwise pep-
tide assembly according to the standard Fmoc solid-phase
peptide synthesis protocol. A triethylene glycol spacer was
incorporated at the N terminus of the glycopeptides to have
additional spacing for later immobilization on an N-
hydroxyACHTUNGTRENNUNG
succinimde (NHS)-activated microarray surface.[8]
The choice of the protecting groups on the glycosylated
amino acid is critical for a successful glycopeptide synthesis.
Here, O-acetyl protection of the saccharide portion was se-
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A Unified Strategy for the Synthesis of Mucin Cores 1–4 Saccharides
FULL PAPER
group and the N-Troc protection contributed positively by
stereoelectronic effects to the increased reactivity of the
common donor 4, which in the later deprotection steps were
easily removed under mild conditions.[11] Furthermore, the
Troc group directs the stereochemistry of the glycosylation
by neighboring group participation, resulting in the forma-
tion of the desired b-glycosidic bond.[11a,b] To obtain good
yields and avoid negative steric effects during the key glyco-
sylation steps, a strategy was selected making use of the in-
creased reactivity of the 3- and 6-position on the Gal and
GalNAc residues. By disconnections A-, D-, and F-selective
glycosylation would take place on the more reactive primary
hydroxyl group in the 6-position of GalNAc, without protec-
tion of the neighboring 4-position, yielding the core 2 hexa-
saccharide 17, the core 2 tetrasaccharide 22, and the core 4
pentasaccharide 37 starting from the acceptors core 1 tetra-
saccharide 11, the T antigen disaccharide 18, and the core 3
trisaccharide 30, respectively. Accordingly, the core 1 tetra-
saccharide 11 (disconnection B), is prepared by selective
glycosylation in the 3-position of acceptor 8 without protec-
tion of the less reactive 2- and 4-hydroxyl groups. The disac-
charide acceptors 8 and 18 as well as the core 3 trisaccharide
30 are all obtained from the common precursor GalNAc-
Thr amino acid 5 by disconnections C, E and G (Figure 1B).
The chemical synthesis commenced with the preparation
of the common disaccharide donor 4, which in later steps is
responsible for the N-acetyllactosamine extension of the dif-
ferent glycan core structures. Starting from the known
GlcN-Troc-protected thioglycoside 1,[12] removal of the
acetyl protecting groups by using HCl/MeOH was followed
by tert-butyldimethylsilyl protection of the 6-position to
form compound 2. Regioselective glycosylation at the more
reactive 4-position of the GlcN-Troc acceptor 2 with tri-
chloroacetimidate (3)[13] activated by trimethylsilyltriflate
(TMSOTf)[14] at ꢀ408C gave disaccharide 4 (Scheme 1A).
For preparation of the core 1 tetrasaccharide 13 and
core 2 hexasaccharide 17 Fmoc-protected amino acids, two
regioselective glycosylations were carried out starting from
the thioglycoside donor 4 and the T-antigen Gal-GalNAc-
Thr disaccharide acceptor 8, respectively. Initially, the T-an-
tigen Thr conjugate 8 was formed by reaction of the
GalNAc-Thr compound 5[15] with the donor thioglycoside
6,[16] activated by NIS/TfOH[17] to give the disaccharide 7.
Removal of the galactose acetyl protecting groups by using
NaOMe in methanol at pH 9.5 was followed by reintroduc-
tion of the partially cleaved Fmoc group yielding the disac-
charide acceptor 8. By NIS/TfOH activation at 08C, glyco-
ing group manipulations. Initially, the TBS groups of the tet-
rasaccharide 9 were removed by using tetrabutylammonium-
fluoride buffered with acetic acid to avoid cleavage of the
base-sensitive Fmoc group. The TBS removal was followed
by acetylation yielding compound 10. Hydrolysis of the ben-
zylidene acetal by 80% aqueous acetic acid gave compound
11. Reductive elimination of the N-Troc group and subse-
quent acetylation followed. In a final step, the tBu ester was
cleaved by treatment with TFA in dichloromethane to give
the core 1 tetrasaccharide-containing Fmoc-protected build-
ing block 13 in a total yield of 29% over seven steps starting
from the common T-antigen precursor 8 (Scheme 1B).
For the preparation of the core 2 hexasaccharide 17,
a second glycosylation of the core 1 tetrasaccharide inter-
mediate 11 was performed by using the thioglycoside donor
4 activated with NIS/TfOH. The hexasaccharide coupling
product 14 was formed with the desired stereo- and regiose-
lectivity. A few protecting group manipulations followed to
generate the Fmoc-protected core 2 hexasaccharide building
block 17 in a total yield of 17% over ten steps starting from
the common T-antigen precursor 8 (Scheme 1C).
Starting from the known T-antigen amino acid intermedi-
ate 18[6d] and the thioglycoside donor 4 activated by NIS/
TfOH at 08C, the core 2 tetrasaccharide 19 was obtained. In
accordance with the procedures described above, the
formed coupling product 19 was converted to the Fmoc-pro-
tected core 2 tetrasaccharide building block 22, which was
obtained in a total yield of 37% over six steps starting from
the common T-antigen precursor 18 (Scheme 1C).
In the initial attempts to generate the core 3 Fmoc-SPPS
building block starting from the Tn amino acid acceptor 5
and thioglycoside donor 4 promoted by NIS/TfOH at 08C,
the coupling reaction proceeded very slowly and gave a mix-
ture of two compounds; the desired trisaccharide coupling
product 23 and byproduct 24. Supported by NMR spectro-
scopic and mass spectrometry data, the formed byproduct
was found to contain an extra phenyl-sulfenyl group at the
N-Troc amine. A plausible mechanism for the formation of
compound 24 suggests that the nitrogen atom in the oxa-
AHCTUNGTREGzNNNU oline intermediate obtained from thioglycoside donor 4
acted as a nucleophile on the phenyl-sulfenyl iodide inter-
mediate to form the PhS-N-Troc product 24 (Scheme 2A).
A number of conditions were investigated to suppress the
formation of compound 24, however the generation of 24
could not be completely avoided. Because the Troc group
ꢀ
and the labile phenyl-sulfenyl N S bond could be cleaved
during reductive zinc treatment used in later steps, condi-
tions with increased amounts of NIS were selected instead,
thus promoting the formation of product 24 exclusively. In
a few steps, the coupling product 24 was converted to the
desired Fmoc-SPPS building block 27 in a total yield of
41% over four steps starting from the Tn precursor 5
(Scheme 2B). In accordance with the synthesis of the core 2
hexasaccharide, the two core 3 products 23 and 24 were con-
ACHTUNGTRENNUNGsylACHTUNGTRENNUNGation took place providing the core 1 tetrasaccharide 9 in
good yield and with complete regio- and stereoselectivity re-
sulting from the higher reactivity of the 3-position of the
galactose in the T-antigen acceptor, compared with the hy-
droxyl groups in 2- and 4-position. Avoiding steric hindrance
from neighboring protecting groups of the acceptor during
disaccharide extension further contribute to the good yield
of the coupling product 9 (Scheme 1B). The conversion of
tetrasaccharide 9 to a stable building block compatible with
solid-phase peptide synthesis, was achieved by a few protect-
verted to accepACTHUNTGRNEUNGtors 30 and 31, respectively, suitable for fur-
ther disaccharide extension resulting in the pentasaccharide
core 4 coupling products 32 and 33, respectively. After ex-
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U. Westerlind et al.
Scheme 1. A) Synthesis of the disaccharide donor elongation building block. B) Synthesis of extended core 1 and core 2, tetra- and hexasaccharide Fmoc-
protected amino acids. C) Synthesis of an extended core 2 tetrasaccharide Fmoc-protected amino acid. Pyr=pyridine, Tf=triflate, Su=succinimidyl,
TBAF=tetrabutylammonium fluoride, DMAP=4-dimethylaminopyridine.
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A Unified Strategy for the Synthesis of Mucin Cores 1–4 Saccharides
FULL PAPER
Scheme 2. A) A plausible mechanism for the formation of the PhS-N-Troc product 24. B) Synthesis of extended core 3 and core 4 tri- and pentasaccha-
ride Fmoc-protected amino acids.
ACHTUNGTRENNUNG
change of the TBS group for an acetyl group the removal of
the Troc groups to give the common intermediate 36 fol-
lowed. The reduction of the Troc groups by treatment with
zinc or cadmium in acetic acid[18] were very slow for both
compounds and did not affect the PhS-N-Troc product 35.
Instead, samarium iodide (SmI2) was investigated as a reduc-
ing agent; it has previously been employed to break labile
19 (via 23) and 15% (via 24) over ten steps starting from
the Tn precursor 5. (Scheme 2B).
To optimize the synthesis of multivalent mucin glycopep-
AHCTUNGTREGtNNNU ides, the shorter T-antigen disaccharide 38 and the core 3
trisaccharide 27 Fmoc-SPPS building blocks were used. Cou-
pling of the standard Fmoc-protected amino acids (8 equiv)
was carried out by using O-(benzotrialzol-1-yl)-tetrameth-
[19]
ꢀ
heteroatom linkages like N S bonds.
An earlier study
A
hexafluorophosphate/1-hydroxybenzotriazole
showed that SmI2 in THF cleaves 2-chloroethoxy carbamate
groups.[20] In a modified procedure that might have broader
applications for Troc deprotection of complex carbohydrates
or natural products, treatment of compound 35 with SmI2 in
THF/tBuOH removed the Troc groups and PhS adducts
within a few hours at room temperature. After subsequent
acetylation compound 36 was obtained in a good yield. Final
tBu removal by using TFA in dichloromethane gave the
Fmoc-protected core 4 pentasaccharide 37 in a total yield of
(HBTU/HOBt),[21] the glycosylated Fmoc-protected threo-
nine building blocks (1.5 equiv) were coupled with O-(7-aza-
benotriazol-1-yl)-tetramethyluronium hexafluorophosphate/
1-Hydroxy-7-azabenzotriazole (HATU/HOAt).[22] The glyco-
sylated amino acids were pre-activated in a smaller volume
of solvent and added manually to the resin. Furthermore,
the reaction times of the glycosylated amino acids were ex-
tended and the two amino acids followed were double cou-
pled. After assembly of the full mucin tandem repeat pep-
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U. Westerlind et al.
Scheme 3. Synthesis of multivalent MUC1 and MUC5B glycopeptides containing core 1 and core 3 glycans. Overall yields in parenthesis starting from
preloaded Fmoc-Ala or Fmoc-Pro trityl resin. DIPEA=diisopropylethylamine
Experimental Section
tides on the resin, the peptides were terminated with cou-
pling of a triethylene glycol spacer and subsequent detach-
General methods and the synthesis of compounds 2–4, 7, 8, 19—37, and
ment from the resin by applying a mixture of trifluoracetic
acid/triisopropylsilane (TIPS)/H2O (90:5:5). After a desalting
step on a C-18 cartridge, the O-acetyl groups were removed
by treatment with catalytic amounts of NaOMe in methanol
at pH 9.5. Finally, the deprotected T-antigen and core 3 gly-
copeptides were purified by preparative HPLC to obtain
compounds 39–66 (Scheme 3).
39–66 are described in the Supporting Information.
Synthesis of the type-2 core 1 tetrasaccharide building block
N-9-Fluorenylmethyloxycarbonyl-O-(2-acetamido-2-deoxy-4,6-O-para-me-
thoxybenzylidenacetal-3-O-{6-tert-butyldimethylsilyl-3-O-[3-O-acetyl-2-de-
oxy-6-tert-butyldimethyldimethylsilyl-2-N-(2,2,2-trichloroethoxycar
amino)-4-O-(2,3,4,6-tetra-O-acetyl-b-d-galactopyranosyl)-b-d-glucopyra
osyl]-b-d-galactopyranosyl}-a-d-galactopyranosyl)-l-threonine-tert-butyl-
ester (Fmoc-Thr(bAc4Gal-
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
A
E
bTBDMS-Gal-(1!3)-aPMP-GalNAc)-OtBu) (9): Compounds 8 (3.98 g,
4.00 mmol) and 4 (4.88 g, 5.23 mmol) were dissolved in anhydrous di-
chloromethane (60 mL) followed by addition of activated molecular
sieves 4 ꢁ (5 g). The mixture was stirred for 1 h under an argon atmo-
Conclusion
AHCTUNGERTGsNNUN phere, before it was cooled down to 08C. Then slow addition of N-iodo-
succinimide (1.18 g, 5.23 mmol) and a suspension of trifluoromethanesul-
fonic acid (53 mL, 91 mg, 0.61 mmol) in dry dichloromethane (1 mL) fol-
In conclusion, we have developed an efficient methodology
for the synthesis of extended mucin core-glycosylated amino
acids. Based on a convergent approach with common pre-
cursor building blocks, the core 1–4 Fmoc-protected building
blocks were obtained in excellent yields. Key glycosylation
reactions proceeded with complete regio- and stereoselectiv-
ity without protection of neighboring hydroxyl groups
taking advantage of the increased reactivity of the galactose
3- and 6-position. The described methodology opens up the
way for the syntheses of other extended core amino acid
building blocks by using similar strategies. With the first N-
acetyllactosamine-extended core amino acids in hand, differ-
ent multivalent mucin glycopeptides can be prepared, which
will enable future biological studies. The first syntheses of
mucin multivalent peptides were optimized by using the di-
and trisaccharide core 1- and core 3-glycosylated amino
acids.
lowed. After 2.5 h additional portions of the thioglycoside donor
4
(1.12 g, 1.20 mmol) and N-iodosuccinimide (270 mg, 1.2 mmol) were
added and the reaction was stirred for 1.5 h at 08C. Then it was diluted
with dichloromethane (40 mL) and the molecular sieve was filtered off
and washed with dichloromethane. The mixture was diluted with addi-
tional dichloromethane to reach a total volume of 200 mL. The organic
phase was washed with a 0.5m aqueous solution of sodium thiosulfate,
a saturated aqueous solution of sodium bicarbonate, water and brine
(80 mL each). Then the solution was dried over sodium sulfate, filtered,
and concentrated in vacuum. The residue was purified by silica column
chromatography (cyclohexane/EtOAc gradient from 2:1 to 1:1) to give
compound 9 (4.71 g, 65% 2.59 mmol) as a colorless solid. Rf =0.45 (cyclo-
hexane/EtOAc 2:3); [a]D20 = +42.22 (c=0.99 in CHCl3); 1H NMR
(600 MHz, [D6]DMSO, 308C, internal DMSO d(H)=2.50 ppm): d=7.90–
7.88 (m, 2H; H-4-Fmoc, H-5-Fmoc), 7.76–7.73 (m, 2H; H-1-Fmoc, H-8-
Fmoc), 7.63 (d, 3J
(NH,Ta)=9.8 Hz, 1H; NH-Fmoc), 7.47 (d, 3J
NH-Ac), 7.42 (t, 3J(H-2,H-1)=3J(H-2,H-3)=3J(H-7,H-6)=3J
7.4 Hz, 2H; H-2-Fmoc, H-7-Fmoc), 7.37 (d, 3J(H-2,H-3)=3J
8.8 Hz, 2H; H-2-PMP, H-6-PMP), 7.31 (t, 3J(H-3,H-4)=3J(H-3,H-2)=3J-
(H-6,H-7)=3J(H-6,H-5)=7.4 Hz, 2H; H-3-Fmoc, H-6-Fmoc), 6.93 (d, 3J-
(H-3,H-2)=3J
(H-5,H-6)=8.8 Hz, 2H; H-3-PMP, H-5-PMP), 5.52 (s, 1H;
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
A
E
N
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
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A Unified Strategy for the Synthesis of Mucin Cores 1–4 Saccharides
FULL PAPER
CH-(PMP)), 5.24 (d, 3J(H-4’’’,H-3’’’)=3.6 Hz, 1H; H-4’’’), 5.11 (dd, 3J(H-
3’’’,H-4’’’)=3.5, 3J(H-3’’’,H-2’’’)=10.3 Hz, 1H; H-3’’’), 4.98 (d, 3J(H-1’’,H-
2’’)=8.5 Hz, 1H; H-1’’), 4.93 (t, 3J(H-3’’,H-2’’)=3J(H-3’’,H-4’’)=6.6 Hz,
1H; H-3’’), 4.89 (d, 3J(CH2a,CH2b)=12.4 Hz, 1H; CH2a-(Troc)), 4.85–4.82
3H; Tg); 13C NMR (150.9 MHz, [D6]DMSO): d=170.3, 170.0, 169.9,
169.5, 169.2, 169.1, 168.7 (C=O-(Ac), C=O-(tBu)), 159.5 (C1-PMP), 156.8
(C=O-(Fmoc)), 154.0 (C=O-(Troc)), 143.7 (C-1a-Fmoc, C-8a-Fmoc), 140.8
(C-4a-Fmoc, C-5a-Fmoc), 130.8 (C-4-PMP), 127.7 (C-2-PMP, C-6-PMP),
127.0 (C-3-Fmoc, C-6-Fmoc, C-2-Fmoc, C-7-Fmoc), 125.3, 125.1 (C-1-
Fmoc, C-8-Fmoc), 120.2 (C-4-Fmoc, C-5-Fmoc), 113.4 (C-3-PMP, C-5-
PMP), 101.5 (C-1’), 100.6 (C-1’’), 100.0 (C-1’’’), 99.8 (CH-(PMP)), 99.1
(C-1), 96.1 (Cquart-(Troc)), 81.5 (Cquart-(tBu)), 78.1 (C-3’), 76.0 (C-4’’), 75.1,
75.0 (Tb, C-3), 73.4 (C-5, CH2ab-(Troc)), 72.8 (C-3’’), 71.5 (C-5’’), 70.7 (C-
5), 70.3 (C-3’’’), 69.6 (C-5’’), 69.4 (C-4’), 69.1, 68.9 (C-2’, C-2’’), 68.6 (C-
ACTHNUTRGNEUNG
(m, 1H; H-2’’’), 4.77 (d, 3J(H-1,H-2)=3.3 Hz, 1H; H-1), 4.72 (d, 3J(H-
1’’’,H-2’’’)=7.9 Hz 1H; H-1’’’), 4.68–4.65 (m, 2H; CH2b-(Troc), OH-2’),
4.48–4.40 (m, 3H; CH2ab-(Fmoc), OH-4’), 4.36–4.26 (m, 5H; H-2, H-5, H-
1’, H-9-(Fmoc), Tb), 4.15–4.10 (m, 2H; H-5’’’, Ta), 4.06–3.97 (m, 4H; H-
00
6ab, H-60a0b0 ), 3.83–3.62 (m, 11H; H-3, H-4, H-4’, H-4’’, H-0ab, H-6 , CH3-
ab
(PMP)), 3.45–3.41 (m, 3H; H-2’, H-2’’, H-5’’), 3.36–3.30 (m, 2H; H-5’, H-
6
6
ab), 67.0 (C-4’’’), 65.6 (CH2ab-(Fmoc)), 62.9 (C-4), 62.5 (C-60ab), 61.8 (C-
3’), 2.09–1.86 (m, 18H; CH3-(Ac)), 1.39 (s, 9H; tBu-(Thr)), 1.12 (d, 3J-
00
000
), 60.8 (C-6 ), 59.3 (Ta), 55.8 (C-2’’), 55.1 (CH3-(PMP)), 46.9, 46.8 (C-
ACHTUNGTRENNUNG
(Tg,Tb)=6.2 Hz, 3H; Tg), 0.87 (s, 9H; tBu-(TBS)), 0.83 (s, 9H; tBu-
ab
ab
2, C-9-(Fmoc)), 27.6 (tBu), 22.9, 20.8, 20.7, 20.6, 20.5, 20.4, 20.3 (CH3-
(TBS)), 0.05 (s, 6H; 2ꢂMe
ACHUTGTNRENNUG(TBS)), 0.04 (s, 3H; MeACHTUNGTREN(NUNG TBS)), 0.03 ppm (s,
+
(Ac)), 19.3 ppm (Tg); HRMS (ESI) (pos): m/z calcd for C78H97Cl3N3O36
:
:
3H; Me
ACHTUNGTRENNUNG
2+
1756.4917 [M+H]+; found: 756.4922; calcd for C78H97Cl3KN3O36
d(C)=39.51 ppm): d=169.9, 169.5, 169.3, 169.0 (C=O-(Ac), C=O-(tBu)),
159.5 (C-1-PMP), 156.7 (C=O-(Fmoc)), 154.4 (C=O-(Troc)), 143.8, 143.5
(C-1a-Fmoc, C-8a-Fmoc), 140.8 (C-4a-Fmoc, C-5a-Fmoc), 130.8 (C-4-
PMP), 127.6 (C-2-Fmoc, C-7-Fmoc, C-2-PMP, C-6-PMP), 127.0 (C-3-
Fmoc, C-6-Fmoc), 125.2 (C-1-Fmoc, C-8-Fmoc), 120.2, 120.1 (C-4-Fmoc,
C-5-Fmoc), 113.3 (C-3-PMP, C-5-PMP), 105.4 (C-1’), 100.3 (C-1’’), 2ꢂ
99.8, 99.7 (C-1, C-1’’’, CH-(PMP)), 96.2 (Cquart-(Troc)), 81.4 (Cquart-(tBu-
(Thr))), 80.4 (C-5’’), 75.8 (Tb), 75.6 (C-4’’), 75.1, 75.0 (C-3, C-3’, C-5’),
74.2 (C-5), 73.5 (C-3’’), 73.4 (CH2ab-(Troc)), 70.3 (C-3’’’), 69.9 (C-2’), 69.8
897.7277 [M+K+H]2+; found: 898.7227.
N-9-Fluorenylmethyloxycarbonyl-O-(2-acetamido-2-deoxy-3-O-{2,4,6-tri-
O-acetyl-3-O-[3,6-di-O-acetyl-2-deoxy-2-N-(2,2,2-trichloroethoxycarbon-
AHCTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
A
1.39 mmol) in a 80% aqueous solution of AcOH (50 mL) was stirred for
1 h at 408C. The solvent was removed by co-evaporation with toluene in
vacuum. The crude product was purified by silica column chromatogra-
phy (100% EtOAc) to give compound 11 (2.05 g, 90%, 1.25 mmol) as
a colorless solid. Rf =0.27 (EtOAc); [a]2D0 = +36.86 (c=0.51 in CHCl3);
1H NMR (600 MHz, [D6]DMSO, 308C): d=7.92–7.90 (m, 2H; H-4-Fmoc,
(C-5’’), 69.0 (C-2’’’), 68.4 (C-6ab), 67.1 (C-4’), 67.0 (C-4’’’), 65.6 (CH2ab
-
(Fmoc)), 63.1 (C-4), 62.1 (C-6a0 b), 61.6 (C-6 ), 60.9 (C-6 ), 59.3 (Ta), 55.8
00
000
ab
ab
(C-2’’), 55.1 (CH3-(PMP)), 47.3, 46.8 (C-2, C-9-(Fmoc)), 27.6 (tBu-(Thr)),
25.8, 25.6 (2ꢂtBu-(TBS)), 23.2, 20.6, 20.5, 20.4, 20.3 (6ꢂCH3-(Ac)), 19.3
(Tg), 18.0, 17.9 (2 Cquart-(TBS)), ꢀ5.3, ꢀ5.4 ppm (4ꢂMe
ACHTUNGTRENUN(NG TBS)); HRMS
+
(ESI) (pos): m/z calcd for C82H117Cl3N3O32Si2
:
1816.6224 [M+H]+;
H-5-Fmoc), 7.81 (d, 3J
AHCTUNGTRENNUNG
(NH,H2’’)=9.1 Hz, 1H; NH-Troc), 7.75 (t, 3J-
found: 1816.6224.
G
(H8,H7)=7.7 Hz, 2H; H-1-Fmoc, H-8-Fmoc), 7.46–7.41 (m,
4H; H-2-Fmoc, H-7-Fmoc, NH-Fmoc, NH-Ac), 7.34–7.29 (m, 2H; H-3-
Fmoc, H-6-Fmoc), 5.31 (d, 3J(H-4’,H-3’)=3.5 Hz, 1H; H-4’), 5.21 (d,
3J(H-4’’’,H-3’’’)=3.6 Hz, 1H; H-4’’’), 5.16 (dd, 3J(H-3’’’,H-2’’’)=10.3 Hz,
3J(H-3’’’,H-4’’’)=3.5 Hz, 1H; H-3’’’), 4.99–4.97 (m, 2H; H-3’’, CH2a-
N-9-Fluorenylmethyloxycarbonyl-O-(2-acetamido-2-deoxy-4,6-O-para-me-
thoxybenzylidene-3-O-{2,4,6-tri-O-acetyl-3-O-[3,6-di-O-acetyl-2-deoxy-2-
N-(2,2,2-trichloroethoxycarbonylamino)-4-O-(2,3,4,6-tetra-O-acetyl-b-d-
galactopyranosyl)-b-d-glucopyranosyl]-b-d-galactosylpyranosyl}-a-d-gal-
3
(Troc)), 4.86 (t, J(H-2’,H-3’)=3J(H-2’,H-1’)=8.2 Hz, 1H; H-2’), 4.82 (dd,
actopyranosyl)-l-threonine-tert-butylester
(Fmoc-Thr
(bAc4Gal-
N
3J(H-2’’’,H-3’’’)=10.3 Hz, 3J(H-2’’’,H-1’’’)=7.9 Hz, 1H; H-2’’’), 4.70–4.58
(m, 4H; H-1, H-1’, H-1’’, H-1’’’), 4.52–4.49 (m, 2H; CH2b-(Troc), CH2a-
bAc2GlcNHTroc-(1!3)-bAc3Gal-(1!3)-aPMP-GalNAc)-OtBu) (10): A
solution of compound 9 (3.80 g, 2.09 mmol) in tetrahydrofuran (60 mL)
was cooled to 08C. Then a mixture of tetrabutylammoniumfluoride trihy-
drate (6.59 g, 20.9 mmol) and acetic acid (2.39 mL, 2.51 g, 41.8 mmol) in
tetrahydrofuran (20 mL) was added. After 1 h the ice bath was removed
and the reaction was stirred for 16 h at room temperature. The mixture
was diluted with ethyl acetate (400 mL) and the organic phase was
washed twice with an aqueous saturated solution of bicarbonate/brine
(1:1, 450 mL) and once with brine (200 mL). The organic phase was dried
over sodium sulfate, filtered, and concentrated. The residue was dissolved
in pyridine (60 mL) followed by addition of N,N-dimethylaminopyridine
(25 mg, 0.20 mmol) and acetic anhydride (30 mL, 32.4 g, 317 mmol). The
reaction was stirred for 24 h. The solvent was removed by co-evaporation
with toluene and the crude material was purified by silica column chro-
matography (cyclohexane/EtOAc 1:2) to give compound 10 (2.50 g, 68%,
1.44 mmol) as a colorless solid. Rf =0.43 (toluene (Tol)/EtOAc 1:3);
[a]2D0 = +56.94 (c=0.49 in CHCl3); 1H NMR (600 MHz, [D6]DMSO,
308C, internal DMSO): d=7.92–7.90 (m, 2H; H-4-Fmoc, H-5-Fmoc),
(Fmoc)), 4.46–4.30 (m, 1H; CH2b-(Fmoc)), 4.39 (d, 3J
ACTHNUGRTENUNG(OH-4,H-4)=
5.4 Hz, 1H; OH-4,), 4.32 (t, 3J(H-9,CH2ab)=6.7 Hz, 1H; H-9-(Fmoc)),
4.26–4.20 (m, 4H; H-2, H-5’’’, H-60a0, Tb), 4.08–4.06 (m, 1H; Ta), 4.01–3.91
000
(m, 6H; H-5’, H-60ab, H-6b00, H-6 ), 3.89–3.87 (m, 1H; H-4), 3.80–3.78 (m,
ab
1H; H-3’), 3.68 (t, 3J(H-4’’,H-3’’)=3J(H-4’’,H-5’’)=9.5 Hz 1H; H-4’’),
3.63–3.61 (m, 1H; H-5), 3.55–3.82 (m, 2H; H-3, H-5’’), 3.48–3.46 (m, 2H;
H-6ab), 3.33–3.29 (m, 2H; OH-6, H-2’’), 2.09- 1.85 (m, 30H; CH3-(Ac)),
1.35 (s, 9H; tBu), 1.16–1.15 ppm (m, 3H; Tg); 13C NMR (150.9 MHz,
[D6]DMSO): d=170.3, 170.0, 169.9, 169.6, 169.5, 169.2, 169.1, 168.8, 168.6
(C=O-(Ac), C=O-(tBu)), 156.8 (C=O-(Fmoc)), 154.0 (C=O-(Troc)), 143.7
(C-1a-Fmoc, C-8a-Fmoc), 140.8 (C-4a-Fmoc, C-5a-Fmoc), 127.7 (C-2-
Fmoc, C-7-Fmoc), 127.0 (C-3-Fmoc, C-6-Fmoc), 125.3, 125.2 (C-1-Fmoc,
C-8-Fmoc), 120.2 (C-4-Fmoc, C-5-Fmoc), 101.4 (C-1’), 100.6 (C-1’’), 100.0
(C-1’’’), 98.9 (C-1), 96.1 (Cquart-(Troc)), 81.4 (Cquart-(tBu)), 78.3 (C-3’), 77.4
(C-3), 75.9 (C-4’’), 73.5 (Tb), 73.4 (CH2ab-(Troc)), 72.8 (C-3’’), 71.7 (C-5),
71.5 (C-5’’), 70.7 (C-5’), 70.3 (C-3’’’), 69.6 (C-5’’’), 69.3 (C-4’), 69.1 (C-2’),
68.9 (C-2’’’), 67.4 (C-4), 67.0 (C-4’’’), 65.6 (CH2ab-(Fmoc)), 62.2 (C-6ab),
7.81 (d, 3J
ACHTUNGTRENNUNG(NH,H-2)=9.1 Hz, 1H; NH-Troc), 7.77–7.34 (m, 2H; H-1-
00
000
61.8 (C-6 ), 60.8 (C-6 ), 60.5 (C-6ab), 59.4 (Ta), 55.8 (C-2’’), 47.0 (C-2),
Fmoc, H-8-Fmoc), 7.43–7.41 (m, 4H; H-2-Fmoc, H-7-Fmoc, NH-Ac,
NH-Fmoc), 7.34–7.30 (m, 4H; H-2-PMP, H-6-PMP, H-3-Fmoc, H-6-
ab
ab
46.8 (C-9-(Fmoc)), 27.6 (tBu), 22.9, 20.8, 20.7, 20.6, 20.5, 20.4, 20.3 (10ꢂ
CH3-(Ac)), 19.1 ppm (Tg); HRMS (ESI) (pos): m/z calcd for
C70H91Cl3N3O35+: 1638.4499 [M+H]+; found: 1638.4480.
Fmoc), 6.93 (d, 3J(H-3,H-2)=3J
ACHTUNGTRENNUNG ACHTUNGTREN(NUNG H-5,H-4)=8.8 Hz, 2H; H-3-PMP, H-5-
PMP), 5.44 (s, 1H; CH-(PMP)), 5.34–5.32 (m, 1H; H-4’), 5.22 (d, 3J(H-
4’’’,H-3’’’)=3.2 Hz, 1H; H-4’’’), 5.16 (dd, 3J(H-3’’’,H-2’’’)=10.3, 3J(H-
3’’’,H-4’’’)=3.3 Hz, 1H; H-3’’’), 4.98–4.97 (m, 2H; H-3’’, CH2a-(Troc)),
4.84–4.80 (m, 2H; H-2’, H-2’’’), 4.71–4.69 (m, 2H; H-1, H-1’’’), 4.63–4.61
(m, 2H; H-1’, H-1’’), 4.53–4.45 (m, 3H; CH2ab-(Fmoc), CH2b-(Troc)), 4.32
N-9-Fluorenylmethyloxycarbonyl-O-(2-acetamido-4,6-O-acetyl-2-deoxy-3-
O-{2,4,6-tri-O-acetyl-3-O-[2-acetamido-3,6-O-acetyl-2-deoxy-4-O-(2,3,4,6-
tetra-O-acetyl-b-d-galactopyranosyl)-b-d-glucopyranosyl]-b-d-galactosyl-
pyranosyl}-a-d-galactosylpyranosyl)-l-threonine-tert-butylester
(Fmoc-
(1!4)-bAc2GlcNAc-(1!3)-bAc3Gal-(1!3)-aAc2GalNAc)-
ACHTUNGTRENNUNG
(t, 3J(H-9,CH2)=6.7 Hz, 1H; H-9-(Fmoc)), 4.28–4.26 (m, 3H; H-5, H-600,
Thr(bAc4Gal-
G
ACHTUNGTRENNUNG
Tb), 4.23–4.17 (m, 2H; H-2, H-5’’’), 4.09–3.99 (m, 6H; H-6ab, H-6a0 , H-60a0ba0
,
OtBu) (12): Zinc dust was activated by treatment with 1n hydrochloric
acid followed by washing with water, methanol, and diethyl ether. The
activated zinc (393 mg, 6.0 mmol) was added to a solution of compound
11 (492 mg, 0.29 mmol) in acetic acid (6 mL). The reaction was stirred at
408C for 18 h and then the mixture was filtered over celite and the fil-
trate was co-evaporated several times with toluene. The residue was dis-
Ta), 3.93–3.89 (m, 3H; H-5’, H-6b0 H-6b00), 3.97 (dd, 3J(H-3’,H-4’)=4.0,
,
3J(H-3’,H-2’)=10.2 Hz, 1H; H3’), 3.75–3.72 (m, 4H; H-3, CH3-(PMP)),
3.70–3.65 (m, 2H; H-4, H-4’’), 3.54–3.51 (m, 1H; H-5’’), 3.29 (q, 3J(H-
2’’,H-1’’)=3J(H-2’’,NH’’)=3J(H-2’’,H-3’’)=9.1 Hz 1H; H-2’’), 2.09–1.83
(m, 30H; CH3-(Ac)), 1.36 (s, 9H; tBu), 1.15 ppm (d, 3J
ACHTNUTRGNEUNG(Tg,Tb)=6.2 Hz,
Chem. Eur. J. 2013, 19, 17001 – 17010
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
17007

U. Westerlind et al.
solved in pyridine/acetic anhydride (2:1, 9 mL). N,N-dimethylaminopyri-
dine (3.7 mg, 0.03 mmol) was added to the solution and the reaction mix-
ture was stirred for 20 h. The solvent was removed by co-evaporation
with toluene and the crude product purified by silica column chromatog-
raphy (100% EtOAc) to give compound 12 (389 mg, 82%, 0.25 mmol) as
a colorless solid. Rf =(EtOAc/MeOH 20:1); [a]2D0 = +41.40 (c=0.50 in
61.8, 61.7 (C-6ab, C-6a00b), 60.8 (C-6a00b0 ), 58.5 (Ta), 54.0 (C-2’’), 47.7 (C-2),
46.8 (C-9-(Fmoc)), 22.8, 22.6, 20.64–20.38 (CH3-(Ac)), 18.4 pm (Tg);
HRMS (ESI) (pos): m/z calcd for C69H88N3O36+: 1534.5148 [M+H]+;
found: 1534.5139.
Synthesis of the type-2 core 2 hexasaccharide building block
N-9-Fluorenylmethyloxycarbonyl-O-(2-acetamido-2-deoxy-3-O-{2,4,6-tri-
O-acetyl-3-O-[3,6-O-acetyl-2-N-(2,2,2-trichloroethoxycarbonylamino)-4-
O-(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)-2-deoxy-b-d-glucopyraACHTUNGTRENNUNGn-
osyl]-b-d-galactosylpyranosyl}-6-O-[3-O-acetyl-2-deoxy-6-O-tert-butyldi-
methylsilyl-2-N-(2,2,2-trichloroethoxycarbonylamino)-4-O-(2,3,4,6-tetra-
CHCl3); 1H NMR (600 MHz, [D6]DMSO, 308C): d=7.77 (d, 3J
3)=3J
(H-5,H-6)=9.1 Hz, 2H; H-4-Fmoc, H-5-Fmoc,), 7.62–7.59 (m, 2H;
ACHTUNGTRNE(NUNG H-4,H-
ACHTUNGTRENNUNG
H-1-Fmoc, H-8-Fmoc), 7.49–7.46 (m, 2H; H-2-Fmoc, H-7-Fmoc), 7.39–
7.35 (m, 2H; H-3-Fmoc, H-6-Fmoc), 6.22 (d, 3J
N
3
NH-Ac), 5.92 (d, J
(NH,Ta)=9.1 Hz, 1H; NH-Fmoc), 5.60–5.54 (m, 1H;
O-acetyl-b-d-galactopyranosyl)-b-d-glucopyranosyl]-a-d-galactosylpyra
osyl)-l-threonine-tert-butylester(Fmoc-Thr(bAc4Gal-
(1!4)-bAc2GlcNHTroc-
(1!3)-bAc3Gal-(1!3)-[bAc4Gal-(1!4)-bAc-TBS-GlcNHTroc-(1!6)]-
aGalNAc)-OtBu) (14): suspension of compound 11 (694 mg,
ACHTUNGTRENNUNGn-
NH’’-Ac), 5.32–5.29 (m, 3H; H-4, H-4’, H-4’’’), 5.15 (t, 3J(H-3’’, H-2’’)=
3J(H-3’’,H-4’’)=9.5 Hz, 1H; H-3’’), 5.07 (dd, 3J(H-2’’’,H-1’’’)=8.0, 3J(H-
2’’’,H-3’’’)=10.3 Hz, 1H; H-2’’’), 4.98–4.95 (m, 2H; H-2’, H-3’’’), 4.83
(sbr, 1H; H-1), 4.76–4.68 (m, 2H; H-1’’, H-60a0), 4.57 (d, 3J(H-1’,H-2’)=
7.8 Hz, 1H; H-1’), 4.51–4.49 (m, 2H; H-1’’’, H-2), 4.43 (m, 1H; CH2a-
(Fmoc)), 4.35–4.31 (m, 1H; CH2b-(Fmoc)), 4.25–4.17 (m, 3H; Tb, Ta, H-
9-(Fmoc)), 4.12–4.02 (m, 6H; H-5, H-6ab, H-60a, H-6a00b0 ), 3.99–3.90 (m, 3H;
A
R
ACHTUNGTRENNUNG
A
0.42 mmol), compound 4 (515 mg, 0.55 mmol), and 4 ꢁ molecular sieves
(0.6 g) in dry dichloromethane (15 mL) was stirred under argon at room
temperature for 1 h before it was cooled to 08C. Then N-iodosuccinimide
(124 mg, 0.55 mmol) was added followed by the dropwise addition of tri-
fluoromethanesulfonic acid (7.5 mmol, 13 mg, 0.09 mmol) in dry dichloro-
H-3, H-60b H-60b0), 3.86–3.50 (m, 3H; H-3’, H-5, H-5’), 3.77 (t, 3J(H-4’’,H-
,
3’’)=3J(H-4’’,H-5’’)=9.3 Hz, 1H; H-4’’), 3.58–3.52 (m, 2H; H-2’’, H-5’’),
2.11–1.87 (m, 39H; CH3-(Ac)), 1.43 (s, 9H; tBu), 1.32–1.28 ppm (m, 3H;
Tg); 13C NMR (150.9 MHz, [D6]DMSO): d=170.9, 170.6, 170.5, 170.3,
170.2, 170.1, 170.0, 169.7, 169.3 (C=O-(Ac), C=O-(tBu)), 156.6 (C=O-
(Fmoc)), 143.8 (C-1a-Fmoc, C-8a-Fmoc), 141.5 (C-4a-Fmoc, C-5a-Fmoc),
128.0 (C-2-Fmoc, C-7-Fmoc), 127.3 (C-3-Fmoc, C-6-Fmoc), 125.1 (C-1-
Fmoc, C-8-Fmoc), 120.2 (C-4-Fmoc, C-5-Fmoc), 101.2 (C-1’’’), 100.6 (C-
1’), 100.5 (C-1’’), 100.1 (C-1), 83.6 (Cquart-(tBu)), 76.6 (Tb), 76.1 (C-5), 75.9
(C-4’’), 72.7 (C-5’’), 71.9 (C-3, C-3’’), 71.3 (C-5’), 71.0 (C-3’’’), 70.8 (C-3’),
methane (100 mL). After 3 h another portion of donor
4 (119 mg,
0.13 mmol) and N-iodosuccinimide (28 mg, 0.13 mmol) was added. The
reaction was stirred for additional 3.5 h under argon and at 08C before
the molecular sieve was filtered off and washed with dichloromethane.
More dichloromethane was added to give a total volume of 120 mL. The
organic phase was washed with a 0.5m aqueous solution of sodium thio-
sulfate, as saturated aqueous solution of sodium bicarbonate, water, and
brine (30 mL each). The organic phase was dried over sodium sulfate, fil-
tered, and concentrated and the residue was purified by silica column
chromatography (cyclohexane/EtOAc 1:1!1:3) to give compound 14
(722 mg, 69%, 0.29 mmol) as a colorless solid. Rf =0.39 (Tol/EtOAc 1:3);
[a]2D0 = +20.27 (c=0.49 in CHCl3); 1H NMR (600 MHz, [D6 DMSO,
308C): d=7.92–7.88 (m, 2H; H-4-Fmoc, H-5-Fmoc), 7.81–7.73 (m, 4H;
H-1-Fmoc, H-8-Fmoc, 2ꢂNH-Troc), 7.46–7.38 (m, 4H; H-2-Fmoc, H-7-
Fmoc, NH-Ac, NH-Fmoc), 7.34–7.30 (m, 2H; H-3-Fmoc, H-6-Fmoc),
70.7 (C-2’), 69.3 (C-4, C-4’), 69.2 (C-2’’’), 68.2 (C-5’’’), 67.2 (CH2ab
-
000
(Fmoc)), 66.8 (C-4’’’), 63.2 (C-60ab), 61.9 (C-6 ), 60.9 (C-6ab, C-6a00b), 59.2
ab
(Ta), 55.3 (C-2’’), 48.7 (C-2), 47.4 (C-9-(Fmoc)), 28.2 (tBu), 23.3–20.7
(CH3-(Ac)), 18.8 ppm (Tg); HRMS (ESI) (pos): m/z calcd for
+
C73H96N3O36
:
1590.5774 [M+H]+; found: 1590.5763; calcd for
C73H96KN3O362+: 814.7706 [M+K+H]2+; found: 814.7661.
N-9-Fluorenylmethyloxycarbonyl-O-(2-acetamido-4,6-O-acetyl-2-deoxy-3-
O-{2,4,6-tri-O-acetyl-3-O-[2-acetamido-3,6-O-acetyl-2-deoxy-4-O-(2,3,4,6-
tetra-O-acetyl-b-d-galactopyranosyl)-b-d-glucopyranosyl]-b-d-galactosyl-
3
5.32 (sbr, 1H; H-4’), 5.25 (d, J(H-4’’’’’,H-3’’’’’)=3.6 Hz, 1H; H-4’’’’’), 5.22
(d, 3J(H-4’’’,H-3’’’)=3.6 Hz, 1H; H4’’’), 5.16 (dd, 3J(H-3’’’,H-4’’’)=3.6,
3J(H-3’’’,H-2’’’)=10.2 Hz, 1H; H3’’’), 5.08 (dd, 3J(H-3’’’’’,H-4’’’’’)=3.6,
3J(H3’’’’’,H2’’’’’’)=10.3 Hz, 1H; H-3’’’’’), 4.98–4.88 (m, 4H; H-3’’, H-3’’’’,
pyranosyl}-a-d-galactosylpyranosyl)-l-threonine
(Fmoc-ThrACTHNUGTRENNU(G bAc4Gal-
(1!4)-bAc2GlcNAc-(1!3)-bAc3Gal-(1!3)-aAc2GalNAc)-OH) (13):
A
00
0000
2a
CH -(Troc), CH -(Troc)), 4.86–4.80 (m, 3H; H-2’, H-2’’’, H-2’’’’’), 4.72–
2a
solution of compound 12 (718 mg, 0.45 mmol) in dichloromethane
(3 mL), anisole (0.5 mL), and trifluoroacetic acid (9 mL) was stirred for
2.5 h. The solvent was removed by co-evaporation with toluene and the
residue was purified by silica column chromatography (EtOAc!EtOAc/
MeOH 25:1) to give compound 13 (616 mg, 89%, 0.40 mmol) as a color-
less solid. Rf =0.21 (EtOAc/MeOH/AcOH/H2O 50:3:3:2); [a]2D0 = +48.57
(c=1.00 in CHCl3); 1H NMR (600 MHz, [D6]DMSO): d=12.90
4.69 (m, 2H; H-1’’’, H-1’’’’’), 4.63–4.59 (m, 2H; H-1’’, CH020b00-(Troc)), 4.57–
4.54 (m, 2H; H-1, H-1’), 4.52–4.44 (m, 4H; H-1’’’’, CH020b-(Troc), CH2ab
-
(Fmoc)), 4.32 (t, 3J(H-9,CH2ab)=6.6 Hz, 1H; H-9-(Fmoc)), 4.27–4.25 (m,
1H; H-60a0), 4.23–4.19 (m, 2H; H-2, H-5’’’), 4.09–3.91 (m, 10H; H-5’’’’’, H-
60ab, H-60b0, H-6 , H-6 , Ta, Tb), 3.88–3.85 (m, 1H; H-5’’), 3.84–3.73 (m,
000
00000
ab
ab
8H; H-3’, H-4, H-4’’’’, H-5, H-5’, H-6a, H-60a0b00), 3.68 (t, 3J(H-4’’,H-3’’)=
3J(H-4’’,H-2’’)=9.3 Hz, 1H; H-4’’), 3.56–3.48 (m, 3H; H-3, H-5’’, H-6b),
3.42–3.37 (m, 1H; H-2’’’’), 3.33–3.28 (m, 2H; H-2’’, H-5’’’’), 2.10–1.84 (m,
(COOH), 7.91, 7.90 (2ꢂd, 3J(H-4,H-3)=3J
ACTHNUGRTNENUG AHCTUNGTRNEN(UGN H-5,H-6)=7.4 Hz, 2ꢂ1H; H-
4-Fmoc, H-5-Fmoc), 7.83 (d, 3J(NH’’,H-2’’)=9.0 Hz, 1H; NH’’-Ac), 7.77–
45H; CH3-(Ac)), 1.35 (s, 9H; tBu-(Thr)), 1.09 (d, JACTHNUGRTENUNG(Tg,Tb)=6.1 Hz, 3H;
7.72 (m, 2H; H-1-Fmoc, H-8-Fmoc), 7.45–7.30 (m, 6H; H-2-Fmoc, H-3-
Tg), 0.88 (s, 9H; tBu-(TBS)), 0.07 (s, 3H; 2ꢂMe-(TBS)), 0.06 ppm (s,
3H; 2ꢂMe-(TBS)); 13C NMR (150.9 MHz, [D6]DMSO): d=170.3, 169.9,
169.7, 169.5, 169.4, 169.3, 169.1, 169.0, 168.8, 168.7 (C=O-(Ac), C=O-
(tBu)), 156.8 (C=O-(Fmoc)), 154.1, 154.0 (2ꢂC=O-(Troc)), 143.7 (C-1a-
Fmoc, C-8a-Fmoc), 140.8 (C-4a-Fmoc, C-5a-Fmoc), 127.7 (C-2-Fmoc, C-7-
Fmoc), 127.0 (C-3-Fmoc, C-6-Fmoc), 125.2, 125.1 (C-1-Fmoc, C-8-Fmoc),
120.2 (C-4-Fmoc, C-5-Fmoc), 101.5 (C-1’), 100.5 (C-1’’), 100.0 (C-1’’’, C-
1’’’’), 99.6 (C-1’’’’’), 99.1 (C-1), 96.2, 96.1 (Cquart-(Troc)), 81.4 (Cquart-(tBu)),
3
Fmoc, H-6-Fmoc, H-7-Fmoc, NH-Fmoc, NH-Ac), 5.28 (d, J(H-4’,H-3’)=
ACTHNUTRGNEUNG
3.7 Hz, 1H; H-4’), 5.27 (d, 3J(H4,H3)=3.4 Hz, 1H; H-4), 5.22 (d, 3J(H-
3
3
4’’’,H-3’’’)=3.5 Hz, 1H; H-4’’’), 5.17 (dd, J(H-3’’’,H-4’’’)=3.5, J(H-3’’’,H-
2’’’)=10.3 Hz, 1H; H-3’’’), 5.00 (t, J(H-3’’,H-4’’)=3J(H-3’’,H-2’’)=9.5 Hz,
3
1H; H-3’’), 4.83 (dd, 3J(H-2’’’,H-3’’’)=10.3, 3J(H-2’’’,H-1’’’)=8.0 Hz, 1H;
H-2’’’), 4.74–4.69 (m, 4H; H-1, H-2, H-1’’, H-1’’’), 4.59 (d, J(H-1’,H-2’)=
8.0 Hz, 1H; H-1’), 4.52–4.43 (m, 2H; CH2ab-(Fmoc)), 4.32–4.30 (m, 2H;
H-9-(Fmoc), H-60a0), 4.27–4.25 (m, 1H; Tb), 4.21 (t, 3J(H-5’’’,H-6’’’)=
6.8 Hz, 1H; H5’’’), 4.15–4.08 (m, 4H; H-2, H-5, H-60a, Ta), 4.00–3.98 (m,
2H; H-60a0b0 ), 3.94–3.79 (m, 6H; H-3, H-3’, H-5’, H-6ab, H-6b0 , H-60b0), 3.65 (t,
3J(H-4’’,H-3’’)=3J(H-4’’,H-5’’)=9.5 Hz, 1H; H-4’’), 3.57–3.54 (m, 1H; H-
5’’), 3.41–3.37 (m, 1H; H-2’’), 2.09–1.70 ppm (m, 39H; CH3-(Ac));
13C NMR (150.9 MHz, [D6]DMSO): d=170.3, 170.1, 170.0, 169.9, 169.7,
169.5, 169.4, 169.1, 169.0, 168.8 (C=O-(Ac)), 156.8 (C=O-(Fmoc)), 143.8,
143.7 (C-1a-Fmoc, C-8a-Fmoc), 140.8 (C-4a-Fmoc, C-5a-Fmoc), 127.7 (C-2-
Fmoc, C-7-Fmoc), 127.1 (C-3, C-6-Fmoc), 125.3, 125.1 (C-1-Fmoc, C-8-
Fmoc), 120.2 (C-4-Fmoc, C-5-Fmoc), 100.6 (C-1’), 100.0, 98.7 (C-1, C-1’’,
C-1’’’), 76.9 (C-3’), 76.1 (C-4’’), 74.5 (Tb), 73.4 (C-3), 72.7 (C-3’’), 71.4 (C-
5’’), 70.3 (C-5’, C-3’’’), 69.7 (C-4), 69.6 (C-5’’’), 69.5 (C-2’), 69.0 (C-4’),
68.9 (C-2’’’), 67.3 (C-5), 67.0 (C-4’’), 65.6 (CH2ab-(Fmoc)), 63.1 (C-60ab),
78.2 (C-3’), 76.8 (C-3), 75.9 (C-4’’), 74.8 (C-4’’’’), 74.5 (C-5’’’’), 74.2 (Tb),
0000
73.4 (CH020ab-(Troc)), 73.3 (CH -(Troc)), 72.8 (C-3’’, C-3’’’’), 71.5 (C-5’’),
2ab
70.7 (C-5’), 70.3 (C-3’’’, C-3’’’’’, C-5), 70.0 (C-6ab), 69.9 (C-5’’’’’), 69.6 (C-
5’’’), 69.2 (C-4’), 69.0, 68.9 (C-2’, C-2’’’, C-2’’’’’), 68.6 (C-4), 67.1 (C-4’’’’’),
00
0000
67.0 (C-4’’’), 65.6 (CH2ab-(Fmoc)), 61.9 (C-60ab), 61.8 (C-6 ), 61.1 (C-6
,
ab
ab
C-60a0b000), 60.8 (C-6 ), 59.6 (Ta), 55.8 (C-2’’), 55.6 (C-2’’’’), 46.9 (C-2), 46.8
000
ab
(C-9-(Fmoc)), 27.6 (tBu-(Thr)), 25.8 (tBu-(TBS)), 23.0, 20.7–20.3 (CH3-
(Ac)), 18.9 (Tg), 18.0 (C
HRMS (ESI) (pos): m/z calcd for
[M+K+H]2+; found: 1250.7862.
(TBS), ꢀ5.2, ꢀ5.3 ppm (2ꢂMe
ACHTUNGTRENNUNG
+
C101H137Cl6KN4O51Si2 :
N-9-Fluorenylmethyloxycarbonyl-O-(2-acetamido-4-O-acetyl-2-deoxy-3-
O-{2,4,6-tri-O-acetyl-3-O-[3,6-O-acetyl-2-deoxy-2-N-(2,2,2-trichloro-
17008
www.chemeurj.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 17001 – 17010

A Unified Strategy for the Synthesis of Mucin Cores 1–4 Saccharides
FULL PAPER
A
E
1-Fmoc, H-8-Fmoc), 7.52 (d, JACTHGNUTERNNU(G NH,H-2)=9.7 Hz, 1H; NH-Ac), 7.45–7.41
b-d-glucopyranosyl]-b-d-galactopyranosyl}-6-O-[3,6-O-acetyl-2-deoxy-2-
N-(2,2,2-trichloroethoxycarbonylamino)-4-O-(2,3,4,6-tetra-O-acetyl-b-d-
(m, 2H; H-2-Fmoc, H-7-Fmoc), 7.40–7.38 (m, 1H; NH-Fmoc), 7.34–7.29
(m, 2H; H-3-Fmoc, H-6-Fmoc), 5.27 (d, 3J
A
3
ga
A
5.23–5.21 (m, 2H; H-4’’’, H-4’’’’’), 5.19 (d, J
tert-butylester
N
U
5.18–5.15 (m, 2H; H-3’’’, H-3’’’’’), 5.00 (t, 3J(H-3’’,H-4’’)=3J(H-3’’,H-2’’)=
9.3 Hz, 1H; H-3’’), 4.91 (t, 3J(H-3’’’’,H-4’’’’)=3J(H-3’’’’,H-2’’’’’)=9.3 Hz,
1H; H-3’’’’), 4.85–4.81 (m, 2H; H-2’’’, H-2’’’’’), 4.73–4.67 (m, 4H; H-1’’,
bAc3Gal-(1!3)-[bAc4Gal-(1!4)-bAc2-GlcNHTroc-(1!6)]-aAcGal-
NAc)-OtBu) (15): Compound 14 (1.66 g, 0.67 mmol) was dissolved in
80% aqueous AcOH (18 mL) and stirred for 20 h at 408C. The solvent
was removed by co-evaporation with toluene in vacuum. The residue was
dissolved in pyridine (20 mL) and cooled to 08C. Addition of N,N-dime-
thylaminopyridine (8 mg, 0.07 mmol) and acetic anhydride (10 mL) fol-
lowed and the solution was stirred at room temperature for 18 h. The sol-
vent was removed by co-evaporation with toluene and the residue was
purified by silica column chromatography (cyclohexane/EtOAc 3:2!4:3)
to give compound 15 (1.48 g, 91%, 0.61 mmol) as a colorless solid. Rf =
0.48 (Tol/EtOAc 1:2); [a]2D0 = +24.30 (c=1.00 in CHCl3); 1H NMR
(600 MHz, [D6]DMSO, 308C): d=7.91–7.89 (m, 2H; H-4-Fmoc, H-5-
Fmoc), 7.76–7.72 (m, 4H; H-1-Fmoc, H-8-Fmoc, 2ꢂNH-Troc), 7.45–7.41
(m, 4H; H-2-Fmoc, H-7-Fmoc, NH-Ac, NH-Fmoc), 7.33–7.31 (m, 2H;
H-3-Fmoc, H-6-Fmoc), 5.29 (sbr, 1H; H-4’), 5.23–5.21 (m, 3H; H-4, H-
4’’’, H-4’’’’’), 5.17–5.14 (m, 2H; H-3’’’, H-3’’’’’), 4.97–4.89 (m, 3H; H-3’’, H-
H-1’’’, H-1’’’’’, H-2’), 4.60 (d, 3J
ACTHNUTRGNEU(GN H-1,H-2)=4.1 Hz 1H; H-1), 4.56 (d,
3J(H-1’,H-2’)=8.0 Hz, 1H; H-1’), 4.52–4.45 (m, 3H; H-1’’’’, CH2ab
-
(Fmoc)), 4.33–4.26 (m, 3H; H-60a0, H-6a0000, H-9-(Fmoc)), 4.22–4.19 (m, 2H;
H-5’’’, H-5’’’’’), 4.15–4.11 (m, 2H; H-2, Tb), 4.08–4.05 (m, 1H; H-6b00),
4.02–3.98 (m, 6H; H-5’’’’’, H-60a0b0 , H-6 , Ta), 3.94–3.89 (m, 3H; H-6a0 b, H-
00000
ab
3
60b000), 3.84 (t, J(H-5’,H-6’)=5.9 Hz, 1H; H-5’), 3.80–3.74 (m, 3H; H-3’, H-
3’’’’’, H-6a), 3.70–3.63 (m, 4H; H-2’’’’, H-4’’, H-4’’’’, H-5’’), 3.56–3.54 (m,
1H; H-5’’’’), 3.39 (q, 3J(H-2’’,H-1’’)=3J(H-2’’,H-3’’)=3J
ACHTUNGTRENNUNG
JACHTUNGTRENNUNG
13C NMR (150.9 MHz, [D6]DMSO): d=170.3, 170.2, 170.0, 169.9, 169.6,
169.5, 169.4, 169.3, 169.1, 169.0, 168.9, 168.8 (C=O-(Ac), C=O-(tBu)),
156.8 (C=O-(Fmoc)), 143.7 (C-1a-Fmoc, C-5a-Fmoc), 140.8 (C-4a-Fmoc,
C-5a-Fmoc), 127.7 (C-2-Fmoc, C-7-Fmoc), 127.0 (C-3-Fmoc, C-6-Fmoc),
125.3, 125.1 (C-1-Fmoc, C-8-Fmoc), 120.2 (C-4-Fmoc, C-5-Fmoc), 100.5
(C-1’), 100.4 (C-1’’’’), 100.0, 99.9 (C-1’’, C-1’’’, C-1’’’’’), 98.4 (C-1), 81.4
(Cquart-(tBu)), 76.8 (C-3’), 76.5 (C-4’’’’), 76.1 (C-4’’), 73.8 (Tb), 73.4 (C-3,
C-3’’’’), 72.7 (C-3’’), 71.7 (C-5’’), 71.4 (C-5’’’’), 70.2, 70.0 (C-3’’’, C-3’’’’’, C-
4, C-5’), 69.6 (C-5’’’, C-5’’’’’), 69.5 (C-2’), 69.1 (C-6ab), 69.0 (C-4’), 68.9 (C-
2’’’, C-2’’’’’), 68.6 (C-5), 67.0 (C-4’’’, C-4’’’’’), 65.6 (CH2ab-(Fmoc)), 62.2 (C-
00
0000
2a
3’’’’, CH -(Troc)), 4.91–4.89 (m, 1H; CH -(Troc)), 4.83–4.80 (m, 2H; H-
2a
2’’’, H-2’’’’’), 4.73–4.70 (m, 3H; H-1’’’, H-1’’’’’, H-2’), 4.63–4.55 (m, 4H; H-
00
1, H-1’, H-1’’, CH020b00-(Troc)), 4.51–4.47 (m, 4H; H-1’’’’, CH -(Troc),
2b
CH2ab-(Fmoc)), 4.32–4.27 (m, 3H; H-9-(Fmoc), H-600, H-6a0000), 4.22–4.19
(m, 2H; H-5’’’, H-5’’’’’), 4.14–4.09 (m, 3H; H-2, H-60b0a, Tb), 4.05–3.99 (m,
000
00000
6H; H-5’’’’’, H-6 , H-6 , Ta), 3.94–3.88 (m, 3H; H-60ab, H-60b000), 3.81–3.76
ab
ab
00
0000
000
00000
(m, 3H; H-3, H-3’, H-6a), 3.71–3.65 (m, 2H; H-4’’, H-4’’’’), 3.61–3.58 (m,
1H; H-5’’), 3.53–3.50 (m, 1H; H-5’’’’), 3.44 (q, 3J(H-2’’’’,H-3’’’’)=3J(H-
2’’’’,H-4’’’’)=3J(H-2’’’,NH)=11.5 Hz, 1H; H-2’’’’), 3.35–3.27 (m, 2H; H-
2’’, H-6b), 2.09–1.82 (m, 51H; CH3-(Ac)), 1.35 (s, 9H; tBu), 1.13 ppm (d,
6
), 61.6 (C-60ab, C-6 ), 60.8 (C-6 , C-6 ), 59.5 (Ta), 54.0 (C-2’’), 53.1 (C-
ab
ab
ab
ab
2’’’’), 47.7 (C-2), 46.8 (C-9-(Fmoc)), 27.6 (tBu), 22.8, 22.7, 22.6, 20.6–20.3
(CH3-(Ac)), 19.0 ppm (Tg); HRMS (ESI) (pos): m/z calcd for
C97H130N4O512+: 1083.8868 [M+2H]2+; found: 1083.8872.
JACHTUNGTRENNUNG
(Tg,Tb)=7.7 Hz, 3H; Tg); 13C NMR (150.9 MHz, [D6]DMSO): d=
N-9-Fluorenylmethyloxycarbonyl-O-(2-acetamido-2-deoxy-4-O-acetyl-3-
O-{2,4,6-tri-O-acetyl-3-O-[2-acetamido-3,6-O-acetyl-2-deoxy-4-O-(2,3,4,6-
tetra-O-acetyl-b-d-galactopyranosyl)-b-d-glucopyranosyl]-b-d-galactopyra-
nosyl}-6-O-[2-acetamido-3,6-O-acetyl-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-
b-d-galactopyranosyl)-b-d-glucopyranosyl]-a-d-galactopyranosyl)-l-threo-
170.3, 170.0, 169.9 169.6, 169.5, 169.3, 169.2, 169.1, 168.8 (C=O-(Ac), C=
O-(tBu)), 156.8 (C=O-(Fmoc)), 154.2, 154.0 (2ꢂC=O-(Troc)), 143.8,
143.7 (C-1a-Fmoc, C-8a-Fmoc), 140.82 (C-4a-Fmoc, C-5a-Fmoc), 127.7 (C-
2-Fmoc, C-7-Fmoc), 127.0 (C-3-Fmoc, C-6-Fmoc), 125.2, 125.1 (C-1-
Fmoc, C-8-Fmoc), 120.2 (C-4-Fmoc, C-5-Fmoc), 100.6 (C-1’’), 100.5 (C-
1’), 100.1 (C-1’’’’), 100.0, 99.9 (C-1’’’, C-1’’’’’), 99.8 (C-1), 96.1, 96.0 (2
Cquart-(Troc)), 81.5 (Cquart-(tBu)), 77.6 (C-3’), 76.4 (C-4’’’’), 75.9 (C-4’’),
nine
(Fmoc-Thr
G
(1!4)-bAc2GlcNAc-(1!3)-bAc3Gal-(1!3)-
[bAc4Gal-(1!4)-bAc2-GlcNAc-(1!6)]-aAcGalNAc)-OH) (17): A solu-
tion of compound 16 (1.82 g, 0.84 mmol) in dichloromethane (5 mL), an-
00
0000
74.3 (Tb), 73.4, 73.3 (C-3, CH -(Troc), CH -(Troc)), 72.7 (C-3’’, C-3’’’’),
2ab
2ab
ACHUTGTNRENNiGU sACTHNGUTRENNUoG le (2 mL) and trifluoroacetic acid (15 mL) was stirred at room temper-
71.8 (C-5’’), 71.5 (C-5’’’’), 70.2 (C-4, C-3’’’, C-3’’’’’, C-5’), 69.7, 69.6 (C-5’’’,
ature for 3 h. The solvent was removed by co-evaporation with toluene.
The residue was purified by silica column chromatography (EtOAc!
EtOAc/MeOH/AcOH/H2O 50:3:3:2) to give compound 17 (1.63 g, 92%,
0.77 mmol) as a colorless solid. Rf =0.23 (EtOAc/MeOH/AcOH/H2O
50:3:3:2); [a]2D0 = +27.60 (c=1.00 in CHCl3); 1H NMR (600 MHz,
[D6]DMSO, 308C): d=12.87 (sbr, 1H; COOH), 7.91–7.89 (m, 2H; H-4-
Fmoc, H-5-Fmoc), 7.83–7.79 (m, 2H; NH’’-Ac, NH’’’’-Ac), 7.77–7.73 (m,
2H; H-1-Fmoc, H-8-Fmoc), 7.45–7.41 (m, 3H; NH-Ac, H-2-Fmoc, H-7-
Fmoc), 7.34–7.31 (m, 3H; NH-Fmoc, H-3-Fmoc, H-6-Fmoc), 5.27 (d,
C-5’’’’’), 69.3 (C-2’), 69.2 (C-4’, C-6ab), 68.9 (C-2’’’, C-2’’’’’, C-5), 67.0 (C-
0000
4’’’, C-4’’’’’), 65.6 (CH2ab-(Fmoc)), 62.2 (C-60a0b), 61.8, 61.7 (C-60ab, C-6 ),
ab
00000
60.9, 60.8 (C-60a0b0 , C-6 ), 59.5 (Ta), 55.8 (C-2’’), 55.6 (C-2’’’’), 47.6 (C-2),
ab
46.8 (C-9-(Fmoc)), 27.6 (tBu), 22.8, 20.6–20.3 (CH3-(Ac)), 18.9 ppm (Tg);
2+
HRMS (ESI) (pos): m/z calcd for C99H128Cl6N4O53
: 1216.7790
[M+2H]2+; found: 1216.7809.
N-9-Fluorenylmethyloxycarbonyl-O-(2-acetamido-4-O-acetyl-2-deoxy-3-
O-{2,4,6-tri-O-acetyl-3-O-[2-acetamido-3,6-O-acetyl-2-deoxy-4-O-(2,3,4,6-
tetra-O-acetyl-b-d-galactopyranosyl)-b-d-glucopyranosyl]-b-d-galactopyra-
nosyl}-6-O-[2-acetamido-3,6-O-acetyl-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-
b-d-galactopyranosyl)-b-d-glucopyranosyl]-a-d-galactopyranosyl)-l-threo-
3J(H-4’,H-3’)=3.5 Hz, 1H; H-4’), 5.23–5.22 (m, 2H; H-4’’’, H-4’’’’’), 5.19
3
(d, J
G
3
3
(t, J(H-3’’,H-2’’)=3J(H-3’’,H-4’’)=9.5 Hz, 1H; H-3’’), 4.90 (t, J(H-3’’’’,H-
nine-tert-butylester
(Fmoc-Thr
(bAc4Gal-
N
2’’’’)=3J(H-3’’’’,H-4’’’’)=9.5 Hz, 1H; H-3’’’’), 4.84–4.81 (m, 2H; H-2’’’, H-
bAc3Gal-(1!3)-[bAc4Gal-(1!4)-bAc2-GlcNAc-(1!6)]-aAcGalNAc)-
OtBu) (16): Zinc dust was activated by treatment with 1n hydrochloric
acid followed by washing with water, methanol, and diethyl ether. Com-
pound 15 (2.20 g, 0.90 mmol) was dissolved in acetic acid (30 mL) and
then the pre-activated zinc (1.18 g, 18.0 mmol) was added. The reaction
was stirred at 408C for 36 h and then the solid was filtered off and
washed with acetic acid. The filtrate was co-evaporated several times
with toluene. The obtained residue was dissolved in pyridine/acetic anhy-
dride (2:1, 30 mL) and N,N-dimethylaminopyridine (11 mg, 0.09 mmol)
was added. The mixture was stirred for 18 h at room temperature. The
solvent was removed by co-evaporation with toluene and the crude prod-
uct was purified by silica column chromatography (EtOAc!EtOAc/
MeOH 20:1) to give compound 16 (1.46 g, 75%, 0.68 mmol) as a colorless
solid. Rf =0.21 (EtOAc/MeOH 15:1); [a]2D0 = +21.05 (c=0.51 in CHCl3);
1H NMR (600 MHz, [D6]DMSO, 308C): d=7.91–7.89 (m, 2H; H-4-Fmoc,
H-5-Fmoc), 7.81–7.78 (m, 2H; NH’’-Ac, NH’’’’-Ac), 7.76–7.72 (m, 2H; H-
2’’’’’), 4.72–4.67 (m, 4H; H-1’’, H-1’’’, H-1’’’’’, H-2’), 4.63 (d, J
4.1 Hz, 1H; H-1), 4.55 (d, J(H-1’,H-2’)=7.9 Hz, 1H; H-1’), 4.51–4.47 (m,
3H; H-1’’’’, CH2a-(Fmoc)), 4.44 (m, 1H; CH2b-(Fmoc)), 4.32–4.30 (m,
2H; H-60a000, H-9-(Fmoc)), 4.26 (m, 1H; H-60a0), 4.23–4.18 (m, 3H; H-5’’’, H-
5’’’’’, Tb), 4.11–4.03 (m, 3H; H-2, H-60b0, Tb), 4.01–3.97 (m, 4H; H-6 , H-
60a0b000), 3.94–3.83 (m, 5H; H-6a0 b, H-6b0000, H-5’), 3.80–3.76 (m, 2H; H-3, H-3’),
3.75–3.71 (m, 1H; H-6a), 3.70–3.61 (m, 4H; H-2’’’’, H-4’’, H-4’’’’, H-5’’),
3.56–3.53 (m,1H; H-5’’’’), 3.40–3.35 (m, 1H; H-2’’), 3.31–3.27 (m, 1H; H-
6b), 2.09–1.69 (m, 57H; CH3-(Ac)), 1.09 ppm (d, 3J
ACTHNUTRGNEUNG(Tg,Tb)=6.4 Hz, 3H;
Tg); 13C NMR (150.9 MHz, [D6]DMSO): d=170.3, 170.0, 169.9, 169.6,
169.5, 169.4, 169.1, 169.0, 168.8 (C=O-(Ac)), 156.8 (C=O-(Fmoc)), 143.8,
143.7 (C-1a-Fmoc, C-8a-Fmoc), 140.8 (C-4a-Fmoc, C-5a-Fmoc), 127.7 (C-2-
Fmoc, C-7-Fmoc), 127.1 (C-3-Fmoc, C-6-Fmoc), 125.3, 125.2 (C-1-Fmoc,
C-8-Fmoc), 120.2 (C-4-Fmoc, C-5-Fmoc), 100.6 (C-1’), 100.4 (C-1’’’’),
100.0, 99.9 (C-1’’, C-1’’’, C-1’’’’’), 98.5 (C-1’’’’), 76.8 (C-3’), 76.6 (C-4’’’’),
76.2 (C-4’’), 74.1 (Tb), 73.5 (C-3), 73.4 (C-3’’’’), 72.7 (C-3’’), 71.7 (C-5’’),
Chem. Eur. J. 2013, 19, 17001 – 17010
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U. Westerlind et al.
71.5 (C-5’’’’), 70.3, 70.2, 70.1 (C-4, C-3’’’, C-3’’’’’, C-5’), 69.6 (C-5’’’, C-5’’’’’),
69.3 (C-2’), 69.0, 68.9 (C-4’, C-2’’’, C-2’’’’’, C-6), 68.6 (C-5), 67.0 (C-4’’’, C-
Chem. 1997, 109, 629–631; Angew. Chem. Int. Ed. Engl. 1997, 36,
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Pieczyk, C. Jones, M. Wild, D. Vestweber, H. Kunz, Angew. Chem.
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o) O. Seitz, C.-H. Wong, J. Am. Chem. Soc. 1997, 119, 8766–8776;
p) K. M. Koeller, M. E. B. Smith, C.-H. Wong, J. Am. Chem. Soc.
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0000
000
4’’’’’), 65.6 (CH2ab-(Fmoc)), 62.3 (C-60a0b), 61.7 (C-6’, C-6 ), 60.8 (C-6 , C-
ab
ab
6a00b000), 58.6 (Ta), 54.0 (C-2’’), 53.1 (C-2’’’’), 47.8 (C-2), 46.8 (C-9-(Fmoc)),
22.8, 22.7, 22.6, 21.0–20.3 (CH3-(Ac)), 18.70 ppm (Tg); HRMS (ESI)
2+
(pos): m/z calcd for C93H122N4O51
: ; found:
1055.8555 [M+2H]2+
1055.8560; calcd for C93H121KN4O512+: 1074.8334 [M+K+H]2+; found:
1074.8311.
General methods for the glycopeptide synthesis: The mucin tandem
repeat peptides were synthesized by stepwise solid-phase peptide synthe-
sis by using the Fmoc strategy, and starting with a preloaded Fmoc-pro-
line or Fmoc-alanine trityl resin (13 mmol scale per peptide). The synthe-
sis employed protected T- and core 3-threonine amino acid building
blocks, which were pre-activated manually by using 1.5 equivalents that
were activated with HATU/HOAt, whereas the other Fmoc-protected
amino acids were coupled automatically on a Multisyntech peptide syn-
thesizer by using eight equivalents of the amino acid and HBTU/HOBt.
Fmoc deprotection was performed according to standard conditions,
20% piperidine in DMF. After assembly of the peptide backbone, a tri-
ethylene glycol amino acid spacer was coupled to the N terminus fol-
lowed by Fmoc deprotection. The peptides were then released from the
resin, and all acid-sensitive side chain protecting groups were simulta-
ACHTUNGTRENNUNGneously removed by treatment with TFA/TIPS/H2O (90:5:5) followed by
solvent concentration, lyophilization, and purification by using a C-18
cartridge (1 g of C-18 material, Waters). For saccharide deprotection the
O-acetyl groups were cleaved by transesterification in methanol by using
catalytic amounts of NaOMe at pH 9–9.5 (defined by using wet pH
paper) for 4–36 h (deprotection followed by analytical HPLC) to yield
peptides 39–66, which were purified by preparative HPLC (Phenomenex
Luna C18 (2), 21.20ꢂ250 mm). Additional experimental and characteri-
zation data for the glycopeptides 39–66 are given in the Supporting Infor-
mation.
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Acknowledgements
This work was supported by ISAS—Leibniz institute for Analytical Sci-
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Fellowship to U.W.) and Deutsche Forschungsgemeinschaftgrant number
WE 4751/2-1. We are grateful to Prof. Dr. Herbert Waldmann (Max-
Planck Institute of Molecular Physiology, Dortmund, Germany) for his
unconditional support.
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Received: July 24, 2013
Published online: November 4, 2013
17010
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