Novel sequential solid-phase synthesis of N-linked glycopeptides from natural sources

Article abstract of DOI:10.1039/a705528e

In the present report a practical and versatile procedure for the solid-phase synthesis of N-linked glycopeptides from natural sources has been demonstrated. The approach is based on the mild hydrazinolysis procedure to release N-linked oligosaccharides in their intact unreduced form from the natural glycoproteins, e.g. fetuin and ribonuclease B and subsequent formation of the corresponding glycosylamines. Treatment of the reducing sugars 1-7 with a saturated solution of ammonium hydrogen carbonate in either water or dimethyl sulfoxide (DMSO) gives in almost quantitative yields the glycosylamines 8-14. Coupling of the unprotected glycosylamines 8-14 to the side-chain-activated aspartic acid derivative Fmoc-Asp(ODhbt)-OBu^t 16 affords the N-glycosylated asparagine derivatives 17-23. Subsequent acetylation of the carbohydrate hydroxy groups and cleavage of the tert-Bu ester by trifluoroacetic acid (TFA) treatment yields the glycosylated N-linked building blocks 31-37. The building blocks 31-37 are then incorporated into the multiple-column peptide-synthesis protocol of the glycopeptide T-cell epitope analogues 40-46 of the mouse haemoglobin-derived decapeptide Hb (67-76), VITAFNEGLK. The decapeptide sequence VITAFNEGLK binds well to the MHC Class II E^k molecule and is non-immunogenic in CBA/J mice. Syntheses of several natural and unnatural glycosylations, e.g. N-acetylglucosamine, N,N′-diacetylchitobiose, glucose, maltotriose, maltoheptose and di- and triantennary complex oligosaccharides on the decapeptide Hb (67-76) affording the N-linked glycopeptides 40-46 are described. The N-linked glycopeptides 40-46 have been fully characterised by 1D- and 2D-¹H and ¹³C NMR spectroscopy and by ES-MS.

Full text of DOI:10.1039/a705528e

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Novel sequential solid-phase synthesis of N-linked glycopeptides from
natural sources
Ernst Meinjohanns,a Morten Meldal,*,a Hans Paulsen,b Raymond A. Dwekc
and Klaus Bocka
a Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby,
Copenhagen, Denmark
b Institut für Organische Chemie, Universität Hamburg, Martin-Luther-King-Platz 6,
D-20146 Hamburg, Germany
c Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road,
Oxford, UK OX1 3QU
In the present report a practical and versatile procedure for the solid-phase synthesis of N-linked
glycopeptides from natural sources has been demonstrated. The approach is based on the mild
hydrazinolysis procedure to release N-linked oligosaccharides in their intact unreduced form from the
natural glycoproteins, e.g. fetuin and ribonuclease B and subsequent formation of the corresponding
glycosylamines. Treatment of the reducing sugars 1–7 with a saturated solution of ammonium hydrogen
carbonate in either water or dimethyl sulfoxide (DMSO) gives in almost quantitative yields the
glycosylamines 8–14. Coupling of the unprotected glycosylamines 8–14 to the side-chain-activated
aspartic acid derivative Fmoc-Asp(ODhbt)-OBut 16 affords the N-glycosylated asparagine derivatives
17–23. Subsequent acetylation of the carbohydrate hydroxy groups and cleavage of the tert-Bu ester
by trifluoroacetic acid (TFA) treatment yields the glycosylated N-linked building blocks 31–37. The
building blocks 31–37 are then incorporated into the multiple-column peptide-synthesis protocol of the
glycopeptide T-cell epitope analogues 40–46 of the mouse haemoglobin-derived decapeptide Hb (67–76),
VITAFNEGLK. The decapeptide sequence VITAFNEGLK binds well to the MHC Class II Ek molecule
and is non-immunogenic in CBA/J mice. Syntheses of several natural and unnatural glycosylations,
e.g. N-acetylglucosamine, N,NЈ-diacetylchitobiose, glucose, maltotriose, maltoheptose and di- and tri-
antennary complex oligosaccharides on the decapeptide Hb (67–76) affording the N-linked glycopeptides
40–46 are described. The N-linked glycopeptides 40–46 have been fully characterised by 1D- and 2D-1H
and 13C NMR spectroscopy and by ES-MS.
fully protected peptide containing an activated aspartic acid
Introduction
side-chain either in solution or on a solid-phase. An orthogonal
side-chain-protecting group compatible with the fluoren-9-
ylmethoxycarbonyl (Fmoc) methology was required during
peptide assembly for the aspartyl β-carboxy group. The protect-
ing group was selectively removed to obtain the free β-carboxy
group which could be activated and subsequently coupled with a
simple glycosylamine to give the desired N-glycopeptide. The
major problem which has not been solved efficiently yet is the
intramolecular aspartimide formation with the C-terminal
amide group upon activation of the aspartic acid side-chains,
particularly when the active ester is N-terminal to a small resi-
due like glycine or alanine (Scheme 1). This side-reaction com-
petes with glycosylation and practically limits the efficacy of the
convergent approach to very small glycan structures. Several
approaches have been described which reduce aspartimide for-
mation.12,13 However, all of these methods remain problematic.
Alternatively, the versatile and general building block
approach14,15 has been found to be advantageous in many
respects. It involves the incorporation of preformed glyco-
sylated Nα-Fmoc-protected asparagine derivatives to which the
target sugar is N-linked through the β-carboxamide in standard
or multiple-synthesis protocols. This method currently offers
the most versatile and general approach for the preparation of a
large variety of complex homogeneous glycopeptides with well
defined and predetermined structures15 and even larger libraries
of biologically active glycopeptides.
Many secreted and cell-surface proteins are heavily glycosylated
with both N- and O-linked oligosaccharides. Asparagine-linked
glycosylation of proteins is the predominant modification in
eukaryotic cells. The N-linked oligosaccharides are covalently
attached to the asparagine (Asn, N) side-chain via a β-N-
glycosidic linkage. The structures of N-linked oligosaccharides
fall into three categories: high mannose, complex and hybrid
type.1,2 All N-linked glycoproteins share the common penta-
saccharide core structure (Man)3-(GlcNAc)2 but do vary in the
nature of the outer residues. The effects of these sugars on the
physical properties,3 immunogenicity,4 folding5 and stability of
the glycoproteins are important issues. Protein glycosylations
can effect pharmacological parameters including circulating
life-time,6 solubility,3 proteolytic stability7 and immunogenic-
ity.4 The N-glycopeptides are often used as models for studying
the interactions between carbohydrates and peptides.8,9 Owing
to the difficulties in expressing or isolating well defined glyco-
proteins, a convenient synthetic route to these glycopeptides
would be of great value. Investigations of protein–carbohydrate
interactions involving complex N-linked glycopeptides have
been performed with only little success due to difficulties in
their preparation.
Owing to the biological importance of protein glycosylation,
the solid-phase synthesis of N-linked glycopeptides has fre-
quently been attempted. Currently, there are two approaches
to synthesise N-linked glycopeptides: (i) The convergent
approach10,11 and (ii) the building-block approach.
The release of N-linked oligosaccharides in small amounts
has previously been accomplished by enzymic16,17 and chemical
methods. Enzymic methods have a high degree of specificity;
The former is based on the coupling of a glycosylamine to a
J. Chem. Soc., Perkin Trans. 1, 1998
549

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however, the enzyme must be validated for every new applic-
ation. An important endoglycosidase which has been very
useful in the analysis of N-linked glycans was identified in
Flavobacterium meningosepticum. However, success of the
enzymic method for preparative purposes is unfortunately
highly dependent on the substrate specificity and on the avail-
ability of the enzyme. Chemical methods have the advantage
of broad specificity towards the substrate and include a large
variety of chemical reagents, e.g. hydrazine,18 β-elimination by
mild alkaline treatment or under strongly alkaline conditions,19
and lithium aluminium borohydride.20 The reducing agent con-
verts the reducing sugars to a sugar alditol residue. On the other
hand, hydrazine18 as cleaving reagent offers the possibility of
obtaining the released oligosaccharides in their unreduced
hemiacetal form. It can be used to release O- and N-glycans
from proteins, and the method can be optimized to permit the
selective removal of only O-linked residues.
Bovine fetuin, the major glycoprotein in foetal calf serum, is
an α-globulin of Mr 48 000.21 It is a globular 341-amino acid
protein with six internal disulfide bridges and six carbohydrate
moieties per molecule, three O-glycosidically linked to Ser/Thr
and three N-glycosidically linked to Asn.22 The majority of the
Asn-linked oligosaccharides (80%) on fetuin have three peri-
pheral branches23 with the desialylated structure presented in
Fig. 1a. A minor population of dibranched Asn-linked oligo-
saccharides, lacking the branch arising from the peripheral
β-1,4-linked GlcNAc residue, has also been detected.
O
R
O
R
H
H
N
N
Activation
Cyclisation
N
N
H
H
O
O
CO2H
CO2X
α-peptide
X = activation catalyst
Sugar-NH2
O
R
O
R
H
N
H
N
N
N
H
O
O
CONH
O
Sugar
N-Glycopeptide
Aspartimide
The isolation of high-mannose structures was achieved by
hydrazinolysis of bovine ribonuclease B.24,25 Bovine ribo-
nuclease B is a glycoprotein with a relative molecular mass of
15 500 daltons. It contains 124 amino acid residues and one
N-glycosylation site at Asn 34.24 Ribonuclease B contains high-
mannose type oligosaccharides with five to nine mannosyl
residues [(Man)5(GlcNAc)2→(Man)9(GlcNAc)2] as presented in
Fig. 1b.
H
N
CO2H
O
H
N
O
R
β-peptide
The N-linked synthetic target has been selected to allow the
investigation of the activation of T-cells by glycopeptides
bound to MHC-class II molecules as an important event in
the triggering of the immune response. Owing to lack of avail-
Scheme 1 Aspartimide formation during convergent N-glycopeptide
synthesis. This side-reaction is depending on the chemical structure of
the adjacent amino acid towards the C-terminal. For large oligosac-
charides, aspartimide formation is predominant.
β-Gal-(1→4)-β-GlcNAc-(1→2)-α-Man-(1→6)
β-Gal-(1→4)-β-GlcNAc-(1→4)
α-Man-(1→3)
β-Man-(1→4)-β-GlcNAc-(1→4)-GlcNAc-Asn
β-Gal-β-(1→4)-β-GlcNAc-(1→2)
a
α-Man-(1→6)
α-Man-(1→6)
α-Man-(1→3)
β-Man-(1→4)-β-GlcNAc-(1→4)-GlcNAc-Asn
α-Man-(1→3)
α-Man-(1→2)0–4
b
OH
OH
O
HO
HO
OH
O
H
NAc
OH
O
O
HO
H
O
HO
O
HO
N
O
O
O
NHAc
OH
NH
O
OH
O
OH
OH
OH
Fig. 1 (a) Desialylated triantennary complex-type oligosaccharide structure from bovine fetuin. (b) Branched high-mannose-type oligosaccharide
structure from ribonuclease B. (c) The common core of N-linked oligosaccharides.
550
J. Chem. Soc., Perkin Trans. 1, 1998

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OH
ability the glycan-specific immunogenicity of glycoproteins and
glycopeptides is described in only a few reports.26,27 Studies of
glycopeptide epitopes, mimicking post-translational modifi-
cation of proteins, is therefore of significant interest. Recently
it was found that O-glycosylation, as found in mucins, can be
specifically recognized by T-cells when linked to the MHC-Class
II binding CBA/J mouse haemoglobin Hb (67–76) peptide.26
This decapeptide, VITAFNEGLK, binds well to the MHC-
Class II Ek molecule and is itself non-immunogenic in CBA/J
mice.28
NHAc
O
O
HO
RO
H
N
O
HO
O
NHAc
NH
O
OH
N-linked glycoprotein
Hydrazinolysis NH2NH2
OH
The aim has been to release and recover N-linked oligo-
saccharides from the natural sources fetuin and ribonuclease B
in their non-reduced form by employing the mild conditions of
hydrazinolysis18,29 in order to synthesise N-linked glycosyl-
asparagine building blocks for use in the multiple-column
peptide syntheses (MCPS) of N-linked glycopeptides. Both
fetuin and ribonuclease B are commercially available, well char-
acterised, affordable and offer the complex-type (fetuin) or the
high-mannose (ribonuclease B) oligosaccharide structures,
respectively. The N-linked glycopeptides 40–46 were prepared
as putative immunogenic epitopes binding to the Major Histo-
compatibility Complex (MHC) class II Ek molecule in CBA/J
mice and eliciting a T-cell response.
NH2
H
N
O
HO
RO
O
H
HO
O
N
H
NH2
OH
β-glycosylhydrazine
N-acetylation Ac2O
OH
NHAc
O
H
N
O
HO
RO
O
HO
Ac
N
H
NHAc
OH
Results and discussion
β-glycosyl-2-acetylhydrazine
In order to investigate the fine specificity of T-cell response
against synthetic glycopeptides further, a series of N-glyco-
peptide analogues of immunogenic epitope structures has been
synthesised. In the present paper, the synthesis of N-glyco-
sylated asparagine building blocks with extended triantennary
chains was demonstrated and they were applied in the solid-
phase synthesis of N-linked glycopeptides derivatised at the
asparagine in the decapeptide VITAFNEGLK. The versatility
of the present method was demonstrated by variation of the
glycan structure to include glucose, maltotriose, maltoheptose,
N-acetylglucosamine and chitobiose as model compounds as
well as high-mannose and the di- and tri-antennary complex
oligosaccharide structures.
Cu(OAc)2
OH
NHAc
O
HO
RO
O
HO
O
OH
NHAc
OH
Reducing oligosaccharide
Scheme 2 Hydrazinolysis to obtain N-linked oligosaccharides as the
reducing sugars from the natural glycoprotein sources, bovine fetuin
and ribonuclease B
The results of preparative hydrazinolysis of fetuin and
ribonuclease B indicate that hydrazinolysis performed under
controlled and optimised conditions can be used to release
intact, unreduced N-linked oligosaccharides on a preparative
scale (100–500 mg) in high overall yields. In the case of fetuin
hydrazonolysis the N-linked oligosaccharides could be separ-
ated from the small O-linked disaccharides by simple separ-
ation on cellulose. The construction of the N-glycosyl linkage
by the present method proceeds through conversion of the
isolated oligosaccharides into their corresponding glyco-
sylamines followed by coupling with the 3,4-dihydro-4-oxo-
1,2,3-benzotriazin-3-yl (Dhbt)-activated side-chain Fmoc-
Asp(ODhbt)-OBut 16. The hydroxy groups of the product
were O-acetylated and the But ester protecting group was
removed by TFA treatment. The purified N-linked asparagine
building blocks 31–37 have been directly employed in MCPS.
MCPS was performed in a Teflon block with 20 synthesis
columns utilizing standard Fmoc-based solid-phase peptide-
synthesis procedures. This procedure has previously been
shown to be suitable for successful N-glycopeptide synthesis.
The N-glycopeptide syntheses were performed either on
Macrosorb or preferably on poly(ethylene glycol)–poly-
(dimethylacrylamide) copolymer (PEGA) resin.30 The resin was
derivatised by the acid-labile hydroxymethylphenoxyacetic acid
(HMPA) by activation with 2-(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium tetrafluoroborate (TBTU) and 4-ethyl-
morpholine (NEM). The first amino acid, lysine, was coupled
as Fmoc-Lys(Boc)-OH by activation with 1-mesitylenesulf-
onyl)-3-nitro-1,2,4-triazole (MSNT) and N-methylimidazole.
Other amino acids were coupled as (pentafluorophenol) OPfp
esters with addition of Dhbt-OH as a catalyst.
Hydrazinolysis procedure
Hydrazinolysis of glycoproteins on an analytical scale has
become a popular method for the isolation of N-linked oligo-
saccharides.29,31 The large-scale hydrazinolysis for the release
and preparative use of N-linked oligosaccharides in the syn-
thesis of N-glycopeptides has not yet been reported. The use of
hydrazinolysis on a large scale for the isolation of N-linked
oligosaccharides from glycoproteins according to Scheme 2
offers a possibility to obtain larger quantities of N-linked
oligosaccharides to be used in synthetic reaction sequences. The
hydrazinolysis procedure was performed under controlled and
optimised conditions. After exhaustive dialysis and lyophiliz-
ation of fetuin and ribonuclease B, respectively, 5 g portions
were heated with anhydrous hydrazine in a sealed tube by an
optimised hydrazinolysis procedure29 at 85 ЊC for 12 h. Hydra-
zine was then removed by repeated condensation to dryness,
with several additions of toluene, under reduced pressure. After
subsequent N-acetylation with acetic anhydride in a saturated
DMSO or aq. sodium hydrogen carbonate solution and ion-
exchange chromatography on a Dowex AG-50 X12 column the
pool of oligosaccharides was lyophilised. Subjecting the crude
product to cellulose column chromatography separated the
N-linked oligosaccharides from the peptides and amino acid
contaminants and smaller O-linked saccharides (disaccharides).
To cleave the formed oligosaccharide hydrazides and residual
sialic acids the oligosaccharide pool was first treated with
copper() acetate for 30 min and then with 25 m sulfuric acid
at 80 ЊC for 1 h. After column purification the neutral desialyted
oligosaccharides were filtered off and lyophilized. Exhaustive
1H NMR spectroscopy, laser-desorption mass spectrometry
J. Chem. Soc., Perkin Trans. 1, 1998
551

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O
O
O
OH
O
O
N
H
15
N
O
O
N
N
O
O
O
O
N
H
O
OH
OH
OH
16
H
N
O
ii
O
O
O
i
R′O
HO
R′O
HO
R′O
HO
NH2
OBut
OH
R
R
R
O
NHFmoc
1–7
8–14
17–23
1, 8, 17 R = NHAc, R′ = H
2, 9, 18 R = NHAc, R′ = GlcNAc
3, 10, 19 R = OH, R′ = H
4, 11, 20 R = OH, R′ = (Glc)2
5, 12, 21 R = OH, R′ = (Glc)6
iii
6, 13, 22 R = NHAc, R′ = (Gal)2(GlcNAc)3(Man)3
7, 14, 23 R = NHAc, R′ = (Gal)3(GlcNAc)4(Man)3
OAc
OAc
H
N
O
H
N
O
O
O
iv
R′O
AcO
R′O
AcO
OH
OBut
R
R
O
NHFmoc
O
NHFmoc
31–37
24–30
24, 31 R = NHAc, R′ = Ac
25, 32 R = NHAc, R′ = Ac3GlcNAc
26, 33 R = OAc, R′ = Ac
27, 34 R = OAc, R′ = (Ac4Glc)(Ac3Glc)
28, 35 R = OAc, R′ = (Ac4Glc)(Ac3Glc)5
29, 36 R = NHAc, R′ = (Ac4Gal)2(Ac2GlcNAc)3(Ac3Man)2(Ac2Man)
30, 37 R = NHAc, R′ = (Ac4Gal)3(Ac2GlcNAc)4(Ac3Man)2(Ac2Man)
Scheme 3 Procedure for the synthesis of the oligosaccharide–asparagine building blocks 31–37. Reagents and conditions: i, (NH4)HCO3; ii, 16
(2 mol equiv.), DIPEA (1.2 mol equiv.), DMSO; iii, Ac2O–pyridine; iv, TFA (1 h).
(LD-MS) and P-4 gel chromatography studies proved the pres-
ence of two structures: the diantennary-complex structure 6
(20%) and the triantennary-complex structure 7 (80%). The
hydrazinolysis procedure with ribonuclease B afforded the iso-
lation of a mixture of exclusively high-mannose-type structures
from Man5 to Man9. P-4 gel chromatography afforded five dif-
ferent high-mannose structures (Man5 to Man9) in sufficient
antennary structure 7 and the high-mannose oligosaccharide
structures (Man5 to Man9) of ribonuclease B, were treated with
saturated aq. ammonium hydrogen carbonate to produce the
corresponding β--glycosylamines 8–14. A solution of the sac-
charide (1%, w/v) in saturated aq. NH4HCO3 was kept for 24 h
at 45 ЊC. Conversion of the reducing sugars 1–7 into the corre-
1
sponding β-glycosylamines 8–14 was followed by H and 13C
1
quantities for H NMR characterisation. Owing to capacity
NMR spectroscopy in D2O and the reducing sugar was con-
verted stereoselectively into the β-anomer in all cases. Alter-
natively, DMSO could be used as a solvent, which increased the
yield. The typical strong upfield shift displacement of the
anomeric proton H-1 signal for the reducing terminus upon
amination was observed. The H-1 of the glycans were all
detected at δ ~4.3–4.1. The oligosaccharides were converted
into the glycosylamines in high yields (95%) with no detectable
formation of di-glycosylamine. After the conversion, the sam-
ples were diluted with water, evaporated and this procedure was
repeated five times to remove all residual ammonium hydrogen
carbonate. The samples were frozen and lyophilized for 24 h.
Owing to the instability of the glycosylamines, purification by
size-exclusion chromatography is not desirable and, thus, the
lyophilized crude products were used directly for coupling to
the Fmoc-Asp(ODhbt)-OBut derivative 16. Compound 16
can be easily synthesised by esterification of the commercially
problems the P-4 gel chromatography method is inadequate for
preparative purposes. Therefore the synthesis was continued
with the mixture of Man5–Man9 high-mannose structures. The
isolation and purification of the specific high-mannose struc-
tures was performed after coupling to Fmoc-Asp(ODhbt)-
OBut 16 by semi-preparative high-performance liquid chrom-
atography (HPLC) purification. This provided high overall
yields of the desired glycosylated building blocks.
Synthesis of the glycosylated blocks 31–37
The conversion of the unprotected mono- and oligo-
saccharides 1–7 to their corresponding β-glycosylamines 8–14
was carried out by modification of the Kochetkov method.32
The reducing sugars N-acetylglucosamine 1, N,N-diacetyl-
chitobiose 2, glucose 3, maltotriose 4, maltoheptose 5, the
asialo-fetuin-diantennary structure 6, the corresponding tri-
552
J. Chem. Soc., Perkin Trans. 1, 1998

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Table 1 Selected 1H NMR chemical shifts (ppm) and coupling constants (Hz) of building blocks measured at 250 MHz on solutions in CDCl3 or
[2H6]DMSO at 300 K
31a
32a
33
34
35
Asn
NH
H-α
H-β
HЈ-β
7.53 (8.0)
4.78
2.88 (6.8, 16.0)
2.70 (7.0)
7.69 (8.2)
4.59
2.89
6.18
5.06
3.11
2.91
6.21
5.05
3.08
2.89
6.20
5.03
3.07
2.92
2.72
Fmoc
CH2
CH2Ј
H-9
4.38 (6.8, 10.5)
4.31
4.22
4.32
4.26
4.18
8.70 (9.2)
4.53
4.42
4.28
6.44
4.52
4.38
4.41
6.39
4.51
4.39
4.38
6.41
NH
8.61 (9.0)
Glc or GlcNAc
H-1
H-2
H-3
H-4
5.18 (9.4)
3.88 (9.8, 9.4)
5.10 (9.6)
4.81 (10.0)
3.84 (4.0, 2.5)
4.16 (12.5)
3.95
5.3 (9.3)
4.02
5.20
3.94
3.77
4.24
3.94
4.90 (8.3)
3.76 (9.2)
5.37 (9.0)
5.04 (9.0)
3.77
5.30 (9.5)
4.96 (9.5)
5.38 (9.4)
5.11 (9.4)
3.85 (4.5, 2.0)
4.36 (12.5)
4.12
5.28 (0.5)
4.86 (9.2)
5.34 (9.5)
4.01 (9.5)
3.82 (3.5, 2.0)
4.48 (12.0)
4.28
5.38 (3.8)
4.9 (10.0)
5.38 (9.8)
4.15 (10.0)
3.92
5.29 (9.5)
4.86 (9.0)
5.32 (9.4)
4.07 (9.5)
3.81 (4.0, 2.0)
4.41 (12.5)
4.28
5.40 (4.0)
4.92 (10.0)
5.41 (9.8)
4.07
H-5
Ha-6
Hb-6
H-1Ј
H-2Ј
H-3Ј
H-4Ј
H-5Ј
Ha-6Ј
Hb-6Ј
NH
3.89
4.19
4.04
4.02
3.87
4.28
4.10
8.21 (9.2)
8.19 (9.0),
8.02 (9.2)
1.90 (NHAc),
1.94 (NHAc),
2.11, 2.12, 2.15,
2.17, 2.19
Ac
1.89 (NHAc),
1.95, 1.98, 200
1.95, 1.97,
2.00, 2.04
1.92 (2×), 1.95
(2×), 1.97 (3×),
2.00, 2.01,
2.04
1.95 (2×), 1.97
(4×), 1.99 (2×),
2.01 (3×), 2.02
(3×), 2.04 (3×),
2.05 (3×), 2.07
(3×)
a 1H NMR spectrum was recorded in [2H6]DMSO.
available amino acid Fmoc-Asp-OBut 15 with Dhbt-OH and
N,N-dicyclohexylcarbodiimide (DCCI) in dry tetrahydrofuran
(THF) as previously described for the α-esters.33 The lyo-
philized glycosylamines 8–14 were dissolved in DMSO, two mol
equiv. of N,N-diisopropylethylamine (DIPEA) were added and
the glycosylamines 8–14 were then selectively N-acylated with 2
mol equiv. of Fmoc-Asp(ODhbt)-OBut 16 at room temp. yield-
ing the glycosylated asparagine derivatives 17–23 (Scheme 3).
The development of the reaction was followed by TLC and
analytical reversed-phase (RP) HPLC. It was found that all the
glycosylamines 8–14, even the triantennary glycan from asialo-
fetuin 14, coupled almost quantitatively to the activated
asparagine derivative 16. The crude coupling mixtures were
lyophilised and purified by RP-HPLC. The free hydroxy groups
of the sugar moieties were O-acetylated with acetic anhydride–
pyridine (1:2) for 12 h at 22 ЊC to afford the peracetylated
derivatives. Simple chromatographic filtration on silica gel 60
resulted in the purification of the building blocks 24–30. The
But protection were removed by treatment with neat TGA for
1 h and the final building blocks 31–37 were purified by prepar-
ative HPLC. They were obtained in overall yields of 45–75%.
It should be mentioned that Fuc-α-(1–6) linkages previously
observed not to be stable were not examined. However, in the
O-acetylated form of glycans the Fuc moiety is known to be
more stable, and glycopeptides containing Fuc can therefore
be prepared by the present method.
A similar procedure was performed with the high-mannose
structures from ribonuclease B; however, although separated
analytically, problems in the preparative separation of the
more-than-5 glycoform building blocks were observed as will be
reported elsewhere.
Solid-phase synthesis of the N-glycopeptides 40–46 by the
building-block approach
Building blocks 31–39 were used in an efficient and practical
solid-phase synthesis of the N-glycopeptides 40–46 starting
from the reducing oligosaccharides 1–7. The building blocks
31–39 can be used directly in MCPS of N-glycopeptides omit-
ting further chemical transformation on the solid phase. The
MCPS was carried out on either Macrosorb or PEGA resin
modified with the acid-labile HMPA linker utilizing preacti-
vated Fmoc-amino acid-OPfp esters with Dhbt-OH catalysis
and subsequent Fmoc deprotection with 20% piperidine in
dimethylformamide (DMF). The glycosylated Fmoc-Asn-OH
building blocks were coupled using the coupling reagents
TBTU or 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) and only a small excess of the
building blocks (1.1–1.6 mol equiv.) was used. The coupling
reagents TBTU and HBTU were both evaluated and it was
found that, compared with TBTU, HBTU afforded an overall
insignificantly higher coupling yield between the glycosylated
Fmoc-Asn-OH building blocks 31–39 and the resin-bound
peptide. Despite the large size of the heptasaccharide building
1
The yields and the H and 13C NMR spectroscopy data of the
glycosylated building blocks 31–37 are presented in Tables 1
and 2 and in the Experimental section. The unprotected build-
ing blocks 38 and 39 were obtained by omission of the O-
acetylation procedure. Compounds 31–39 were subsequently
successfully used as building blocks for the solid-phase syn-
thesis of the N-glycopeptides 40–46. The results demonstrated
further the stability of the N- and O-glycosidic bonds in the
extended sugar chains during the TFA treatment. No cleavage
of N- or O-glycosidic bonds in the oligosaccharides was
1
observed during analytical HPLC, H NMR and MS studies.
J. Chem. Soc., Perkin Trans. 1, 1998
553

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Table 2 Selected 13C NMR chemical shifts (ppm) of the building blocks 31–35 measured at 62.9 MHz at 300 K on solutions in [2H6]DMSO or
CDCl3
31a
32a
33
34
35
Asn
C-α
C-β
49.8
36.7
50.9
37.6
50.2
37.7
50.1
37.6
50.2
37.6
Glc or GlcNAc
C-1
C-2
C-3
C-4
C-5
C-6
C-1Ј
C-2Ј
C-3Ј
C-4Ј
C-5Ј
C-6Ј
78.1
78.7
78.2
70.8
73.7
67.8
72.5
61.4
77.9
71.3
74.7
72.4
74.3
62.7
95.8
70.1
68.9
70.9
68.6
61.5
77.8
71.4
74.8
71.9
74.2
62.8
96.0
70.2
69.2
70.8
68.4
61.2
52.2
73.3
68.3
72.2
61.8
53.0
74.8
71.4
76.3
62.8
100.94
54.5
73.1
69.2
74.2
61.9
Fmoc
C-9
CH2
47.6
65.9
47.5
66.5
47.1
67.4
47.0
67.5
47.0
67.5
C᎐O
᎐
155.8
155.9
155.9
156.0
156.0
Ar
144.5, 141.5,
128.4, 127.8,
126.0, 120.9
144.6, 141.6,
128.5, 127.9,
126.1, 120.9
144.1, 141.7,
127.8, 127.1,
125.0, 120.2
144.2, 141.6,
127.9, 127.3,
125.2, 120.4
144.1, 141.7,
127.8, 127.2,
125.1, 120.3
a 13C NMR spectrum was recorded in [2H6]DMSO.
Table 3 Results of the SPPS of the N-linked glycopeptides 40–46 utilising the building blocks 31–39. Comparison of expected and observed
molecular mass for the individual glycopeptides 40–46 analysed by MALDI and ES-MS
Compound
Molecular formula
Relative molecular mass
ES-MSa
Yields (%)b
40
41
42
43
44
45
46
C58H95N13O20
C66H108N14O25
C56H92N12O20
C68H112N12O30
C92H152N12O50
C112H184N16O60
C126H207N17O70
1294.47
1497.68
1253.41
1577.70
2226.27
2714.76
3080.10
1295.00
1498.10
1254.17
1578.01
2227.73
2715.41
3080.56c
76
62
78
62
46
n.d.
35
a Glycopeptides were detected as [glycopeptide ϩ H]ϩ ions. b Yields were determined based on the loading of the resin (0.2 mmol gϪ1) and obtained
after semipreparative RP-HPLC. c Glycopeptide was detected as [glycopeptide ϩ H]ϩ ions by MALDI-MS. n.d. = not determined.
block 35, coupling yields were not significantly lower than
Conclusions
observed with the smaller building blocks 31–34. In the case of
large building blocks 36–39 the PEGA resin was used because
of its good swelling capacity in DMF and adequate pore size,
which provides good access for the large building blocks 36–39
to the resin-bound peptide.
In the present work an efficient and practical MCPS protocol
which allows the simultaneous and parallel synthesis of the N-
linked glycopeptides 40–46 with a significant variety of carbo-
hydrate sizes and structures has been described. The large
oligosaccharides presented in Fig. 1 were obtained by hydra-
zinolysis (Scheme 2) of fetuin and ribonuclease B, respectively.
The results indicate that hydrazinolysis performed under con-
trolled and optimised conditions can be used to release intact,
non-reduced, N-linked oligosaccharides from naturally occur-
ring glycoproteins in overall high yields and purity. The N-linked
glycosylated building blocks 31–39 can be synthesised by use of
the four-step procedure presented in Scheme 3. Starting with
the oligosaccharides 1–7, aminolysis, coupling of the glycosyl-
amines to the side-chain of activated Fmoc-Asp(ODhbt)-
OBut, O-acetylation of the carbohydrate moiety, and finally
cleavage of the tert-Bu ester afforded the building blocks 31–39
after final HPLC purification with high purity and yields. The
building blocks 31–39 have been shown to be well suited for
the MCPS of the N-linked glycopeptides 40–46. Furthermore,
we have demonstrated the efficient solid-phase assembly of
N-glycopeptides 40–46 by coupling the glycosylated asparagine
building blocks 31–39 to the resin-bound peptide. TBTU and
HBTU were efficient coupling reagents. The N-linked building
blocks 31–39 were fully compatible with Fmoc-based solid-
After successful coupling of the glycosylated building blocks
31–39 and Fmoc cleavage employing 20% piperidine in DMF,
synthesis was completed using Fmoc-amino acid-OPfp esters
and Dhbt-OH as an auxiliary nucleophile. The glycopeptide
was deprotected and cleaved from the resin by treatment with
TFA–water (96:5). Removal of the O-acetyl groups from the
carbohydrate part was achieved by Zemplen O-de-acetylation
with sodium methoxide in methanol at pH 8–9. The crude N-
glycopeptides 40–46 were directly subjected to semipreparative
RP-HPLC purification. Compounds 40–46 were characterised
by matrix-assisted laser-desorption time-of-flight (MALDI-
1
TOF), ES-mass spectrometry and 1D and 2D H NMR spec-
troscopy. After lyophilization the pure N-glycopeptides were
obtained in good yields from 35% in the case of the tribranched
complex oligosaccharide derivative 46 up to 78% for the N-
acetylglucosamine derivative 42. The yields are based on the
loading of the resin (0.2 mmol gϪ1) (see Table 3). The solid-
phase synthesis of the N-glycopeptides 40–46 proceeded with-
out problems even with the large building blocks 35–39 and no
deletion peptides were observed.
554
J. Chem. Soc., Perkin Trans. 1, 1998

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OH
OH
O
O
HO
O
HO
O
NHAc
OH
OH
HO
O
HO
HO
OH
O
NHac
O
OH
O
O
H
N
HO
O
HO
HO
O
O
O
NHAc
OH
OH
OH
O
OH
OH
OH
FmocN
H
O
AcNH
O
OH
O
HO
O
O
HO
OH
OH
38
OH
OH
O
O
HO
O
HO
O
NHAc
OH
OH
HO
O
HO
HO
OH
O
NHac
O
OH
O
O
H
N
HO
O
HO
HO
O
O
O
NHAc
OH
OH
OH
O
O
OH
OH
AcNH
O
FmocN
H
O
OH
O
HO
O
O
O
NHAc
O
HO
HO
OH
OH
OH
O
OH
HO
HO
HO
39
Scheme 4 Structures of di- and tri-antennary building blocks 38 and 39 ready for use in solid-phase peptide synthesis (SPPS)
phase chemistry. The difference in size of the building blocks
31–39 did not significantly influence the yields in the solid-
phase coupling reactions. PEGA resins with appropriate pore
size provide equal access for both small and large building
blocks to the site of reaction. Applying the multiple-column
glycopeptide synthesis protocol, the N-glycopeptides 40–46
could be obtained in good yields and with high purity (see
Tables 3 and 4). Immunological studies of the N-glycopeptides
40–46 will be reported elsewhere.
light, when applicable. DMF was freshly distilled by fractional
distillation at reduced pressure and kept over molecular sieves
(3 Å). Dichloromethane was distilled from phosphorus penta-
oxide and kept over molecular sieves (3 Å). Pyridine was dis-
tilled and kept over molecular sieves (3 Å). Light petroleum was
the 60–80 ЊC fraction. Concentrations were performed under
reduced pressure at <40 ЊC. Suitable protected Nα-Fmoc-amino
acid-OPfp esters were purchased from Bachem (Bubendorf,
Switzerland), the coupling reagents TBTU and HBTU from
NovaBiochem (Switzerland) and MSTN from Fluka. Macro-
sorb SPR 250 resin was from Sterling Organics, NEM, N-
acetylglucosamine, octa-acetylchitobiose, maltotriose, malto-
heptose, fetuin and ribunuclease B from Sigma, Fmoc-Asp-
OBut from Bachem, Dhbt-OH from Fluka and DIPEA from
Sigma. Dowex AG50X 12 (Hϩ), Dowex AG 3X 4A (OHϪ),
Chelex 100 (Naϩ) and Biogel P4 (400 mesh) were purchased
from Bio-Rad (UK). ES-MS was performed in the positive
mode on a VG Fisons Quattro Instrument. MALDI-TOF MS
was performed on a Finnigan MAT 2000 spectrometer using a
Experimental
General procedures
All solvents were purchased from Labscan Ltd. (Dublin,
Ireland). Vacuum liquid chromatography (VLC) was performed
on Merck silica gel 60 H (0.040–0.060 mm) and chrom-
atography under dry conditions was performed on dried silica
gel (120 ЊC; 24 h) with solvents dried over molecular sieves.
TLC was performed on Merck Silica Gel 60 F254 aluminium
sheets with detection by charring with sulfuric acid and by UV
1
matrix of α-cyano-4-hydroxycinnamic acid. H and 13C NMR
J. Chem. Soc., Perkin Trans. 1, 1998
555

View Article Online
OH
OH
NHAc
O
O
O
HO
O
HO
HO
HO
NH
NH
RO
AcNH
AcNH
OH
O
O
H
Glu-Gly-Leu-Lys
H
Glu-Gly-Leu-Lys
Val-lle-Thr-Ala-Phe-N
Val-lle-Thr-Ala-Phe-N
O
O
40
41 R = H
45 R = (β-D-Gal)2(β-D-GlcNAc)2(α-D-Man)2(β-D-Man)
OH
O
RO
HO
NH
H
OH
O
Glu-Gly-Leu-Lys
Val-lle-Thr-Ala-Phe-N
O
42 R = H
43 R = (α-D-Glc)2
44 R = (α-D-Glc)6
HO
HN
HN
HN
HN
O
O
O
O
O
H2N
NH
NH
NH
NH
O
O
O
O
OH
OH
O
O
HO
O
HO
O
NHAc
HO
OH
OH
OH
O
O
HO
HO
OH
O
NHAc
O
OH
O
O
H
HO
O
HO
HO
N
O
O
NHAc
OH
O
OH
O
O
OH
OH
AcNH
O
OH
O
HO
O
O
NHAc
O
HO
HO
OH
OH
OH
OH
O
O
HO
HO
NH2
46
HO
Scheme 5 N-Linked glycopeptides synthesised by the present method. Peptides were assembled using Fmoc-amino acid-OPfp esters. The products
45 and 46 containing complex glycans could be used as substrates for 1–6-sialyl transferase.
spectra were recorded on Bruker AMX 600 or AM 500 spectro-
meters. Chemical shifts are given in ppm and referenced to
internal SiMe4 (δH 0.00) or to external 1,4-dioxane (δH 3.76 and
δC 67.40, respectively) for solutions in D2O at 300 K. For spec-
tra recorded in CD3CO2D–water the HOAc signal at δH 2.03
was used as internal reference. For the assignment of signals 1H,
1H–1H chemical-shift correlation spectroscopy (COSY), 1H–1H
double quantum-filtered phase-sensitive COSY, nuclear Over-
Synthesis of Fmoc-Asp(ODhbt)-OBut 16
Fmoc-Asp-OBut 15 (1.5 g, 3.65 mmol) was dissolved in freshly
distilled THF (20 cm3) and the solution was cooled to Ϫ35 ЊC.
DCCI (0.752 mg, 3.65 mmol) was added and the mixture was
stirred for 10 min. Dhbt-OH (595 mg, 3.65 mmol) was added
and the mixture was stirred for 2 h at Ϫ35 ЊC before being
allowed to warm up to ambient temperature, filtered through
Celite and concentrated. The residue was redissolved in THF
(15 cm3), filtered through Celite and concentrated. Chrom-
atography on silica gel [ethyl acetate–light petroleum (1:4)]
yielded the title compound 16 (1.78 g, 88.1%) [Found:
(M ϩ Na)ϩ ES-MS, 579.22. C30H28N4O7 requires M, 556.7];
δH(250 MHz; CDCl3) 8.35–7.15 (H, Fmoc, Dhbt), 6.05 (1 H,
NH, NHFmoc), 4.65 (1 H, ddd, Hα), 4.33 (2 H, t, Fmoc CH2),
4.18 (1 H, m, Fmoc CH), 3.32 (2 H, m, H2-β), 1.46 (9 H, s, But);
δC 135.93, 133.24, 129.48, 127.50, 126.23, 125.66, 120.35 (arom.
Fmoc and Dhbt carbons), 67.83 (Fmoc CH2), 51.16 (C-α),
47.51 (Fmoc CH), 35.02 (C-β) and 28.23 (s, But).
1
hauser effect (NOE) in rotating frame (ROESY) and H–13C
correlation spectroscopy experiments were used. Analytical and
semi-preparative RP-HPLC separations were performed on a
Waters HPLC system using switchable analytical RCM
(8 × 100 mm) and Delta PAK (15 µm; 300 Å; 25 × 200 mm)
C-18 columns with a flow rate of 1 cm3 minϪ1 and 10 cm3
minϪ1, respectively. Detection was at 215 and 280 nm with a
photodiode array detector (Waters M 991). Solvent System A:
0.1% TFA in water; B: 0.1% TFA in 90% acetonitrile–10%
water.
556
J. Chem. Soc., Perkin Trans. 1, 1998

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Table 4 1H NMR data of the N-linked glycopeptides 40–46 measured in CD3CO2D–D2O (1:) at 600 MHz and 300 K
40
41
42
43
44
45
46
40
41
42
43
44
45
46
Val
Lys
NH
α-H
β-H
γ-H3
NH
α-H
β-H2
8.11
4.45
1.94/
1.79
1.47
1.72
3.05
7.45
8.15
4.42
1.91/
1.76
1.43
1.68
3.01
7.47
8.17
4.38
1.90/
1.77
1.42
1.68
3.00
7.46
8.16
4.38
1.90/
1.77
1.40
1.68
2.99
7.47
8.17
4.40
1.90/
1.75
1.44
1.68
3.01
7.46
8.11
4.46
1.90/
1.78
1.46
1.71
3.00
7.51
8.14
4.40
1.92/
1.79
1.48
1.70
3.02
7.48
3.97
2.24
1.00,
1.02
3.92
2.22
1.00,
0.99
3.93
2.19
1.00,
0.99
3.92
2.18
1.00,
0.99
3.92
2.17
1.01,
0.99
3.93
2.18
1.01,
0.99
3.92
2.23
1.01,
1.00
γ-H2
δ-H2
ε-H2
NH2
Ile
NH
α-H
β-H
γ-H2
8.37
4.34
1.82
1.52/
1.14
0.86
0.91
8.40
4.32
1.83
1.51/
1.14
0.89
0.84
8.41
4.32
1.81
1.50/
1.14
0.88
0.83
8.41
4.31
1.80
1.50/
1.14
0.88
0.84
8.40
4.30
1.82
1.51/
1.15
0.86
0.84
8.39
4.32
1.83
1.52/
1.14
0.89
0.84
8.39
4.33
1.83
1.52/
1.13
0.88
0.85
GlcNAc
1-H
N-H
5.07
7.95
5.05
8.01
5.02
5.04
γ-H3
δ-H3
2-H
3-H
4-H
5-H
6-H
6-HЈ
3.88
3.69
3.51
3.51
3.91
3.75
3.86
3.77
3.52
3.52
3.81
3.81
3.84
3.72
n.d.
n.d.
n.d.
n.d.
3.88
3.75
3.55
n.d.
n.d.
n.d.
Thr
NH
α-H
β-H
γ-H3
8.21
4.47
4.24
1.14
8.19
4.41
4.20
1.12
8.16
4.39
4.18
1.15
8.17
4.39
4.18
1.14
8.14
4.38
4.17
1.13
8.20
4.39
4.21
1.15
8.21
4.36
4.24
1.14
GlcNAc
Ala
1-H
N-H
2-H
3-H
4-H
5-H
6-H
6-HЈ
4.58
8.17
3.79
3.602
3.50
3.50
3.94
3.74
4.64
4.64
NH
α-H
β-H3
8.05
4.36
1.29
8.08
4.41
1.27
8.09
4.32
1.28
8.08
4.31
1.28
8.07
4.35
1.28
8.09
4.38
1.29
8.02
4.35
1.27
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
Phe
NH
α-H
β-H2
7.99
4.60
3.14/
2.99
8.01
4.56
3.11/
2.98
8.01
4.56
3.11/
2.99
8.02
4.57
3.12/
2.99
8.01
4.57
3.13/
3.00
7.98
4.59
3.12/
2.98
8.00
4.60
3.11/
2.99
Glc
1-H
J1,2
2-H
3-H
4-H
5-H
6-H
6-HЈ
other 1Ј-H
4.97
(8.5)
3.43
3.60
3.42
3.51
3.90
3.72
4.98
(8.5)
3.45
3.59
3.44
3.53
3.88
3.71
5.30
(2×)
4.97
(8.4)
3.47
3.59
3.41
3.51
3.89
3.73
5.31
(6×)
Asn
NH
8.23
4.73
2.87/
2.75
8.47
8.22
4.67
2.84/
2.71
8.40
8.24
4.68
2.91/
2.81
8.68
8.23
4.69
2.90/
2.82
8.68
8.22
4.69
2.91/
2.82
8.67
8.24
4.72
2.90
8.21
4.68
2.91/
2.80
8.58
α-H
β-H/
β-HЈ
NЈH
8.61
Glu
NH
α-H
β-H2
7.98
4.39
2.18/
2.00
2.47
7.98
4.34
2.16/
1.97
2.45
8.09
4.36
2.13/
1.96
2.46
8.09
4.32
2.13/
1.95
2.47
8.07
4.36
2.12/
1.97
2.49
8.06
4.38
2.18/
1.96
2.45
8.08
4.39
2.12/
1.95
2.48
Man-3
Man-4
Man-4Ј
GlcNAc-5
Gal-6
Gal-6Ј
GlcNAc-7
Gal-8
4.77
5.12
4.94
4.58
4.48
4.47
4.79
5.12
4.91
4.58
4.47
4.47
4.55
4.47
γ-H2
Gly
NH
α-H2
8.21
3.97
8.22
3.94
8.24
3.96
8.22
3.92
8.23
3.96
8.26
3.98
8.24
3.99
NHAc
2.02
2.03,
2.02
2.04,
2.05(2), 2.05(2),
2.07
2.04
Leu
2.07(2)
NH
α-H
β-H2
γ-H
δ-H3
7.87
4.43
1.62
1.63
0.87/
0.92
7.86
4.39
1.60
1.62
0.85/
0.90
7.87
4.36
1.60
1.59
0.90/
0.86
7.88
4.38
1.61
1.58
0.90/
0.85
7.88
4.36
1.60
1.60
0.90/
0.87
7.86
4.41
1.62
1.58
0.90/
0.88
7.88
4.42
1.61
1.60
0.90/
0.87
ArH
7.30,
7.25,
7.22
7.31,
7.25,
7.21
7.30,
7.24,
7.21
7.31,
7.25,
7.21
7.31,
7.25,
7.24
7.30,
7.26,
7.24
7.31,
7.26,
7.22
n.d. = not determined.
ature was raised to 85 ЊC at 10 ЊC hϪ1 and was then maintained
at 85 ЊC for a further 12 h. Hydrazine was removed by concen-
tration under reduced pressure at 25 ЊC followed by repeated
co-concentration with anhydrous toluene. N-Acetylation of
primary amino groups was performed by the addition of a five-
fold excess of acetic anhydride (25 cm3; 0.5  final concen-
tration) in saturated aq. NaHCO3 (200 cm3) first at 0 ЊC for 20
min, followed by further addition of acetic anhydride (25 cm3)
and reaction at ambient temperature for 30–60 min. Sodium ions
were removed by passage through a Dowex AG-50 X12 (Hϩ,
200–400 mesh) column. The water eluate was reduced in volume
by rotary evaporation and was then exhaustively lyophilized.
Isolation of the N-linked oligosaccharides from fetuin and
ribonuclease B by hydrazinolysis
Bovine fetuin or ribonuclease B (5 g) was exhaustively dialysed
with a 5–10-kDa-cutoff dialysis membrane against 0.1% (v/v)
aq. TFA and was then lyophilised followed by drying in vacuo
for 96 h in a lyophilisation flask. The sample was then sus-
pended in freshly distilled anhydrous hydrazine (80 cm3; triple-
vacuum-distilled at room temp.) with a water content of <1%
v/v [as determined by direct GLC analysis with free induction
decay (FID) quantitation]. The hydrazinolysis was performed
under controlled conditions of time and temperature29 in a
sealed (melted) glass vessel under argon. The reaction temper-
J. Chem. Soc., Perkin Trans. 1, 1998
557

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Purification of the oligosaccharides
oligosaccharide was dissolved in DMSO (20 mg/5 cm3) and
The combined and freeze-dried fractions were applied to a
column of microcrystalline cellulose (E. Merck, Darmstadt,
Germany) (1.5 × 12 cm) equilibrated in butan-1-ol–ethanol–
water (4:1:1 by volume). The oligosaccharide sample was
applied to the cellulose column by dissolution in water (1 vol)
and mixing with ethanol (1 vol) on the surface of the cellulose
matrix. Butan-1-ol (4 vol) was subsequently added and mixed
with the sample components. The peptide impurities were first
eluted with 10 column volumes of butan-1-ol–ethanol–water
(4:1:1 by vol). Oligosaccharides larger than disaccharides do
not elute in the solvent system under these conditions. The
oligosaccharide pool was recovered by elution with water (100
cm3) after using methanol as transient solvent (1 vol). The
oligosaccharide samples, determined by the phenol/sulfuric
acid assay, were then filtered (0.45 µm Teflon filter) and then
lyophilised. To cleave residual oligosaccharide hydrazides the
oligosaccharide pool was dissolved in a fivefold molar excess
of 22 m copper() acetate in 0.1  acetic acid and incubated
at ambient temperature for 30 min. After passage through a
tandem column of Chelex 100 (Naϩ) and Dowex AG 50W-X12
(Hϩ), the oligosaccharide pool was then filtered through a 0.45
µm Teflon filter (Millex SR) and lyophilized. For the cleavage of
the residual sialic acids on the fetuin-oligosaccharides the
oligosaccharide pool was dissolved in a small amount of 25 m
sulfuric acid and kept for 1 h at 80 ЊC. This cleavage step was
not necessary in the case of the purification of the neutral ribo-
nuclease B oligosaccharide pool. To separate the cleaved
sialic acid monosaccharides from neutral oligosaccharides
the pool was passed through a column composed of Chelex 100
(Naϩ), Dowex AG50X-12 (Hϩ), Dowex AG3-X4A (OHϪ) and
QAE Sephadex A-25 (four-layer column). The pool of neutral
asialo-fetuin oligosaccharides was finally filtered through a 0.45
ammonium hydrogen carbonate was added until saturation.
The mixture was kept for 24–28 h at 45 ЊC. Solid NH4HCO3
was added frequently to ensure saturation. The conversion was
1
followed by H NMR analysis in [2H6]DMSO. After complete
conversion the solution was lyophilised. Utilizing DMSO pro-
hibits the hydrolysis of the glycosylamines in aqueous solutions.
In the case of very unstable glycosylamines the use of DMSO
as a polar solvent system may prohibit the hydrolysis of already
formed glycosylamines. Overall higher yields and more reliable
NMR spectra could be obtained avoiding aqueous solutions for
the preparation of glycosylamines.
General procedure for the syntheses of the N-linked building
blocks 31–37
In a typical experiment, N,NЈ-diacetylchitobiosylamine 9 (200
mg, 472 µmol) was dissolved in DMSO (10 cm3) and DIPEA
(97 µl, 566 µmol, 1.2 mol equiv.) was added. The mixture
was stirred at ambient temperature for 10 min and Fmoc-
Asp(ODhbt)-OBut (262 mg, 472 µmol, 1 mol equiv.) was added
subsequently. The solution turned immediately deep yellowish,
indicating that the coupling reaction occurred instantaneously.
The coupling reaction was monitored by RP-HPLC and TLC
[chloroform–methanol–water (10:4:1)]. After complete disap-
pearance of the starting glycosylamine 9 (30 min), the crude
mixture was directly lyophilized to remove DMSO. The crude
mixture was stirred for 6 h with an excess of acetic anhydride
in pyridine (1:2) at ambient temp. to obtain the O-acetylated
building block 25. This was concentrated and co-concentrated
with toluene and purified by column chromatography on silica
gel 60 [VLC:CHCl3–MeOH (30:1 → 10:1)]. The But-ester
was removed by treatment with neat TFA for 1 h at ambient
temp. Finally, preparative RP-HPLC [gradient 70:30 (A:B) for
5 min, then to 0:100 (A:B) in 60 min] yielded the building
block 32 in an overall yield of 65% (based on N,NЈ-diacetyl-
chitobiose 2).
Synthesis of the building blocks 31 and 33–37 was achieved
similarly. In the case of the large oligosaccharide building
blocks 35–37 O-acetylation with acetic anhydride and pyridine
was performed at 50 ЊC for 12 h. Final purification of the build-
ing blocks was obtained by preparative RP-HPLC [gradient
70:30 (A:B) for 5 min, then to 0:100 (A:B) in 60 min]. The
pure glycosylated N-linked building blocks 31–37 were fully
characterised by electrospray mass spectroscopy (ES-MS) and
1H and 13C NMR spectroscopy. The retention times by ana-
lytical HPLC [gradient: 90:10 (A:B) for 5 min, then to 0:100
(A:B) in 30 min] were Fmoc-Asn(Ac3GlcNAc)-OH 31, tR 21.33
min, Fmoc-Asn(Ac3GlcNAcAc2GlcNAc)-OH 32, tR 21.08 min,
Fmoc-Asn(Ac4Glc)-OH 33, tR 20.25 min, Fmoc-Asn(Ac10Glc3)-
OH 34, tR 26.25 min, Fmoc-Asn(Ac22Glc7)-OH 35, tR 29.03 min.
The purified N-linked building blocks 31–37 were fully charac-
terized by ES-MS, and 1H and 13C NMR spectroscopy. 1H and
13C NMR data are presented in Tables 1 and 2, respectively.
1
µm filter and lyophilized. Samples for H NMR spectroscopy
were prepared by repeated dissolution in D2O followed by con-
centration. Finally, the samples were dissolved in 0.5 cm3 of
D2O and transferred into an NMR tube. 1D 1H NMR spectra
were recorded at 500 MHz at a probe temperature of 300 K.
General method for the preparation of the glycosylamines 8–14
The glycosylamines 8–14 were prepared using a modification of
the method by Likhosherstov et al.32 The parent saccharides 1–7
were dissolved in saturated aq. ammonium hydrogen carbonate
(10 mg/1 ml) and the solution was stirred at 45 ЊC for 1–2 days.
Solid NH4HCO3 was added in fractions during the course of
glycosylamine formation to ensure saturation. The amine form-
ation was followed both by TLC (propan-1-ol–ethyl acetate–
water, 6:1:3, detection with ninhydrin reagent) and 1H and 13
C
NMR spectroscopy in D2O. In all cases conversions were found
in the range of 80–90% by comparison of the anomeric 1-H
signal of the exclusively β-glycosylamine with the anomeric 1-H
signals of the reducing half acetals. After the successful conver-
sion into the glycosyl amines 8–14 it was diluted with water and
concentrated to half the volume. This procedure was repeated 5
times until no further ammonia could be detected. The glyco-
sylamines 8–14 were repetitively frozen, lyophilized and dis-
solved. Gravimetrically determined yields were almost 100%.
Derivatisation of the mixture of the tri- and the di-antennary
asialo-fetuin oligosaccharides with Fmoc-Asp(ODhbt)-OBut 16
The isolated and purified asialo-fetuin oligosaccharide fraction
(20 mg, 10 µmol) was analyzed by Bio-Gel P-4 gel permeation
chromatography and laser desorption mass spectrometry (LD-
MS) using a Finnigan MAT laser desorption mass spectrometer.
It was determined that the oligosaccharide fraction exclusively
contained 20% of the dibranched complex structure 6 and 80%
of the tribranched complex structure 7. The mixture of the
fetuin oligosaccharides 6 and 7 was dissolved in water (2 cm3)
and ammonium hydrogen carbonate (1.5 g) was added. The
mixture was kept for 30 h at 45 ЊC. The formation of the
1
According to H NMR spectroscopy yields were around 80–
90%; however, yields determined in D2O may be artificially low
due to hydrolysis of the glycosyl amines 8–14 in aqueous solu-
tion. Formation of the diglycosylamine, a typical side-reaction
1
during the amination procedure, was not observed by H and
13C NMR spectroscopy. The amination reaction yields stereo-
specifically the β-glycosylamines 8–14 as white, amorphous
solids which could be stored at Ϫ20 ЊC under anhydrous con-
1
ditions. Yields and analytical data were determined from H
1
NMR spectra in D2O and are calculated on the integral ratio
for the anomeric proton of the glycosyl amines. Owing to the
instability of the glycosylamine in aqueous solutions the amin-
ation procedure was alternatively performed in DMSO. The
oligosaccharide-glycosylamine was carefully monitored by H
NMR spectroscopy by the disappearance of the reducing α/β
1-H signals of the reducing oligosaccharides and the character-
istic formation of the 1-H proton of the β-glycosylamine at δH
558
J. Chem. Soc., Perkin Trans. 1, 1998

View Article Online
4.12 with the coupling constant of J1,2 8.3 Hz. The reaction was
judged complete when the reducing oligosaccharide had been
nearly consumed as determined by 1H NMR analysis. The reac-
tion mixture was then diluted with water (5 cm3) and concen-
trated. This procedure was repeated five times and the formed
oligosaccharide-glycosyl amine was re-dissolved in water (2
cm3) and lyophilized several times. This procedure yielded
quantitatively the glycosyl amines 13 and 14 (20 mg). The
derivatization of the dibranched and the tribranched glyco-
sylamines 13 and 14 was accomplished by dissolution of the
oligosaccharide–glycosylamine mixture (20 mg) in DMSO (2
cm3), adding of DIPEA (1.2 mol equiv.) and twice molar excess
of Fmoc-Asp(ODhbt)-OBut 16. The reaction was allowed to
proceed at ambient temp. for 3 h. The formation of the
oligosaccharide-amino acid building blocks 22 and 23 was
monitored by analytical HPLC and TLC [ethyl acetate–
methanol–water (2:3:3)]. When the reaction was completed,
the mixture was lyophilized exhaustively. Separation of the
derivatised oligosaccharides from the asparagine amino acid
derivates was achieved by preparative HPLC [0→5 min 100:0
(A:B), then from 5→65 min to 50:50 (A:B) and from 65→85
min to 0:100 (A:B)] detected by absorption at 215 and 280
nm. The two N-linked oligosaccharides 22 and 23 eluted sep-
arately as single peaks and their purity was confirmed by ana-
lytical HPLC [0→5 min to 100:0 (A:B), then 5→35 min to
50:50 (A:B) and then 35→45 min to 0:100 (A:B). They were
pooled separately and freeze dried]. Each of the major peaks
were chromatographed again on analytical RP-HPLC and
analyzed by 1H NMR spectroscopy. Chromatographic data
(HPLC retention time) tR and yields after preparative HPLC.
Analytical HPLC [gradient: 100:0 (A:B) for 5 min, 50 to 50
(A:B) in 35 min and then 0 to 100 in 10 min] Fmoc-
Asn(Gal2GlcNAc2Man3GlcNAc2)-OBut 22 tR 16.06 min, Fmoc-
Asn(Gal3GlcNAc3Man3GlcNAc2)-OH 23 tR 14.38 min. For the
synthesis of the N-linked glycopeptides 45 and 46 containing
the dibranched and the tribranched complex oligosaccharides,
respectively, two different synthesis strategies were utilized.
The first strategy used the peracetylated building blocks 36
and 37. The separated oligosaccharide-asparagine derivatives
22 (2 mg) and 23 (8 mg) were acetylated with pyridine–acetic
anhydride (1:1; 2 cm3) at 50 ЊC for 12 h. The mixtures were
concentrated and co-concentrated with toluene and analyzed
GlcNAc2)-OH 39 tR 10.38 min. All four building blocks 36–39
have been used in two different approaches for the synthesis of
the N-linked glycopeptides 45 and 46.
Syntheses of the N-linked glycopeptides 40–46 (table 4):
preparation of the macrosorb SPR 250 and PEGA resins
Syntheses of the glycopeptides 40–46 were performed in DMF
on Macrosorb SPR 250 or alternatively PEGA-resin.30 The
resin (5 g, loading 0.2 mmol gϪ1) was packed into a 50 cm3
disposable syringe (Discardit II, Beckton Dickinson) fitted with
a Teflon filter. The syringe was connected to a suction flask
through a Teflon tube with a manual 2-way Teflon valve and the
resin was swelled in DMF (20 cm3) for 30 min. Excesses of
reagents and DMF were removed by applying vacuum to the
outlet of the syringe. The resin was derivatized with the HMPA-
linker: The HMPA-linker (455 mg, 2.5 mmol), TBTU (750 mg,
2.37 mmol) and NEM (315 µl, 2.5 mmol) were dissolved in
DMF (20 cm3) and after 5 min added to the resin. After 2 h
the resin was washed carefully with DMF (5 × 20 cm3) and
dichloromethane (5 × 10 cm3). Fmoc-Lys(Boc)-OH (1.75 mg,
3.75 mmol) and N-methylimidazole (231 mg, 223 mm3, 2.81
mmol) were dissolved in dichloromethane (20 cm3) and MSNT
(1.11 g, 3.71 mmol) was added. After 5 min the solution was
added to the resin and the mixture was kept for 3 h. The resin
was washed thoroughly with dichloromethane (5 × 10 cm3)
and then with DMF (5 × 10 cm3), and unreactive amino groups
of the resin were acetylated with a solution of 20% acetic
anhydride in DMF (10 cm3). After 20 min the resin was washed
with DMF (5 × 10 cm3) and diethyl ether (5 × 10 cm3) and
dried in vacuo.
General procedures for the solid-phase peptide synthesis (SPPS)
of the N-linked glycopeptides 40–44
The derivatised resin was transferred into a 20-column custom-
made Teflon reactor. (100 mg resin per column, loading 0.2
mmol gϪ1). Throughout the synthesis Nα-Fmoc deprotection
was effected by successive 2 min and 20 min treatments of the
resin with 20% piperidine in DMF (1 cm3/column). The wash-
ing procedure (10 vol of DMF) was repeated after each
coupling/Fmoc deprotection step. Each batch of Nα-Fmoc-
amino acid-OPfp ester (3 mol equiv.) and Dhbt-OH (1 mol
equiv.), added as an auxiliary nucleophile, was dissolved in
DMF (0.6 cm3). In the case of the N-linked glycosylated
asparagine building blocks just 1.1 mol equiv. were employed
and coupled to the free amino group by activation with TBTU
(1.5 mol equiv., this was found to be optimal) and DIPEA (1
mol equiv.). The solutions were added to the resins and then left
for 4 h. In the case of the N-glycosylated building blocks 31–37
the reaction times were elongated to 12 h. After each coupling
step the resin was washed, the Fmoc group removed, and the
resin washed as described above. After coupling of the last
amino acid, valine, the terminal Fmoc group was removed, the
resin was washed successively with DMF (10 vol) and diethyl
ether (10 vol), and dried in vacuo. Cleavage of the N-glyco-
peptides from the HMPA-linker and simultaneous amino acid
deprotection were performed by treatment with 95% aq.
TFA for 2 h. After cleavage, the soluble product filtered from
the resin and the resin was washed three times with 95% aq.
TFA. The combined filtrates were concentrated, then co-
concentrated with toluene, and the glycopeptides were then
precipitated by several triturations with diethyl ether. Residual
solvent was removed under reduced pressure and the acetylated
glycopeptides were dissolved in dry methanol (2 mg cmϪ3) and
O-deacetylated by addition of a catalytic amount of 1% sodium
methoxide in methanol until pH paper indicated pH 8.5. The
mixture was then stirred for 4–8 h at room temp., treated
with solid CO2, and concentrated. The crude glycopeptides
were dissolved in water and purified by semipreparative HPLC,
using a linear gradient 0–50% B in 50 min and then 50–
100% in 10 min. The purified N-glycopeptides 40–44 were then
1
by analytical HPLC and H NMR spectroscopy. Both tech-
niques showed that complete O-acetylation of the building
blocks 22 and 23 could not be achieved. The analytical HPLC
showed a mixture of not-completely acetylated products 29
and 30. However, the synthesis was continued by cleaving the
tert-Bu ester of the oligosaccharide-asparagine derivatives
29 and 30 with treatment with neat TFA for 1 h. TFA was
evaporated and it was co-evaporated with toluene and the
compound was purified by semipreparative HPLC. The not
completely acetylated building blocks 36 (2.6 mg) and 37
(12 mg) were, however, used in the synthesis of the glycopep-
tides 45 and 46. Owing to the difficulties in peracetylating the
oligosaccharide-asparagine building blocks 22 and 23 another
strategy was followed utilizing building blocks with completely
unprotected carbohydrate moieties.
In this second approach the unprotected oligosaccharide-
asparagine derivatives 22 and 23 were purified and subsequent
directly subjected to TFA treatment. Compounds 22 (2 mg) and
23 (8 mg) were kept in neat TFA for 1 h, respectively, in order to
cleave the tert-Bu ester. Purification by semipreparative HPLC
afforded the diantennary- and triantennary-complex building
blocks 38 (1.2 mg) and 39 (5.2 mg), respectively. The pure
oligosaccharide-asparagine building blocks 38 and 39 were
1
characterized by ES-MS and H NMR spectroscopy. HPLC
retention times tR were in analytical semipreparative HPLC
[gradient: 100:0 (A:B) for 5 min, 50:50 (A:B) in 35 min and
then0:100in10min]:Fmoc-Asn(Gal2GlcNAc2Man3GlcNAc2)-
OBut 38 tR = 12.06 min, Fmoc-Asn(Gal3GlcNAc3Man3-
J. Chem. Soc., Perkin Trans. 1, 1998
559

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lyophilized from water and fully characterized by ES-MS and
1D and 2D 1H NMR spectroscopy. Yields and ES-MS data are
presented in Table 3. H NMR data of the N-glycopeptides
Acknowledgements
This project was supported by the EU-Science program (grant
MM-SCI*-CT92-Ϫ765).
1
40–44 are presented in Table 4.
Synthesis of the di- and tri-antennary N-linked glycopeptides 45
and 46
References
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For the synthesis of the glycopeptides 45 and 46 the PEGA
resin with the acid-labile HMPA linker was employed due to its
better swelling and pore-size properties.30 The PEGA resin (500
mg, 0.25 mmol gϪ1) was placed in a syringe and allowed to swell
in DMF (5 cm3). After treatment of the resin with piperidine in
DMF (20%) for 20 min, the resin was rinsed thoroughly with
DMF (10 vol), dried in vacuo, and derivatized with the HMPA
linker. HMPA (45.5 mg, 250 µmol), TBTU (75 mg, 237 µmol)
and NEM (63 mm3, 500 µmol) were dissolved in DMF (3 cm3)
and added to the resin after 5 min. After 2 h the resin was
washed successively with DMF (20 vol) and dichloromethane
(10 vol) and dried in vacuo for 24 h. Fmoc-Lys(Boc)-OH
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dichloromethane (3 cm3) under argon. After 5 min the solu-
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the resin were acetylated with 20% acetic anhydride in DMF.
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incorporation of the first amino acid lysine was determined by
measuring the absorbance, at 290 nm, of the dibenzofullvene
(= Fm)-piperidine adduct and comparing this with a standard
curve. The subsequent amino acids were coupled as their Pfp
esters (3 mol equiv.) with Dhbt-OH (1 mol equiv.) as auxiliary
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blocks 36–39. The acetylated building blocks 36 and 37 were
dissolved in 0.5 and 1 cm3 of DMF, respectively. TBTU (1.5
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rinsing with DMF (10 vol) the peptide synthesis was continued
as described above. Cleavage of the glycopeptide from the resin
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of a catalytic amount of sodium methoxide (1%) in methanol
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HPLC, using a linear gradient 0–50% B in 50 min and then
50–100% B in 10 min. The purified glycopeptides 45 and
46 were then characterized by MALDI-MS and 1H NMR
spectroscopy.
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In the second approach the unprotected oligosaccharide-
asparagine building blocks 38 and 39 were directly utilized in
the MCPS. The building blocks 38 and 39 were dissolved in 0.5
cm3 and 1 cm3 of a DMF–DMSO (1:1) solution, respectively.
DIPEA (1 mol equiv.) and TBTU (1.4 mol equiv.) were added
and the solutions were added to the resin. This mixture was
kept for 16 h and was then rinsed with DMF (10 vol). The
peptide synthesis was continued as described above. Cleavage
and deprotection of the glycopeptides 45 and 46 was achieved
with 95% aq. TFA. The crude glycopeptides 45 and 46 were
dissolved in water and directly purified by semipreparative
HPLC using a linear gradient 0–50% B in 50 min and then 50–
100% B in 10 min. The purified glycopeptides 45 and 46 were
then characterized by MALDI-MS and 1H NMR spectroscopy.
The partial assignment of the 1H NMR data for glycopeptides
45 and 46 is presented in Table 4.
33 E. Atherton, L. H. Jill, M. Meldal, R. C. Sheppard and R. M.
Valerio, J. Chem. Soc., Perkin Trans. 1, 1988, 2887.
Paper 7/05528E
Received 30th July 1997
Accepted 29th September 1997
560
J. Chem. Soc., Perkin Trans. 1, 1998