Prompt Chemoenzymatic Synthesis of Diverse Complex-Type Oligosaccharides and Its Application to the Solid-Phase Synthesis of a Glycopeptide with Asn-Linked Sialyl-undeca- and Asialo-nonasaccharides

Article abstract of DOI:10.1002/chem.200305115

We describe herein the preparation of 24 pure asparagine-linked oligosaccharides (Asn-oligosaccharides) from asparagine-linked biantennary complex-type sialylundecasaccharide [(NeuAc-α-2,6-Gal-β -1,4-GlcNAc-β-1,2-Man-α-1,6/1,3-)₂-Man-β -1,4-GlcNAc-β-1,4-GlcNAc-β-1-asparagine, 2] obtained from egg yolk. Our synthetic strategy aimed at adapting branch specific exo-glycosidases digestion (β-D-galactosidase, N-acetyl-β-D-glucosaminidase and α-D-mannosidase) of the individual asialo-branch after preparation of monosialyloligosaccharides obtained from 2 by acid hydrolysis of NeuAc. In order to perform branch specific exo-glycosidase digestion, isolation of pure monosialyloligosaccharides obtained was essential. However, isolation of two kinds of monosialyloligosaccharides are difficult by HPLC due to their highly hydrophilic nature. Therefore, we examined chemical protection with hydrophobic protecting (Fmoc and benzyl) groups. These chemical protection enabled us to separate the monosialyloligosaccharides by use of a HPLC column (ODS) on synthetic scales. Using these pure monosialiloligosaccharides enable us to obtain 24 Asn-linked oligosaccharides (100 mg scale) within a few weeks by branch specific exo-glycosidase digestions (α-D-neuraminidase, β-D-galactosidase, N-acetyl-β-D-glucosaminidase and α-D-mannosidase). In addition, solid-phase synthesis of glycopeptide having Asn-linked sialyl-undeca- and asialo-nonasaccharides thus obtained, was also performed on an acid labile HMPA-PEGA resin.

Full text of DOI:10.1002/chem.200305115

FULL PAPER
Prompt Chemoenzymatic Synthesis of Diverse Complex-Type
Oligosaccharides and Its Application to the Solid-Phase Synthesis of a
Glycopeptide with Asn-Linked Sialyl-undeca- and Asialo-nonasaccharides
Yasuhiro Kajihara,*[a] Yasuhiro Suzuki,[a] Naoki Yamamoto,[a] Ken Sasaki,[b]
Tohru Sakakibara,[a] and Lekh Raj Juneja[b]
Abstract: We describe herein the prep-
aration of 24 pure asparagine-linked
of NeuAc. In order to perform branch
specific exo-glycosidase digestion, iso-
lation of pure monosialyloligosacchar-
ides obtained was essential. However,
isolation of two kinds of monosialyloli-
gosaccharides are difficult by HPLC
due to their highly hydrophilic nature.
Therefore, we examined chemical pro-
tection with hydrophobic protecting
(Fmoc and benzyl) groups. These
chemical protection enabled us to sepa-
rate the monosialyloligosaccharides by
use of a HPLC column (ODS) on syn-
thetic scales. Using these pure mono-
sialiloligosaccharides enable us to
obtain 24 Asn-linked oligosaccharides
(100 mg scale) within a few weeks by
branch specific exo-glycosidase diges-
tions (a-d-neuraminidase, b-d-galacto-
oligosaccharides
(Asn-oligosacchar-
ides) from asparagine-linked bianten-
nary complex-type sialylundecasacchar-
ide [(NeuAc-a-2,6-Gal-b-1,4-GlcNAc-
b-1,2-Man-a-1,6/1,3-)2-Man-b-1,4-
GlcNAc-b-1,4-GlcNAc-b-1-asparagine,
2] obtained from egg yolk. Our syn-
thetic strategy aimed at adapting
branch specific exo-glycosidases diges-
tion (b-d-galactosidase, N-acetyl-b-d-
glucosaminidase and a-d-mannosidase)
of the individual asialo-branch after
preparation of monosialyloligosacchar-
ides obtained from 2 by acid hydrolysis
sidase,
N-acetyl-b-d-glucosaminidase
and a-d-mannosidase). In addition,
solid-phase synthesis of glycopeptide
having Asn-linked sialyl-undeca- and
asialo-nonasaccharides thus obtained,
was also performed on an acid labile
HMPA-PEGA resin.
Keywords: carbohydrates
¥
glycoproteins ¥ oligosaccharides
Introduction
roles to physicochemical- and chemical-behavior such as
protein folding, resistance to proteolysis, and clearance of
glycoproteins.[1d] N-Glycans are attached to the asparagine
residue of the Asn-X-Thr/Ser (X is any amino acid except
for proline) sequence in the protein backbone as a post-
translational modification[2] and are then transported to the
golgi apparatus for further modification by glycosyltransfer-
ases. However, most glycoproteins have many various oligo-
saccharide structures (glycoforms) depending on the struc-
tural diversity of their N-glycans. N-Glycans show micro-
heterogeneity at the nonreducing terminal and can show
over one hundred of glycoforms. However, there is no evi-
dence for why glycoproteins have glycoforms. In order to in-
vestigate the roles of the oligosaccharides, chemical and che-
moenzymatic syntheses of these oligosaccharides have been
performed.[3] Especially, in the past decade, synthetic tech-
nologies for the synthesis of large oligosaccharides have ad-
vanced considerably.[4] In order to study why the glycopro-
teins show different glycoforms, N-glycans of diverse struc-
ture must be promptly prepared and then tested in bioas-
says. Although N-glycans have been generally synthesized
from the corresponding monosaccharides, their complex
Cell-surface oligosaccharides are responsible for several bio-
logical processes such as cell cell adhesion, differentiation,
inflammation and immune responce.[1] Oligosaccharides are
mainly divided into two groups, glycolipids and glycopro-
teins. In the case of glycoproteins, oligosaccharides are
known to be O-glycans and N-glycans. The N-glycans are
further divided into highmannose-, complex- and hybrid-
types. These N-glycans on the protein surface play essential
[a] Prof. Dr. Y. Kajihara, Y. Suzuki, N. Yamamoto,
Prof. Dr. T. Sakakibara
Graduate School of Integrated Science
Yokohama City University, 22-2, Seto
Kanazawa-ku, Yokohama 236-0027 (Japan)
Fax : (+81)45-787-2370
E-mail: kajihara@yokohama-cu.ac.jp
[b] K. Sasaki, Dr. L. R. Juneja
Taiyokagaku Co, 9-5
Akahori Shinmachi, Yokkaichishi 510-0825 (Japan)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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FULL PAPER
structures result in time consuming syntheses due to repeti-
tive protection and deprotection of hydroxyl groups.
Recently, semisynthetic methods have been developed.[5]
In this approach, oligosaccharides from natural sources such
as commercially available glycoproteins are used to afford
large quantities of pure oligosaccharides. This methodology
has advantages compared with chemical and chemoenzymat-
ic synthesis, because most glycosylation reactions and pro-
tection/deprotection steps can be omitted. On the other
hand, the disadvantage is the inability to introduce branch
specific modification of N-glycan structure. Complex type
N-glycans generally form bi-, tri- and tetra-branch structures
in which the branches consist of NeuAc-a-2,6/3-Gal-b-1,4-
GlcNAc-b-1,2-Man-a sequences. In order to synthesize di-
verse oligosaccharides by the semisynthetic method, branch
specific removal of the sugar residues from a single NeuAc-
a-2,6/3-Gal-b-1,4-GlcNAc-b-1,2-Man-a structure is necessa-
ry. Therefore, we set out to synthesize over 20 pure aspara-
gine-linked oligosaccharides (Asn-oligosaccharides) using
branch-specific exo-glycosidase digestion from the aspara-
gine-linked biantennary complex-type sialylundecasacchar-
ide 2 obtained in large scale
groups to increase the degree of interaction between the oli-
gosaccharides and ODS-HPLC column (step 2 in Figure 2).
Successful purification would enable us to obtain many di-
Figure 1. Structure of sialylglycopeptide.
from egg yolk(Figure 1). If we
could prepare pure monosialy-
loligosaccharides from 2 by se-
lective acid hydrolysis of
a
NeuAc, we would be able to
obtain diverse pure Asn-oligo-
saccharides by subsequent
branch specific exo-glycosidase
digestion
(b-d-galactosidase,
N-acetyl-b-d-glucosaminidase
and a-d-mannosidase) of the
individual
asialo-branches.
However, this simple strategy
has troublesome drawbacks as
shown in Figure 2. Acid hy-
drolysis of the NeuAc may not
be selective and may afford
various products (step 1 in
Figure 2): the disialyloligosac-
charide (substrate), two kinds
of monosialyoligosaccharides
and the asialooligosaccharide
(the over-reaction product). If
these Asn-oligosaccharides can
be purified on a synthetic scale
(>100 mg scale), subsequent
branch specific exo-glycosidase
digestions would afford many
kinds of diverse Asn-oligosac-
charides. However, oligosac-
charides have a highly hydro-
philic nature, therefore the pu-
rification by HPLC is known
to be very difficult on a syn-
thetic scale. In order to purify
these oligosaccharides, we
added hydrophobic protecting
Figure 2. Synthetic strategy of diverse Asn-linked oligosaccharides.
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Complex-Type Oligosaccharides
971 985
verse oligosaccharides by branch specific exo-glycosidase di-
gestion as shown in step 3 and 4 of Figure 2.
obtained was then treated by acid hydrolysis to remove one
of the two NeuAc residues of 2 (Scheme 1). For these acidic
conditions, we have found a 40 mm of HCl solution to be
the most suitable condition for removing the NeuAc resi-
due.[8] However, as expected, this acid hydrolysis afforded
four different Asn-oligosaccharides 2 5 along with 6 and 7
as side-products. In order to use these oligosaccharides, the
Asn-oligosaccharides needed to be purified at this point.
However, since these Asn-oligosaccharides are very hydro-
philic, purification by HPLC with an ODS-column was un-
successful on scales greater than a 1 mg. Indeed, it is known
that large scale separation (mg scale) of oligosaccharide
mixtures prepared from natural sources is very difficult even
using several different HPLC columns. Therefore, we exam-
ined the addition of hydrophobic protecting groups to these
Asn-oligosaccharides in order to increase the interaction be-
tween hydrophobic protected Asn-oligosaccharides and the
ODS column. Among several protecting groups examined,
the 9-fluorenylmethyl (Fmoc) group was found to be suita-
ble for hydrophobicity as well as its application to Fmoc
solid phase glycopeptide synthesis. 9-Fluorenylmethyl-N-suc-
cimidylcarbonate[9] was added to a mixture of Asn-oligosac-
charides 2 7, affording the corresponding Fmoc-Asn-oligo-
In this paper, we report the highly efficient chemoenzy-
matic semisynthesis of 24 different, diverse Asn-oligosac-
charides and its application to the solid-phase synthesis of
sialylglycopetides by use of the asparagine linked oligosac-
charides thus obtained.
Results and Discussion
It is known that sialylglycopeptide 1 can be easily prepared
[6]
from egg yolkwith large scale. As shown in Figure 1, this
oligosaccharide has general biantennary complex-type struc-
ture. If this sialylglycopeptide can be used to make diverse
oligosaccharides, this sialyloligosaccharide would be a valua-
ble source for large scale preparation. However, in order to
use this sialylglycopeptide as a versatile oligosaccharide
block, unnecessary amino acid sequences should be removed
(Figure 1, bottom right). Therefore, we first had to remove
the amino acids in order to convert the asparagine-linked
sialyloligosaccharide 2[7] by protease digestion (Actinase-E).
The asparagine-linked biantennary oligosaccharide 2 thus
Scheme 1.
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Y. Kajihara et al.
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saccharides 8 13 in almost quantitative yields. As shown in
Figure 3, all the Fmoc-Asn-oligosaccharides can be purified
on a HPLC column except for 9 and 10. This purification
scale is limited to a 30 mg mixture 8 13 per purification
(ODS column 120 mmî250 mm). In order to obtain each
Fmoc-Asn-oligosaccharides in a few hundred mg scale, we
performed this purification repeatedly. Since the purification
of a mixture takes less 1 h, this semisynthetic method is
preferable compared with the chemical synthesis which
takes a few months up to a half year. If a large ODS column
can be used for this purification, a large scale purification
could be easily performed. In order to separate monosialyl-
oligosaccharides 9 and 10, further protection with hydropho-
bic groups was then examined. Since NeuAc residues have a
carboxylic group, this functional group should be protected
selectively by esterification. For this protection, we have
found benzylesterification to occur selectively on the car-
boxylic acid of the two NeuAc in 8 without protection of
the asparagine.[10] Therefore, this benzylesterification condi-
tion was applied to the mono-benzyl esterification of the
monosialyloligosaccharides 9 and 10 to afford the corre-
sponding products 14 and 15 in moderate yields. The in-
creased hydrophobicity of the benzyl esters enabled us to
separate Fmoc-Asn-oligosaccharides 14 and 15 (Scheme 2).
The limit of this purification is about 10 mg per purification
(ODS column 120 mmî250 mm). The structure of oligo-
saccharide 14 and 15 was determined by conversion into 16
and 12, respectively, by galactosidase digestion and subse-
quent deprotection. The Fmoc-Asn-oligosaccharide 14 and
15 can also be used in synthesis of glycopeptides with mono-
sialyl biantennary oligosaccharides which are abundant
among cell surface glycoproteins.
Figure 3. HPLC profile (analytical scale) of acid hydrolysis reaction.
Since the limit per purification of 14 and 15 is about
10 mg, we examined another condition to afford more mon-
osialooligosaccharides easily. Therefore we also examined
purification of monosialyloligosaccharides each other after
galactosidase digestion toward mixture of 9 and 10. We per-
formed exo-b-d-galactosidase digestion on a mixture of 9
and 10. We used commercially available b-d-galactosidase
containing less than 5% of other glycosidases as impurities.
This galactosidase digestion afforded monosialyl Asn-oligo-
saccharides 12 and 16 in 48 and 41% yield, respectively
(Scheme 3). Purification of these Asn-oligosaccharides can
also be performed by HPLC as shown in Figure 4. This puri-
fication (ODS column 120 mmî250 mm) can be per-
formed on a 20 mg scale of mixture of 12 and 16. Indeed,
we obtained each monosialyloligosaccharides on a 200 mg
scale by repetitive purification. In this purification, the re-
tention time of 16 was faster than that of 12. The structures
1
of Asn-oligosaccharides 12 and 16 were determined by H
NMR signals of the reporter group,[11] after conversion into
41 and 19, respectively, by glycosidase digestions. Because
the anomeric protons of the three mannosides are empirical-
ly observed with the corresponding chemical shifts in the
NMR spectrum, the structures of 19 and 41 can be identified
based on disappearance of 4H-1 and 4’H-1, respectively. As
1
shown in Figure 5 and Table 1, although differences in H
NMR data of 12 and 16 are only small, the retention time
by HPLC is clearly visible (Figure 4).
In order to modify pure Asn-oligosaccharide 16, sequen-
tial exo-glycosidase digestions were performed as shown in
Scheme 4. These exo-glycosidase digestions can be per-
formed in >100 mg scale with yields in the range 60 90%
and reactions finished within one day depending on the
enzyme quantities added. Each reactions can be easily moni-
tored by HPLC and their products 18 22 were easily puri-
fied by the same HPLC conditions. We also examined de-
protection of the N-Fmoc group by morphorine after isola-
Scheme 2.
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Complex-Type Oligosaccharides
971 985
Since those structures are positional isomers to those in
Schemes 4 and 5, that is, 40 and 18, 41 and 19, the structural-
ly diverse Asn-oligosaccharides shown in Figure 6 were pre-
pared by the same procedure shown in Schemes 4 and 5. For
Jackbeans a-d-mannosidase digestion, large excess of a-d-
mannosidase is essential. Hydrolysis of 1!6 branched a-d-
mannoside in Asn-oligosaccharide 40 (Figure 6) to afford 41
required a 27 fold excess of enzyme over substrate 18
(Scheme 4). This branch specificity of Jackbeans a-d-man-
nosidase digestion is reversed compared with Asn-penta-oli-
gosaccharide 37.[12] Brossmer reported that the 1!6
branched in mannoside in 37 was easier digested than the
1!3-branched mannoside. Attachment of the branched
NeuAc-a-2,6-Gal-b-1,4-GlcNAc-b-1,2 sequence to the man-
noside-4 may alter the substrate specificity of mannosidase
digestion. In the NMR analysis, a remarkable phenomenon
was observed. When GlcNAc-5’ residue of 12, 13 and 34, re-
spectively, was removed by N-acetyl-b-d-glucosaminidase,
the H-2 of Man-4’ was remarkably shifted upfield. This ten-
dency have been also reported by Vliegenthart.[11] As seen
in Schemes 4 and 5, Asn-oligosaccharides were obtained by
glycosidase digestion from the pure Asn-monosialyloligosac-
charide 16. Glycosidase digestion to obtain Asn-oligosac-
charides can also be performed by use of a mixture of 9 and
10 as a starting substrate, and then purified by HPLC as
shown in Scheme 6. In this one-pot procedure, b-d-galactosi-
dase and N-acetyl-b-d-glucosaminidase were added to a
mixture of 9 and 10 and the reaction was monitored by
HPLC. After removal of N-acetyl-b-d-glucosaminide, the re-
action mixture was heated under reflux for 5 min in order to
deactivate glycosidases and then a-d-neuraminidase was
added to the reaction mixture. If b-d-galactosidase and N-
acetyl-b-d-glucosaminidase activities remained after a-d-
neuraminidase digestion, the reaction mixture would afford
Scheme 3.
Figure 4. HPLC profile (analytical scale) of galactosidase digestion of 9
and 10.
tion of the each Fmoc-Asn-oligosaccharides 18 22. Com-
plete deprotection reactions afforded Asn-oligosaccharides
23 27 in good yields.
In order to further modify the Asn-oligosaccharides, we
examined a variety of sequence for glycosidases digestion as
shown in Scheme 5. These digestions can be performed on a
100 mg scale and no over-reaction products were observed
in HPLC and NMR analysis by good commercially available
glycosidase (less than 5% of other glycosidases as contami-
nant). For the asialooligosaccharides 11 and 34 (Scheme 5),
slight branch specificity was observed. However since excess
glycosidase were used, the digestions proceeded smoothly.
The diverse Asn-oligosaccharides shown in Scheme 5 were
also deprotected by morphorine to afford Asn-oligosacchar-
ides 29, 32, 33, 5, 36, and 37 in good yield.
In Figure 6, several Asn-oligosaccharides are shown which
were prepared from pure Asn-monosialyloligosaccharide 12.
Figure 5. 1H NMR spectra of monosialo-nonasaccharides 12 (top) and 16
(bottom).
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Y. Kajihara et al.
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mers can be easily purified by HPLC to afford pure 30 and
38. Although the separation of 9 and 10 is a little difficult,
the other positional isomers can easily be separated from
one another.
As shown in Schemes 2 6 and Figure 6, this procedure
can easily afford more than 100 mg of these Asn-oligosac-
charides by repetitive enzymatic reactions. Since these Asn-
oligosaccharides contain a-sialyl- and b-mannosyl linkages
which are the most difficult during chemical glycosylation,
our semisynthetic method is the first efficient procedure to
prepare such a variety of Asn-oligosaccharides. Although
some Asn-oligosaccharides were prepared by a chemical
method consisting of many steps, our method can easily
yield the desired biantennary Asn-oligosaccharides in a few
weeks. In order to increase the variety of Asn-oligosacchar-
ides, use of glycosyltransferases and sugar nucleotide ana-
logues should be employed.
Solid-phase synthesis of glycopeptide with several Asn-oli-
gosaccharides: Glycoproteins with N-glycan or O-glycan
linkages, have several different oligosaccharides on their
peptide backbones. Therefore, a synthetic technology for
glycopeptides consisting of several oligosaccharides is essen-
tial in order to elucidate the roles of glycopeptides and gly-
coproteins. However, although synthetic methods for glyco-
peptides have been developed,[13] preparation of oligosac-
charides is also time consuming, which hinders further bio-
logical investigations. Especially, the synthesis of Asn-linked
oligosaccharide such as 8 require sophisticated synthetic
techniques and is very time consuming. In addition, solid-
phase synthesis of sialylglycopeptides is very difficult, be-
cause the sialyl linkage in the glycopeptide is very labile
during trifluoroacetic acid (or HF in Boc method) treatment
to release the sialylglycopeptide from the solid phase. In this
respect, a-2,6 sialyl linkages are more labile than a-2,3-sialyl
linkages. In order for a-2 6 sialyloligosaccharide to resist
acid hydrolysis, an electron-withdrawing group such as an
acetyl group should be used as a protecting group.[13n]
Our semisynthetic method solved the above time consum-
ing problems during preparation of oligosaccharide and pro-
vides adequate quantities. In addition, asparagine is already
protected with the Fmoc group which is convenient for
Fmoc solid phase peptide synthesis. However, in order to
use Fmoc-Asn sialyloligosaccharide 8 for the synthesis of
sialylglycopeptides, selective protection of the carboxylic
acid groups in NeuAc is essential. For this problem, we have
already found a selective benzyl esterification method for
the carboxylic acid of NeuAc to afford benzyl esterified
Fmoc-Asn-sialyloligosaccharide 52 (Scheme 7)[10] In addi-
tion, we also found that benzyl esterified Fmoc-Asn-sialylo-
ligosaccharide 52 is stable towards treatment with 95% TFA
for 3 h; this is a conventional condition in solid-phase syn-
thesis to cleave the peptide from the solid phase. These new
finding enabled us to synthesize sialyloligopeptide
(ALLVNSS: N is Asn-oligosaccharide 8) on a solid phase.[10]
Since native proteins have several different oligosacchar-
ides on the peptide backbone, we next examined the synthe-
sis of a glycopeptide with two kinds of large Asn-oligosac-
Scheme 4. a) N-Acetyl-b-d-glycosaminidase; b) a-d-mannosidase; c) a-d-
neuraminidase; d) b-d-galactosidase.
multiple compounds including over-reaction products, such
as 35. The reflux time is dependent on the quantity of glyco-
sidases added. A series of those one-pot glycosidase diges-
tions afforded the structural isomers 30 and 38. These iso-
charides,
8 and 11, prepared using our semisynthetic
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Complex-Type Oligosaccharides
971 985
Scheme 5.
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many peaks appeared simplify in the HPLC analysis. This
indicates that undesired esterification occurred. Therefore,
we terminated the coupling reaction of Val at 85% yield.
The problem may result from steric hindrance between Val
and the oligosaccharide. Coupling of other amino acids
(Leu, Leu, Ala) by the OPfp method occurred in quantita-
tive yield by monitoring the Dhbt color, and no esterifica-
tion of the hydroxyl group of the oligosaccharide was ob-
served. Subsequently the Fmoc-Asn-asialooligosaccharide
11 was attached in 48% yield under the same conditions as
the disialyloligosaccharide 52. Coupling of Val, Leu, Leu,
Ala was also performed by the same pentafluorophenol
method. In this step, the coupling yields of Val, Leu, Leu,
Ala showed quantitative yield. In this case, coupling of a
second valine to asialooligosaccharyl-Asn might be good
yield, since the terminal asialooligosaccharide appear to be
flexible compared with the sialyloligosaccharide due to the
distance from the solid phase. After construction of the gly-
copeptide, the glycopeptide was deprotected and cleaved
from the resin by treatment with TFA/water (95:5) for 3 h.
In this case, the sialyl linkage was stable toward acid treat-
ment. Removal of the benzyl ester was carefully performed
using aqueous sodium hydroxide at pH 11. The crude prod-
ucts were then subjected to RP-HPLC purification. Pure
1
compound 53 was obtained and characterized by H NMR
spectroscopy (2D-TOCSY). WatergateTOCSY shows corre-
sponding amide hydrogens in the 2D NMR spectra (Sup-
porting Information). Since the coupling yield of Asn-oligo-
saccharide and valine was not high on the solid phase, sever-
al glycopeptides fragments were afforded in the final reac-
tion mixture released from solid phase. Nevertheless, the de-
sired glycopeptide with two different large Asn-
oligosaccharides can be synthesized by the solid-phase
method.
Scheme 6.
method. The peptide backbone we selected contains twelve
amino acids, ALLV N(asialooligosaccharide)ALLVN(sialyl-
oligosaccharide)SS. The sequence ALLVNSS is a fragment
of erythropoietin 85 95.[14] Although the natural type eryth-
ropoietin has Asn-linked triantennary oligosaccharide at the
asparagine in ALLVNSS, we added a biantennary oligosac-
charide for use as a model compound.
In addition, we added ALLVN(asialo-biantennary oligo-
saccharide) to the ALLVN(sialyloligosaccharide)SS in order
to perform a model solid-phase synthesis of a glycopeptide
with two different large oligosaccharides. In this amino acid
sequence, valine is attached to the asparagine. Because
steric hindrance between the isopropyl residue of valine and
the Asn-oligosaccharide may occur, this synthesis also gives
the limitations of this method. The synthetic technique is
based on the Meldal procedure.[5b] After two serines were
attached to the HMPA-PEGA resin, protected sialyloligo-
saccharide 52 was attached by 2-(1H-9-azobenzotriazole-1-
Conclusion
In the past decade, many kinds of oligosaccharides have
been synthesized by chemical and chemoenzymatic methods.
However, since asparagine linked oligosaccharides have
complicated structures, synthesis of these oligosaccharides is
very difficult. In addition, in order to examine solid phase
glycopeptide synthesis, at least 100 mg of the oligosacchar-
ide should be necessary to optimize several coupling reac-
tions as well as for obtaining an adequate quantity of target
glycopeptide. Furthermore, in order to record the 1H,15N
HSQC and its HSQC-TOCSY and HSQC-NOESY spectra,
which is a general method in structural biology of peptides
and proteins, a minimum 10 mm concentration of glycopep-
tide should be prepared if natural abundance of 15N is used
for the measurement.[15]
Our semisynthetic method has solved these problems for
the synthesis of biantennary complex type oligosaccharides
and affords 24 different, diverse Asn-oligosaccharides. Al-
though this semisynthetic method is based on a laboratory
HPLC purification procedure for the preparation of Asn-oli-
gosaccharides as a starting materials, we believe that this
method is the most convenient chemoenzymatic method for
yl)-1,1,3,3-tetramethyluronium
hexafluorophosphonate
(HATU) and diisopropylethylamine in DMSO/DMF for
24 h (yield 38%).[10] The peptide was then elongated by ad-
dition of valine, leucine, alanine using Fmoc-amino acid acti-
vated by pentafluorophenol (OPfp) esters in the presence of
3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl (Dhbt) as the cata-
lyst.[5b] Coupling of Fmoc-Val-OPfp was stopped at 85%
yield by monitoring the disappearance of the yellow Dhbt
color. If coupling of Val is continued, esterification by
amino acid toward hydroxyl groups on oligosaccharide oc-
curred. This undesired esterification was observed by HPLC
analysis after release of the glycopeptide from the solid
phase. When the sample was treated with NaOHaq (pH 11),
978
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 971 985

Complex-Type Oligosaccharides
971 985
Figure 6. Structure of oligosaccharide derivatives.
purchased from SIGMA (USA). NMR spectra were measured with
Bruker Avance 400 (308C, internal standard HOD=4.718 ppm; external
(or internal) standard acetone=2.225 ppm)[11] instrument. Pure sialylgly-
copeptide 1 was prepared by the reported method.[6]
diverse Asn-oligosaccharides, which can then be used for
further biological research and synthesis of other glycocon-
jugates. For this application, we demonstrated the solid-
phase synthesis of sialyloligopeptides with two large Asn-oli-
gosaccharides. Research to increase the quantity of several
target glycopeptides is in progress. We have applied our
method to the synthesis of glycoproteins by use of native
chemical ligation or the intein method[16] in order to investi-
gate the influence of oligosaccharides on glycoprotein con-
formation and bioactivity.
Asparagine linked sialylundecasaccharide 2
Method A: Actinase-E (9 mg) was added to a solution of sialylglycopep-
tide 1[6] (60 mg) and NaN3 in a Tris-HCl buffer (50 mm, CaCl2 10 mm,
pH 7.5, 3 mL) and this mixture was incubated for 2 d at 378C. During in-
cubation, the pH was kept at 7.5. This reaction was monitored by TLC
(1m NH4OAc/isopropanol 1:1). After the reaction was finished, the mix-
ture was lyophilized and purification of the residue by gel permeation
(Sephadex-G-25, 12.0 cmî20 cm, H2O) afforded pure asparagine-linked
sialyloligosaccharide 2[7] (42 mg, 86%).
Method B: Crude sialylglycopeptide 1 containing egg yolkpeptides (ca.
1:1 weight%) was also used for the preparation of 2. The crude material
was easily prepared by repetitive purification (2 3 times) with Sephadex
G-25 (H2O) after extraction from ca. 80 egg yolks.[6] Actinase-E (263 mg,
Tris-HCl buffer 8 mL) was added to a solution of this crude sialylglyco-
peptide 1 (809 mg) and NaN3 in a Tris-HCl buffer (50 mm, CaCl2 10 mm,
Experimental Section
General methods: Jackbeans b-d-galactosidase was purchased from Sei-
kagakukogyo Co. Ltd. (Japan). Jack beans N-acetyl-b-d-glucosaminidase,
Jackbeans a-d-mannosidase and vibrio cholera a-d-neuraminidase were
979
Chem. Eur. J. 2004, 10, 971 985
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Y. Kajihara et al.
FULL PAPER
Table 1. 1H NMR data of diverse Asn-oligosaccharides.[a]
8: R =
2: R= 14: R = Fmoc
15: R = Fmoc
12: R =
6: R = 16: R =
17: R = 40: R =
46: R =
Fmoc
H
Fmoc
H
Fmoc
H
Fmoc
H
1 H-1
2 H-1
3 H-1
4 H-1
4’H-1
5 H-1
5’H-1
6 H-1
6’H-1
3 H-2
4 H-2
4’H-2
7 H-3ax 1.72
7 H-3eq 2.67
7’H-3ax 1.72
7’H-3eq 2.67
Asn-
CH2
Ac
5.00
4.60
4.77
5.14
4.95
4.60
4.60
4.45
4.45
4.25
4.20
4.11
5.07
4.61
4.77
5.13
4.95
4.60
4.60
4.44
4.44
4.25
4.20
4.12
1.71
2.67
1.71
2.67
2.94,
2.85
2.07,
2.06
2.06,
2.02
2.02
4.99
4.58
4.76
5.12
4.93
4.58
4.58
4.46
4.33
4.24
4.19
4.11
4.99
4.58
4.76
5.12
4.93
4.58
4.58
4.33
4.47
4.24
4.19
4.11
1.84
2.68
5.00
4.61
4.77
5.14
4.92
4.56
4.56
4.45
5.06
4.61
4.77
5.13
4.91
4.60
4.55
4.44
5.00
4.60
4.77
5.12
4.94
4.55
4.55
5.07
4.62
4.78
5.13
4.95
4.56
4.62
5.00
4.62 4.49
4.78
5.14
4.92
5.06
4.61
4.77
5.12
4.91
4.59
4.62 4.49
4.44
4.43
4.45
4.25
4.19
4.11
4.52
4.25
4.19
4.12
4.25
4.20
4.11
1.72
2.67
4.24
4.19
4.10
1.71
2.66
4.25
4.20
<4.0
1.72
2.67
4.24
4.18
<4.0
1.70
2.66
1.84
2.68
2.72, 2.52
1.79
2.67
2.72, 2.52
1.72
2.68
2.73, 2.52
2.72, 2.52
2.06, 2.05
2.04, 2.02
2.72, 2.54
2.07, 2.07
2.05, 2.03
2.90,
2.80
2.07,
2.06
2.05,
2.02
2.01
2.94, 2.85 2.73, 2.52
2.08, 2.07 2.07, 2.07
2.06, 2.04 2.03, 1.89
2.91, 2.81
2.06, 2.06
2.02, 2.00
2.07, 2.07
2.07, 2.03
2.06, 2.05
2.04, 2.02
2.06, 2.06
2.05, 2.03
1.89
1.89
1.89
1.89
1.89
2.02
Fmoc
other
7.92, 7.72
7.51, 7.44
7.91, 7.71
7.91, 7.71
7.92, 7.71
7.92, 7.71
7.51, 7.44
7.92, 7.72
7.50, 7.44
7.53 7.41 (Fmoc and 7.53 7.41 (Fmoc and 7.51, 7.43
Ph)
5.38, 5.31 (CH2)
Ph)
5.38, 5.31 (CH2)
18: R =
23: R = 41: R =
47: R = 19: R =
24: R = 11: R =
5: R = 34: R =
36: R = 35: R =
37: R =
Fmoc
H
Fmoc
H
Fmoc
H
Fmoc
H
Fmoc
H
Fmoc
H
1 H-1
2 H-1
3 H-1
4 H-1
4’H-1
5 H-1
5’H-1
6 H-1
6’H-1
3 H-2
4 H-2
4’H-2
7H-3ax
7H-3eq
5.00
4.62 4.56
4.77
5.11
4.95
5.08
4.62
4.78
5.11
4.95
5.00
4.63 4.50
4.78
5.07
4.61
4.78
5.14
5.00
4.64 4.53
4.77
5.07
4.61
4.76
5.00
4.58
4.76
5.12
4.93
4.58
4.58
4.47
4.47
4.24
4.19
4.11
5.07
4.62
4.76
5.12
4.92
4.58
4.58
4.47
4.47
4.24
4.19
4.12
4.99
4.55
4.76
5.11
4.91
4.55
4.55
5.07
4.61
4.77
5.11
4.91
4.55
4.55
4.99
4.58 4.52
4.77
5.10
4.91
5.07
4.61
4.78
5.10
4.91
5.14
4.94
4.94
4.59
4.63 4.50
4.45
4.60
4.44
4.62 4.56
4.62
4.64 4.53
4.45
4.25
4.07
4.12
4.45
4.26
4.08
4.12
4.45
4.28
4.44
4.07
4.23
4.20
4.23
4.19
4.24
4.18
4.10
4.24
4.18
4.10
4.24
4.06
3.97
4.25
4.06
3.97
4.11
4.10
1.72
2.67
1.71
2.67
7’H-3ax 1.72
7’H-3eq 2.68
1.72
2.68
2.94,
2.85
2.09,
2.07
2.04,
2.02
1.72
2.68
2.73, 2.55
1.71
2.67
2.93,
2.85
2.08,
2.06
2.02,
2.01
Asn-
CH2
Ac
2.73, 2.53
2.73, 2.52
2.07, 2.05
2.03, 1.89
2.92,
2.83
2.06,
2.06
2.03,
2.01
2.72, 2.52
2.07, 2.05
2.05, 1.88
2.93,
2.84
2.08,
2.05
2.05,
2.01
2.72, 2.51
2.06, 2.05
2.05, 1.89
2.80,
2.63
2.07,
2.05
2.05,
2.01
2.72, 2.53
2.05, 1.88
2.79,
2.61
2.07,
2.01
2.07, 2.07
2.03, 1.89
2.07, 2.07
2.03, 1.89
Fmoc
7.92, 7.72
7.51, 7.44
7.92, 7.72
7.51, 7.44
7.93, 7.72
7.51, 7.44
7.91, 7.70
7.50, 7.43
7.91, 7.71
7.50, 7.43
7.91, 7.70
7.50, 7.43
980
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 971 985

Complex-Type Oligosaccharides
971 985
Table 1. (Continued)
13: R =
7: R = 28: R =
29: R = 38: R =
39: R = 30: R =
32: R = 45: R =
51: R = 31: R =
33: R =
Fmoc
H
Fmoc
H
Fmoc
H
Fmoc
H
Fmoc
H
Fmoc
H
1 H-1
2 H-1
3 H-1
4 H-1
4’H-1
5 H-1
5’H-1
6 H-1
6’H-1
3 H-2
4 H-2
4’H-2
7 H-3ax
7 H-3eq
7’H-3ax
7’H-3eq
Asn-
5.00
4.57
4.76
5.12
4.92
4.55
4.55
4.46
5.06
4.60
4.76
5.11
4.91
4.57
4.55
4.46
4.99
4.58
4.76
5.12
4.92
4.55
4.55
5.06
4.61
4.75
5.11
4.92
4.57
4.55
4.99
4.57
4.77
5.12
4.91
4.57
5.07
4.61
4.77
5.11
4.91
4.57
4.99
4.58
4.76
5.10
4.92
5.07
4.61
4.76
5.10
4.92
4.99
4.55
4.76
5.11
4.91
4.55
5.06
4.60
4.77
5.11
4.91
4.54
4.99
4.55
4.76
5.10
4.91
5.06
4.61
4.76
5.09
4.91
4.58
4.57
4.55
4.54
4.46
4.46
4.47
4.24
4.18
4.11
4.46
4.23
4.18
4.10
4.47
4.24
4.07
4.11
4.47
4.24
4.06
4.10
4.24
4.19
4.11
4.28
4.18
4.10
4.24
4.19
<4.0
4.24
4.18
<4.0
4.24
4.18
3.97
4.24
4.18
3.96
4.24
4.06
4.10
4.24
4.06
4.10
2.72, 2.51
2.06, 2.05
2.05, 1.89
2.88,
2.77
2.07,
2.04
2.04,
2.00
2.72, 2.52
2.06, 2.05
2.04, 1.89
2.87,
2.76
2.07,
2.04
2.04,
2.00
2.72, 2.52
2.06, 2.05
1.89
2.90,
2.80
2.07,
2.04
2.01
2.72, 2.52
2.06, 2.04
1.89
2.90,
2.81
2.07,
2.04
2.01
2.72, 2.52
2.06, 2.05
1.89
2.88,
2.77
2.06,
2.04
2.00
2.72, 2.52
2.06, 2.05
1.89
2.87,
2.76
2.07,
2.04
2.00
CH2
Ac
Fmoc
7.92, 7.72
7.51, 7.44
7.91, 7.71
7.51, 7.43
7.92, 7.71
7.51, 7.43
7.92, 7.71
7.51, 7.44
7.90, 7.70
7.49, 7.42
7.91, 7.70
7.50, 7.43
42: R =
48: R = 20: R =
25: R = 43: R =
49: R = 21: R =
26: R = 44: R =
50: R = 22: R =
27: R =
Fmoc
H
Fmoc
H
Fmoc
H
Fmoc
H
Fmoc
H
Fmoc
H
1 H-1
2 H-1
3 H-1
4 H-1
4’H-1
5 H-1
5’H-1
6 H-1
6’H-1
3 H-2
4 H-2
4’H-2
7 H-3ax
7 H-3eq
7’H-3ax
7’H-3eq
Asn-
4.99
4.57
4.77
5.12
5.06
4.61
4.77
5.11
5.00
4.58
4.75
5.07
4.62
4.75
4.99
4.55
4.76
5.12
5.07
4.61
4.77
5.12
4.99
4.55
nd
5.07
4.62
4.76
4.99
4.57
4.77
5.11
5.06
4.61
4.77
5.10
5.00
4.58
4.76
5.07
4.62
4.76
4.92
4.58
4.92
4.58
4.91
4.55
4.91
4.55
4.91
4.91
4.57
4.46
4.57
4.46
4.55
4.54
4.47
4.07
4.47
4.08
4.22
4.19
4.22
4.18
4.22
4.18
4.22
4.18
4.07
4.09
4.07
4.10
4.22
4.07
4.22
4.07
4.07
3.97
4.08
3.97
4.10
4.09
2.72, 2.52
2.05, 2.05
2.91,
2.82
2.05,
2.04
2.01
2.72, 2.52
2.07, 2.05
2.91,
2.81
2.08,
2.04
2.01
2.72, 2.54
2.05, 2.05
2.89,
2.78
2.06,
2.05
2.01
2.72, 2.56
2.07, 2.05
2.89,
2.79
2.07,
2.05
2.01
2.72, 2.52
2.05, 1.89
2.90,
2.80
2.05,
2.01
2.72, 2.52
2.07, 1.89
2.92,
2.83
2.07,
2.01
CH2
Ac
1.89
1.89
1.88
1.89
Fmoc
7.92, 7.71
7.50, 7.43
7.92, 7.71
7.51, 7.44
7.87, 7.67
7.48, 7.41
7.89, 7.68
7.49, 7.42
7.91, 7.70
7.50, 7.43
7.91, 7.71
7.50, 7.43
[a]
NeuAc,
Gal,
GlcNAc,
Man.
pH 7.5, 32 mL) and this mixture was incubated for 60 h at 378C. During
incubation, the pH was kept at 7.5. After 60 h, to this mixture was added
Actinase-E (25 mg) and this mixture was further incubated for 55 h. This
reaction was monitored by TLC (1m NH4OAc/isopropanol 1:1). After
the reaction was finished, the mixture was lyophilized and purification of
the residue by gel permeation (Sephadex-G-25, 12.5cmî100 cm, H2O)
afforded pure asparagine-linked sialyloligosaccharide 2 (301 mg).
ture was cooled to 48C and neutralized with aq NaHCO3, and lyophi-
lized. Purification of the residue by gel permeation column (Sephadex G-
25: 12.5 cmî100 cm, water) afforded a mixture (778 mg) of disialo sub-
strate 2, monosialyl oligosaccharides 3, 4 and asialooligosaccharide 5[5a]
along with 6 and 7. This mixture was then used for next protecting reac-
tion after lyophilization. To a solution of this mixture (778 mg) in H2O/
acetone (3.8 5.7 mL) was added NaHCO3 (162 mg, 1.93 mmol) and 9-flu-
orenylmethyl-N-succimidylcarbonate (432 mg, 1.28 mmol), and the mix-
ture was stirred at room temperature. After 2 h, the mixture was evapo-
rated to remove acetone, and desalted with ODS-column (120 mmî
Fmoc-oligosaccharides 8 11: An HCl solution (80 mm, 11.4 mL) was
added to a solution of pure Asn-oligosaccharide 2 (1.07 g, 456 mmol) in
water (11.4 mL) and this mixture was stirred at 378C. After 6 h, the mix-
981
Chem. Eur. J. 2004, 10, 971 985
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Y. Kajihara et al.
FULL PAPER
250 mm; eluted with H2O, 100 mL and then 25% MeCN,
200 mL) afforded a mixture of Fmoc-oligosaccharides 8 13
(681 mg). This mixture was further purified by HPLC: ODS
column (YMC packed column D-ODS-5-A 120A, 120 mmî
250 mm, 50 mm NH4OAc/MeCN 82:18, 7.5 mLminꢀ1, moni-
toring at 215 nm) to obtain disialyloligosaccharide 8 (tR
15.7 min), mixture of monosialyloligosaccharide 9, 10 (tR
=
=
19.5 min) and asialooligosaccharide 11[5a] (tR =25.5 min). In
addition, monosialylnonasaccharide 12 (tR =23.3 min) and
asialooctasaccharide 13 (28.2 min) were also obtained. This
purification was performed repeatedly with ca. 30 mg of mix-
ture each time (total ca. 680 mg). The individual oligosac-
charides thus obtained were combined, lyophilized and then
desalted with HPLC (ODS-column: 15 mmî150 mm;
eluted with H2O, 100 mL then 25% MeCN, 200 mL) afforded
pure oligosaccharides 8 (148 mg, 13%), mixture of 9 and 10
(249 mg, 24%), 11 (101 mg, 11%), 12 (68 mg, 7%) and 13
(35 mg, 4%).
Disialooligosaccharide 8: HRMS: calcd for C103H154N8NaO66
2581.8838; found: 2581.8821 [M+Na]+.
:
Asialooligosaccharide 11: HRMS: calcd for C81H120N6NaO50
1999.6930; found: 1999.6939 [M+Na]+.
:
Monosialyloligosaccharide
12:
HRMS:
calcd
for
C86H125N7Na3O53: 2172.6995; found: 2172.7084 [M+Na]+.
Asialooctasaccharide 13: HRMS: calcd for C75H110N6NaO45
1837.6402; found: 1837.6418 [M+Na]+.
:
Benzyl esterification of monosialyloligosaccharides 14 and
15: A solution of a mixture of Fmoc-monosialyldecasacchar-
ide 9 and 10 (5.0 mg) in cold H2O (1 mL, 48C) was passed
through to a column (10.5 cmî5 cm containing Dowex-
50Wî8(H+) resin), and the column was washed with cold
water (10 mL). The eluant and the washing were pooled and
lyophilized. This residue was dissolved in H2O (0.22 mL) and
neutralized (pH 7) by stepwise addition of a solution of
Cs2CO3 (2.5 mgmLꢀ1) monitoring with pH meter, and lyophi-
lized. The residue was dissolved in dry DMF (0.43 mL) and
was mixed with a solution of BnBr (6.6 mL) in DMF (20 mL)
and stirred at room temperature under argon atmosphere.
After 48 h, diethyl ether (5 mL) was added and the precipi-
tate formed was collected. Purification of the precipitated
material by HPLC (ODS column, 120 mmî250 mm, 50 mm
NH4OAc/MeCN 78:22) afforded monobenzyl-sialyloligosac-
charides 14 (91 min) and 15 (88 min). Desalting of the each
product by HPLC (ODS-column: 15 mmî150 mm; H2O
50 mL and then 25% MeCN, 100 mL) afforded pure mono-
benzyl-sialyloligosaccharides 14 (1.6 mg) and 15 (1.8 mg).
Saccharide 14: FAB-MS: calcd for C99H143N17NaO58: 2380.8;
found: 2380.1 [M+H]+.
Saccharide 15: FAB-MS: calcd for C99H143N17NaO58: 2380.8;
found: 2380.5 [M+H]+.
Galactosidase digestion of monosialyldecasaccharide 9 and
10: b-d-Galactosidase (390 mU in 50 mm HEPES buffer,
100 mL, pH 6.0) was added to a mixture of monosialyldeca-
saccharides 9 and 10 (135 mg, 59.4 mmol) in HEPES buffer
(50 mm, pH 6.0, 5.6 mL) containing bovine serum albumin
(1.0 mg) and this mixture was incubated at 378C for 19 h,
and lyophilized. Purification of the residue by HPLC (YMC
packed column D-ODS-5-A120A, 120 mmî250 mm, 50 mm
NH4OAc/MeCN 82:18, 7.5 mLminꢀ1) afforded monosialyloli-
gosaccharide 16 (36 min) and 12 (39.2 min). This purification
was performed repeatedly with ca. 10 20 mg portions of the
reaction mixture (total ca. 120 mg). Desalting of each prod-
uct by HPLC (ODS-column: 15 mmî150 mm; H2O 100 mL
and then 25% MeCN solution 200 mL) yielded pure oligo-
saccharides 16 (51 mg, 41%) and 12 (60 mg, 48%).
Monosialylnonasaccharide
16:
HRMS:
calcd
for
C86H127N7NaO53: 2128.7356; found: 2128.7363 [M+Na]+.
General purification method for exo-glycosidase digestion:
After exo-glycosidase digestion, the mixture was lyophilized,
Scheme 7.
982
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 971 985

Complex-Type Oligosaccharides
971 985
and fractionated by HPLC (YMC packed column D-ODS-5 S-5 120A,
Compound 38: Substrate 40 (47 mg, 25 mmol); a-d-neuraminidase:
369 mU; reaction time: 37 h; 38 (26 mg, 65%); HRMS: calcd for
C67H97N5NaO40: 1634.5608; found: 1634.5644 [M+Na]+.
120 mmî250 mm, eluted with 50 mm NH4OAc/MeCN 8:2, 4.0 mLminꢀ1
,
monitoring at 215 nm) afforded the oligosaccharide. The fraction contain-
ing the desired product was pooled and lyophilized. Removal of
NH4OAc was accomplished with HPLC (ODS-column: 15 mmî
150 mm; H2O, 100 mL then 25% MeCN, 200 mL, monitoring at 215 nm)
afforded the pure oligosaccharide.
Compound 40: Substrate 12 (100 mg, 47 mmol); N-acetyl-b-d-glucosami-
nidase: 1.65 U; reaction time: 20 h; 40 (63 mg, 70%); HRMS: calcd for
C78H114N6NaO48: 1925.6562; found: 1925.6533 [M+Na]+.
Compound 41: Substrate 40 (51 mg, 26 mmol); a-d-mannosidase: 16 U;
General procedure for N-acetyl-b-d-glucosaminidase digestion: N-
Acetyl-b-d-glucosaminidase (ca. 1.6 U in 50 mm HEPES buffer, 100 mL,
pH 6.0) was added to a solution of substrate (48 mmol) in HEPES buffer
(50 mm, pH 6.0, 1.8 mL) containing bovine serum albumin (1 mg) and in-
cubated at 378C. After completion of the digestion (ca. one day) as
monitored by HPLC (ODS: 15 mmî150 mm, 50 mm NH4OAc/MeCN
8:2, monitoring at 215 nm), the mixture was lyophilized.
reaction time: 25 h; 41 (22 mg, 48%); HRMS: calcd for C72H104N6NaO43
:
1763.6034; found: 1763.6041 [M+Na]+.
Compound 42: Substrate 41 (28 mg, 16 mmol); a-d-neuraminidase:
117 mU; reaction time: 17 h; 42 (15 mg, 68%); FAB-MS: calcd for
C61H88N5O35: 1450.5; found: 1450.3 [M+H]+.
Compound 43: Substrate 42 (10 mg, 6.9 mmol); b-d-galactosidase:
205 mU; reaction time: 20 h; 43 (5.6 mg, 64%); FAB-MS: calcd for
C55H78N5O30: 1288.5; found: 1288.3 [M+H]+.
General procedure for a-d-mannosidase digestion: a-d-Mannosidase (ca.
0.5 U in 50 mm HEPES buffer, 50 mL, pH 6.0) was added to a solution of
substrate (24 mmol) in HEPES buffer (50 mm, pH 6.0, 0.9 mL) containing
bovine serum albumin (1 mg) and the solution was incubated at 378C. In
the case of substrate 40, 16 U of enzyme was used. After completion of
the digestion (ca. one day) as monitored by HPLC (ODS: 15 mmî
150 mm, 50 mm NH4OAc/MeCN 8:2, monitoring at 215 nm), the mixture
was lyophilized.
Compound 44: Substrate 43 (3.6 mg, 2.8 mmol); N-acetyl-b-d-glucosami-
nidase: 195 mU; reaction time: 24 h; 44 (2.3 mg, 77%); FAB-MS: calcd
for C47H65N4O25: 1085.4; found: 1085.3 [M+H]+.
Compound 45: Substrate 38 (12 mg, 7.4 mmol); b-d-galactosidase:
297 mU; reaction time: 46 h; 45 (6.6 mg, 61%); FAB-MS: calcd for
C61H88N5O35: 1450.5; found: 1450.3 [M+H]+.
General procedure for Fmoc deprotection: Morpholine (160 mL) was
added to a solution of the Fmoc-substrate (1 mmol) in DMF (240 mL) and
this mixture was stirred at room temperature. After finish the reaction
(ca. 7 10 h), to this mixture was added Et2O (4.0 mL) and then precipi-
tate was collected. Purification of the residue by HPLC (ODS-column:
15 mmî150 mm; H2O, 100 mL then 25% MeCN, 200 mL, monitoring at
215 nm) afforded Asn-oligosaccharide.
General procedure for a-d-neuraminidase digestion: a-d-Neuraminidase
(ca. 130 mU in 50 mm HEPES buffer, 50 mL, pH 6.0) was added to a solu-
tion of substrate (18.4 mmol) in HEPES buffer (50 mm, pH 6.0, 0.72 mL)
containing bovine serum albumin (1 mg) and this solution was incubated
at 378C. After completion of the digestion (ca. one day) as monitored by
HPLC (ODS: 15 mmî150 mm, 50 mm NH4OAc/MeCN 8:2, monitoring
215 nm), the mixture was lyophilized.
Compound 6: FAB-MS: calcd for C71H117N7NaO51: 1906.7; found: 1906.1
[M+Na]+.
General procedure for b-d-galactosidase digestion: b-d-Galactosidase
(ca. 180 mU in 50 mm HEPES buffer, 50 mL, pH 6.0) was added to a solu-
tion of substrate (6.2 mmol) in HEPES buffer (50 mm, pH 6.0, 0.6 mL)
containing bovine serum albumin (1 mg) and this solution was incubated
at 378C. After completion of the digestion (ca. 30 h) as monitored by
HPLC (ODS 15 mmî150 mm, 50 mm NH4OAc/MeCN 8:2, monitoring
at 215 nm), the mixture was lyophilized.
Compound 7: FAB-MS: calcd for C60H101N6O43: 1593.6; found: 1593.8
[M+H]+.
Compound 17: FAB-MS: calcd for C71H118N7O51: 1884.7; found: 1884.5
[M+H]+.
Compound 23: FAB-MS: calcd for C63H104N6NaO46
1703.1 [M+Na]+.
: 1703.6; found:
Compound 13: Substrate 12 (20 mg, 9.4 mmol); a-d-neuraminidase:
141 mU; reaction time: 18 h; 13 (13 mg, 76%).
Compound 24: FAB-MS: calcd for C57H94N6NaO41: 1541.5; found: 1541.3
[M+Na]+.
Compound 18: Substrate 16 (102 mg, 48 mmol); N-acetyl-b-d-glucosami-
nidase: 1.6 U; reaction time: 28 h; 18 (63 mg, 69%); HRMS: calcd for
C78H114N6NaO48: 1925.6562; found: 1925.6539 [M+Na]+.
Compound 25: FAB-MS: calcd for C46H77N5NaO33 1250.4; found: 1250.3
[M+Na]+.
Compound 19: Substrate 18 (45 mg, 24 mmol); a-d-mannosidase:
585 mU; reaction time: 20 h; 19 (28 mg, 69%); HRMS: calcd for
C72H104N6NaO43: 1763.6034; found: 1763.6074 [M+Na]+.
Compound 26: FAB-MS: calcd for C40H67N5NaO28: 1088.4; found: 1088.2
[M+Na]+.
Compound 27:[22] FAB-MS: calcd for C32H55N4O27: 863.3; found: 863.2
[M+H]+.
Compound 20: Substrate 19 (32 mg, 18.4 mmol); a-d-neuraminidase:
134 mU; reaction time: 17 h; 20 (13 mg, 52%); FAB-MS: calcd for
C61H88N5O35: 1450.5; found: 1450.3 [M+H+].
Compound 29: FAB-MS: calcd for C60H100N6NaO43
1615.0 [M+Na]+.
: 1615.6; found:
Compound 21: Substrate 20 (9 mg, 6.2 mmol); b-d-galactosidase: 186 mU;
reaction time: 32 h; 21 (5.4 mg, 68%); FAB-MS: calcd for
C55H77N5NaO30: 1310.5; found: 1310.2 [M+Na]+.
Compound 32: FAB-MS: calcd for C52H88N5O38: 1390.5; found: 1390.1
[M+H]+.
Compound 33:[19] FAB-MS: calcd for C46H78N5O33: 1228.5; found: 1228.3
Compound 22: Substrate 21 (3.4 mg, 2.6 mmol); N-acetyl-b-d-glucosami-
nidase: 144 mU; reaction time: 24 h; 22 (2.1 mg, 75%); FAB-MS: calcd
for C47H65N4O25: 1085.4; found: 1085.3 [M+Na]+.
[M+H]+.
Compound 36:[13q,22c] FAB-MS: calcd for C54H90N6NaO38: 1453.5; found:
1453.2 [M+Na]+.
Compound 28: Substrate 16 (28 mg, 21 mmol); a-d-neuraminidase:
198 mU; reaction time: 20 h; 28 (17 mg, 70%); HRMS: calcd for
C75H110N6NaO45: 1837.6402; found: 1837.6471 [M+Na+].
Compound 37:[23] FAB-MS: calcd for C38H65N4O28: 1025.4; found: 1025.2
[M+H]+.
Compound 39[20]: FAB-MS: calcd for C52H88N5O38: 1390.5; found: 1390.2
[M+H]+.
Compound 30: Substrate 18 (45 mg, 24 mmol); a-d-neuraminidase:
134 mU; reaction time: 14 h; 30 (28 mg, 74%); HRMS: calcd for
C67H97N5NaO40: 1634.5608; found: 1634.5564 [M+Na]+.
Compound 46:[18] FAB-MS: calcd for C63H104N6NaO46: 1703.6; found:
1703.0 [M+Na]+.
Compound 31: Substrate 30 (11 mg, 6.8 mmol); b-d-galactosidase:
275 mU; reaction time: 14 h; 31 (6.3 mg, 64%); FAB-MS: calcd for
C61H88N5O35: 1450.5; found: 1450.4 [M+H]+.
Compound 47: FAB-MS: calcd for C57H94N6NaO41: 1541.5; found: 1541.2
[M+Na]+.
Compound 34:[13q] Substrate 11 (123 mg, 62 mmol); b-d-galactosidase:
612 mU; reaction time: 61 h; 34 (71 mg, 70%); HRMS: calcd for
C69H100N6NaO40: 1675.5873; found: 1675.5841 [M+Na+].
Compound 48: FAB-MS: calcd for C46H78N5O33: 1228.5; found: 1228.3
[M+H]+.
Compound 49:[22c] FAB-MS: calcd for C40H67N5NaO28: 1088.4; found:
1088.3 [M+Na]+.
Compound 35: Substrate 34 (50 mg, 30 mmol); N-acetyl-b-d-glucosamini-
dase: 2.1 U; reaction time: 48 h; 35 (25 mg, 66%); MALDI-TOF: calcd
for C53H75N4O30: 1247.4, found 1248.0 [M+H]+.
Compound 50: FAB-MS: calcd for C32H55N4O23: 863.3; found: 863.3
[M+H]+.
983
Chem. Eur. J. 2004, 10, 971 985
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Y. Kajihara et al.
FULL PAPER
Compound 51:[21] FAB-MS: calcd for C46H78N5O33: 1228.5; found: 1228.3
[M+H]+.
5.11 (d, 2H, GlcNAc1-H1, GlcNAc1’’-H1), 5.03, 5.01 (2s, 2î1H, Man4-
H1, Man4’’-H-1), 4.86 (dd, 2H, Asn-a), 4.69 4.66 (GlcNAc2, 2’’, 5, 5’, 5’’,
5’’’-H1), 4.61 4.48 (Leu-aî4, Ser-aî2, Gal6, 6’, 6’’, 6’’’-H1), 4.33 (brs,
2H, Man3, 3’’-H2), 4.28 (brs, 2H, Man4’, 4’’’-H2), 4.20 (brs, 2H, Man4,
4’’-H2), 4.20 4.17 (Val-aî2, Ala-aî2), 3.00 (dd, 2H, Asn-bî2), 2.83 (dd,
2H, Asn-bî2), 2.76 (dd, 2H, NeuAc7, 7’-H3eq), 2.16 2.10 (Acî10, Val-
bî2), 1.82 (dd, 2H, NeuAc7, 7’-H3ax), 1.70 1.60 (m, Leu-b, -g), 1.60,
1.49 (2d, 2î3H, Ala-b), 1.02 0.96 (m, 36H, Val-gCH3 î4, Leu-CH3 î8);
MALDI-TOF: m/z: calcd for C200H334N24O123: 5040.05, found: 5041.64
[M+H+].
Synthesis of 52: A solution of Fmoc-disialyloligosaccharide 8 (20 mg) in
cold H2O (2 mL, 4oC) was passed through to a pasteur pipette column
(10.5 cmî5 cm) containing resin of Dowex-50Wî8(H+). The eluant
was pooled and lyophilized. To a solution of this residue in H2O was neu-
tralized by addition of a solution of aq Cs2CO3 (2.5 mg in 1 mL H2O) and
this solution was adjusted to pH 6. This solution was then lyophilized. To
a solution of this residue in dry DMF (1.3 mL) was added BnBr (5.1 mL)
and then the mixture was stirred at room temperature under argon at-
mosphere. After 45 h, to a solution of this mixture was added diethyl
ether (10 mL) and then precipitate was collected. Purification of the resi-
due by ODS-column (11.6 cmî14 cm, H2O to 40% MeOH) afforded di-
benzyl-sialyloligosaccharide 52 (18.2 mg, 85%). 1H NMR (400 MHz,
308C in D2O, HOD at 4.81): d = 8.00 (d, 2H, Fmoc), 7.80 (d, 2H,
Fmoc), 7.65 7.50 (m, 12H, Ph, Fmoc), 5.46 (d, 2H, J=11.6 Hz, PhCH2),
5.40 (d, 2H, J=11.6 Hz, PhCH2), 5.21 (s, 1H, Man4-H-1), 5.08 (d, 1H,
J=9.3 Hz, GlcNAc1-H-1), 5.02 (s, 1H, Man4’-H-1), 4.86 (s, 1H, Man3-H-
1), 4.67 (m, 3H, GlcNAc2,5,5’-H-1), 4.41 (brd, 3H, Gal6, 6’-H-1, Fmoc),
4.33 (brd, 1H, Man3-H-2), 4.27 (brd, 1H, Man4’-H-2), 4.20 (d, 1H,
Man4-H-2), 2.79 (brd, 3H, Asn-b-CH2, NeuAc7, 7’-H3eq), 2.61 (brdd,
1H, Asn-b-CH2), 2.15 (s, 3H, Ac), 2.12 (s, 6H, Acî2), 2.10 (s, 6H, Acî
2), 1.98 (s, 3H, Ac), 1.93 (dd, 2H, J=12.2, 12.2 Hz, NeuAc7, 7’-H-3ax);
HRMS: m/z: calcd for C117H165N8Na2O66: 2783.9597, found 2783.9501
[M+Na]+.
Acknowledgement
We thankMasayoshi Kusama (Japan Tobbaco INC), Dr. Mitsuhiro Kino-
sita (Kinki University) and Kazuaki Kakehi (Kinki University) for meas-
urement of the mass spectra. We thankDr. Yutaka Ogasawara, and Dr.
Seiji Shu in Taiyo Kagaku, Co. for support of this research. We would
like to thank Hiroaki Asai, Dr. Michio Sasaoka, Dr. Yuataka Kameyama,
Kazuhiro Fukae, Hiroshi Shibutani, Hiroyasu Hayashi in Otsuka Chemi-
cal Co., Ltd. and Akihiro Kajihara for technical support and encourage-
ment. We thankDr. Katsuji Haneda, the Noguchi Institute for technical
support of Actinase-E.
Glycopeptide 53: Peptide synthesis on solid phase was performed by
Meldal×s group procedure[5b] with the HMPA-PEGA resin. The solid
phase syntheses were performed by manual procedure using a polypropy-
lene column (Tokyo Rika, No. 183470). The HMPA-PEGA resin (35 mg)
was washed with CH2Cl2/DMF and then dried in desiccator. The first
serine was attached by addition of a solution of Fmoc-Ser(OtBu)-OH
(3.8 mg, 10 mmol), MSNT (1-mesitylenesulfonyl-3-nitro-1,2,4-triazole,
2.3 mg, 10 mmol), and N-methyimidazole (0.8 mg, 7 mmol) in CH2Cl2
(1 mL) and the solution was stirred. After 3 h, this resin was washed by
CH2Cl2, isopropanol and DMF and then the resin was treated by capping
reaction with 20% acetic anhydride in DMF for 20 min. The mixture was
washed with DMF and then treated with 20% piperidine/DMF (1.5 mL)
for 20 min. After washing of the resin with DMF, the second serine was
attached to the first serine by the addition of a solution of Fmoc-Ser(Ot-
Bu)-OH (4.6 mg, 12 mmol), HOBt¥H2O (1-hydroxybenzotriazole monohy-
drate, 1.6 mg, 12 mmol) and DIPCDI (diisopopylcarbodiimide, 1.6 mg,
12 mmol) in DMF (1 mL). An identical washing procedure was performed
as for the first serine. Attachment of Asn-linked disialyloligosaccharide
52 (18 mg, 6.3 mmol) was performed with HATU (O-(7-azabenzotriazol-
[1] a) M. Demetriou, M. Granovsky, S. Quaggin, J. W. Dennis, Nature
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1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphonate,
2.4 mg,
9.4 mmol) and DIPEA (diisopropylethylamine, 0.8 mg, 6.3 mmol) in DMF/
DMSO (0.5 0.5 mL) at room temperature. After 24 h, washing, capping
and de-N-Fmoc group were performed by the procedure above descri-
bed. Attachment of valine, leucineî2, alanine was performed with corre-
sponding activated pentafluorophenol amino acid (Fmoc-AA-Opfp;
valine (4 mg, 8.1 mmol), leucine (3.4 mg, 6.5 mmol), alanine (3.1 mg,
6.5 mmol) in the presence of Dhbt (3,4-dihydro-4-oxo-1,2,3-benzotriazin-
3-yl, 0.1 equiv).[5b] The reaction was monitored by the yellow color of
Dhbt. Washing, capping and de-N-Fmoc group were performed by the
above conventional procedure. However, capping procedure was only ex-
amined after attachment of Val. After completion of condensation with
alanine, attachment of asialooligosaccharide 11 was performed using the
same procedure as with 52. Elongation of valine, leucineî2 and alanine
were also examined using the same procedure. After elongation, this gly-
copeptide was treated with 95% TFA (1 mL). After 3 h, the glycopeptide
was eluted from the reaction column by washing with 95% TFA and this
solution was then concentrated in vacuo at room temperature. After lyo-
philization of the residue, saponification (pH 11, NaOH solution) of
benzyl ester afforded disialylglycopeptide. The saponification (20 min)
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1
was performed in NMR tube and monitored by H NMR. After neutrali-
zation of this mixture by acetic acid, the mixture was lyophilized. Purifi-
cation of this residue by HPLC (YMC-PackODS-A 13.0 mmî250 mm,
linear gradient: from 0.1% TFA solution to 0.1% TFA containing
MeCN 90% in 90 min) afforded the desired glycopeptide 53 (3.4 mg,
total 3.2% based on first serine attached). 1H NMR (400 MHz, 308C in
D2O, HOD=d 4.81): d = 5.22, 5.21 (2s, 2î1H, Man4’-H1, Man4’’’-H1),
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984
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Chem. Eur. J. 2004, 10, 971 985

Complex-Type Oligosaccharides
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