Glycopeptide synthesis through endo-glycosidase-catalyzed oligosaccharide transfer of sugar oxazolines: Probing substrate structural requirement

Article abstract of DOI:10.1002/chem.200501196

An array of sugar oxazolines was synthesized and tested as donor substrates for the Arthrobacter endo-ssN-acetylglucosaminidase (Endo-A)-catalyzed glycopeptide synthesis. The experiments revealed that the minimum structure of the donor substrate required for Endo-A catalyzed transglycosylation is a Manss1→4-GlcNAc oxazoline moiety. Replacement of the ss-D-Man moiety with ss-D-Glc, ss-D-Gal, and ss-D-GlcNAc monosaccharides resulted in the loss of substrate activity for the disaccharide oxazoline. Despite this, the enzyme could tolerate modifications such as attachment of additional sugar residues or a functional group at the 3- and/or 6-positions of the ss-D-Man moiety, thus allowing a successful transfer of selectively modified oligosaccharides to the peptide acceptor. On the other hand, the enzyme has a great flexibility for the acceptor portion and could take both small and large GlcNAc-peptides as the acceptor. The studies implicate a great potential of the endoglycosidase-catalyzed transglycosylation for constructing both natural and selectively modified glycopeptides.

Full text of DOI:10.1002/chem.200501196

FULL PAPER
DOI: 10.1002/chem.200501196
Glycopeptide Synthesis through endo-Glycosidase-Catalyzed Oligosaccharide
Transfer of Sugar Oxazolines: Probing Substrate Structural Requirement
Ying Zeng, Jingsong Wang, Bing Li, Steven Hauser, Hengguang Li, and Lai-Xi Wang*[a]
Abstract: An array of sugar oxazolines
was synthesized and tested as donor
substrates for the Arthrobacter endo-b-
N-acetylglucosaminidase (Endo-A)-cat-
alyzed glycopeptide synthesis. The ex-
periments revealed that the minimum
structure of the donor substrate re-
quired for Endo-A catalyzed transgly-
cosylation is a Manb1!4-GlcNAc oxa-
zoline moiety. Replacement of the b-d-
Man moiety with b-d-Glc, b-d-Gal, and
b-d-GlcNAc monosaccharides resulted
in the loss of substrate activity for the
disaccharide oxazoline. Despite this,
the enzyme could tolerate modifica-
tions such as attachment of additional
sugar residues or a functional group at
the 3- and/or 6-positions of the b-d-
Man moiety, thus allowing a successful
transfer of selectively modified oligo-
saccharides to the peptide acceptor. On
the other hand, the enzyme has a great
flexibility for the acceptor portion and
could take both small and large
GlcNAc-peptides as the acceptor. The
studies implicate a great potential of
the endoglycosidase-catalyzed transgly-
cosylation for constructing both natural
and selectively modified glycopeptides.
Keywords:
enzymes
carbohydrates
glycopeptides
·
·
·
glycosylation
Introduction
glycopeptides. We are particularly interested in the endo-b-
N-acetylglucosaminidase (ENGase)-catalyzed oligosacchar-
ide transfer for glycopeptide synthesis, because it allows the
attachment of a large oligosaccharide to a pre-assembled,
unprotected GlcNAc-peptide/protein in a single step in a
regio- and stereospecific manner, thus providing a highly
convergent approach.[6,7] The Endo-A from Arthrobacter
protophormiae[8] and the Endo-M from Mucor hiemalis[9] are
two ENGases that possess significant transglycosylation ac-
tivity and have been widely used for synthesizing large gly-
copeptides.[6,7] However, ENGases are inherently a class of
endoglycosidases that hydrolyze N-glycans by cleaving the
b-1,4-glycosidic bond in the N,N’-diacetylchitobiose core.
Like many glycosidase-catalyzed syntheses,[10] ENGase-cata-
lyzed transglycosylation suffers from the inherent low trans-
glycosylation yield and product hydrolysis. In addition, the
ENGase-catalyzed synthesis has been limited thus far to the
use of only natural N-glycans as the donor substrates, which
themselves are difficult to obtain. To address these prob-
lems, we have recently communicated the use of synthetic
oligosaccharide oxazolines, the presumed transition state an-
alogues, as donor substrates for ENGase-catalyzed glyco-
peptide synthesis, which significantly enhanced the transgly-
cosylation yield and also broadened the substrate availabili-
ty by adopting synthetic substrates.[11] The method was
based on the assumption that the ENGase-catalyzed reac-
tion proceeds via a mechanism of the substrate-assisted cat-
Glycoproteins are an important class of biomolecules that
play crucial roles in many biological events such as cell ad-
hesion, tumor metastasis, pathogen infection, and immune
response.[1] However, a major problem in structural and
functional studies of glycoproteins is their structural micro-
heterogeneity. Natural and recombinant glycoproteins are
typically produced as a mixture of glycoforms that differ
only in the structure of the pendent oligosaccharides. The
difficulty to obtain pure glycoforms from natural source has
thus motivated chemists to take the challenge of assembling
homogeneous glycopeptides (partial structures of glycopro-
teins) and even glycoproteins themselves by chemical/che-
moenzymatic synthesis.[2] The introduction of new techni-
ques, such as native chemical ligation and chemoselective li-
gation,[3] novel solid-phase synthesis,[4] and enzymatic oligo-
saccharide transfer[5–7] has significantly expanded our syn-
thetic repertoire for constructing large homogeneous
[a] Dr. Y. Zeng, Dr. J. Wang, Dr. B. Li, Dr. S. Hauser, Dr. H. Li,
Prof. Dr. L.-X. Wang
Institute of Human Virology
University of Maryland Biotechnology Institute
University of Maryland, Baltimore, MD 21201 (USA)
Fax: ( +1)410-706-5068
E-mail: wangx@umbi.umd.edu
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alysis involving an oxazolinium ion intermediate, as demon-
strated for some chitinases[12] and N-acetyl-b-hexosaminidas-
es.[13] Fujita and co-workers have first demonstrated that
Endo-A and Endo-M did take a disaccharide oxazoline of
Manb1,4GlcNAc as a substrate for enzymatic transglycosyla-
tion.[14] There are also precedents using disaccharide oxazo-
lines for chitinase- and hyaluronidase-catalyzed synthesis of
oligo- and polysaccharides, respectively.[15–17] Our prelimina-
ry studies have shown that the di- and tetrasaccharide oxa-
zolines corresponding to the N-glycan core structure could
serve as donor substrates for a highly efficient chemoenzy-
matic synthesis of large N-glycopeptides.[11] To further ex-
plore the potential of this promising chemoenzymatic
method, we describe in this paper the synthesis and evalua-
tion of an array of oligosaccharide oxazolines (Scheme 1) as
donor substrates for glycopeptide synthesis. The present
study intends to probe the substrate requirement for the
Endo-A catalyzed transglycosylation and to further explore
the potential of the chemoenzymatic method for construct-
ing both natural and modified N-glycopeptides.
4GlcNAc core disaccharide and related derivatives using the
readily available disaccharide 2,3,4,6-tetra-O-acetyl-b-d-glu-
copyranosyl-(1!4)-3,6-di-O-acetyl-d-glucal[19] as the starting
material, which can be easily prepared from cellobiose on a
large scale. The synthesis started with an azidonitration[20] of
the cellobiose glycal to obtain the known 2-azido-2-deoxy-
cellobiose derivative 8.[19] Acetolysis of the isolated b-nitrate
8 with sodium acetate in acetic acid, followed by reduction
of the azide to acetamido group with zinc dust in the pres-
ence of acetic anhydride, gave the 2-acetamido-2-deoxy-cel-
lobiose derivative 9 in 65% yield. Treatment of compound 9
with TMSOTf[21] resulted in the formation of per-O-acetylat-
ed oxazoline derivative 10 (90%), which was de-O-acetylat-
ed with catalytic amount of MeONa in MeOH to give the
oxazoline 5. To synthesize the Manb1!4GlcNAc core disac-
charide and its derivatives, the configuration at C-2’ needs
to be converted from the gluco-type to the manno-type. This
was achieved by a series of selective transformations. First, a
4-pententyl group was introduced at the anomeric position
of the disaccharide moiety, which could be directly trans-
formed to an oxazoline moiety in the later stage by a one-
step oxazoline formation reaction.[18] Thus treatment of 10
with 4-penten-1-ol under the catalysis of 10-camphorsulfonic
acid (CSA) gave pentenyl 2-acetamido-b-glycoside 11
(90%). De-O-acetylation of 11, followed by subsequent ben-
zylidenation, gave compound 12 (53%). Selective benzoyla-
tion[22] of 12 with BzCN in MeCN at low temperature gave
compound 14 in 34% isolated yield, which has a free hy-
droxyl group at the 2’-position. The inversion of the C-2’
configuration was achieved by following the previously de-
scribed SN2 inversion of the C-2 configuration for b-manno-
side synthesis.[22,23] Thus, triflation of compound 14 with trifl-
ic anhydride/pyridine, followed by SN2 substitution with ben-
zoate, gave the protected Manb1!4GlcNAc disaccharide
derivative 15 in 65% yield in two steps. Compound 15 was
converted to the O-acetylated derivative 16, which was then
modified to yield oxazoline derivative 17 by treatment with
NIS/TESOTf.[18] Finally, de-O-acetylation of 17 gave disac-
charide oxazoline 1 (Scheme 2).
The 6’-modified disaccharide oxazoline 4 was prepared
from compound 15 in several steps (Scheme 3). Selective re-
ductive ring-opening of the benzylidene group in 15 by
treatment with Et3SiH/TFA/trifluoroacetic anhydride/
[24]
CH2Cl2 gave compound 18, with a benzyl group attached
to the 6’-O-position. The benzoyl groups were then replaced
by acetyl groups to give compound 19. Treatment of 19 with
NIS/TESOTf resulted in the formation of oxazoline deriva-
tive 20 (78%), which was de-O-acetylated to give 6’-O-
benzyl disaccharide oxazoline 4.
Scheme 1. Structures of oligosaccharide oxazoline derivatives.
To prepare trisaccharide oxazoline 3, the 4’,6’-O-benzyli-
dene group in 15 was selectively removed by treatment with
80% aqueous AcOH at 508C. The resulting compound 21
was then selectively glycosylated at the 6’-OH position with
2,3,4,6-tetra-O-benzoyl-b-d-mannopyranosyl trichloroaceti-
midate under the catalysis of TMSOTf to give trisaccharide
derivative 23 (76%). De-O-benzoylation of 23 with subse-
quent O-acetylation afforded the O-acetylated pentenyl gly-
Results and Discussion
Synthesis of oligosaccharide oxazolines: The Manb1!
4GlcNAc moiety is a core disaccharide of N-glycans. This
core disaccharide moiety was previously synthesized through
stereocontrolled glycosylation of monosaccharides[11,14,18]
Here we describe an alternative synthesis of the Manb1!
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Glycopeptide Synthesis
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Scheme 2. Synthesis of disaccharide oxazolines.
Scheme 3. Synthesis of modified oxazoline derivatives.
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3357

L.-X. Wang et al.
coside 25. Finally, compound 25 was converted to the oxazo-
line derivative by treatment with NIS/TESOTf to give 26,
which was de-O-acetylated to provide trisaccharide oxazo-
line 3 (Scheme 3). On the other hand, the known chitobiose
oxazoline 6[15] and the LacNAc-oxazoline 7[16] were prepared
from O-acetylated N,N’-diacetylchitobiose and N-acetyllac-
tosamine, respectively, following the reported proce-
dure.[15,16]
and acceptor 27 (ratio 3:1) in the presence of Endo-A gave
glycopeptide 30 carrying the N-linked tetrasaccharide
moiety in 72% isolated yield. Interestingly, 6’-O-benzyl dis-
accharide oxazoline 4, which has an aromatic substituent at-
tached at the 6’-position, could still be recognized by Endo-
A as a donor substrate. The Endo-A catalyzed reaction of 4
with acceptor 27 (3:1) afforded the modified glycopeptide
31 in 55% isolated yield (Scheme 4). Although the disac-
charide oxazolines 1 and 4 were recognized by Endo-A as
donor substrates for the transglycosylation, their reactions
were found to proceed more slowly than those reactions of
tetrasaccharide oxazoline 2 and trisaccharide oxazoline 3. A
quick comparison of the transglycosylation under the same
conditions revealed that the relative transglycosylation rates
with acceptor 27 were in the following order: tetrasacchar-
ide oxazoline 2 > trisaccharide oxazoline 3 > disaccharide
oxazoline 1 > Bn-substituted disaccharide oxazoline 4. Al-
though a quantitative comparison of the sugar oxazoline
substrates will await detailed enzyme kinetic studies to
obtain the kinetic data Km and Vmax, these results suggest
that the attachment of additional b-mannosyl moieties at
the 3- and 6-position of the b-mannose core, as present in
the natural N-glycans, enhance the enzymatic recognition of
the donor substrates. The results also suggested that Endo-
A could tolerate modification at the 6’-position of the disac-
Endo-A catalyzed transglycosylation of the synthetic oxazo-
lines with GlcNAc-peptides: To test the transglycosylation
potential of the synthetic oligosaccharide oxazolines, we syn-
thesized a small GlcNAc-tripeptide acceptor, Asn(GlcNAc)-
Ile-Thr, which represents the minimum consensus sequence
for the N24 and N38 N-glycosylation sites in erythropoietin,
an important therapeutic glycoprotein for the treatment of
anemia.[25] Previously we reported that disaccharide oxazo-
line 1 and tetrasaccharide oxazoline 2 were good donor sub-
strates for Endo-A.[11] As expected, the Endo-A catalyzed
reaction of oxazolines 1 and 2 with the GlcNAc-peptide 27
(donor/acceptor 3:1) proceeded smoothly in a phosphate
buffer (pH 6.5) to give the corresponding N-glycopeptides
28 and 29 in 63 and 86% yields, respectively. Trisaccharide
oxazoline 3 was also an efficient donor substrate for the
Endo-A catalyzed transglycosylation. Thus incubation of 3
Scheme 4. ENGase-catalyzed transglycosylation with different oxazolines.
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Glycopeptide Synthesis
FULL PAPER
charide oxazoline, thus allowing the synthesis of modified
glycopeptides. In all cases, the newly formed glycosidic bond
in the resulting glycopeptides 28, 29, 30, and 31 was deter-
mined by 2D NMR analysis (data not shown) to be a b-1,4-
glycosidic linkage, confirming the previous conclusion that
the Endo-A catalyzed transglycosylation using oligosacchar-
ide oxazoline as the donor substrates proceeded in a regio-
and stereospecific manner.[11] We observed that Endo-A
could slowly hydrolyze the oxazolines 1–4 but, in the pres-
ence of a GlcNAc-peptide acceptor, the transglycosylation
was found to be faster than the hydrolysis. The results sug-
gest that the GlcNAc moiety is a better acceptor than water
molecule in the Endo-A catalyzed reaction.
Next, the structurally related disaccharide oxazolines 5–7
were tested. Cellobiose-oxazoline 5, which differs from
Manb-1,4GlcNAc-oxazoline 1 only by the configuration of
the C’-2 hydroxyl group, was inactive in the enzymatic trans-
glycosylation. Similarly, chitobiose-oxazoline 6 and LacNAc-
oxazoline 7, which were substrates of Bacillus chitinase,[15,16]
could not be recognized by Endo-A. These results indicate
that the structure of the core b-mannose moiety is required
for Endo-A recognition. Replacement of the b-Man moiety
in the disaccharide oxazoline substrate 1 with b-Glc, b-
GlcNAc, and b-Gal moiety, respectively, resulted in total
loss of its substrate activity. The experiments revealed that
the Manb1!4GlcNAc oxazoline moiety is the minimum
structure that is recognized by the enzyme Endo-A for
transglycosylation. Despite this, the enzyme could tolerate
selective modification on the mannose moiety, for example,
attachment of additional sugar moieties at the 3’, 6’-posi-
tions and even an aromatic group at the 6’-position.
Scheme 5. Synthesis of glycoforms of gp41 C-peptide C34.
In our preliminary communication,[11] we have shown that
very large GlcNAc-peptides such as the 34-mer peptide
GlcNAc-C34 could serve as an acceptor for Endo-A cata-
lyzed transglycosylation of the di- and tetrasaccharide oxa-
zolines 1 and 2. In the present study, we found that the enzy-
matic transglycosylation of trisaccharide-oxazoline 3 and
modified disaccharide oxazoline 4 to the large acceptor
GlcNAc-C34 also proceeded successfully (Scheme 5). This
led to the synthesis of the large glycopeptide 32 that con-
tains the core N-linked tetrasaccharide (75% yield) and gly-
copeptide 33 which carries a modified trisaccharide (68%)
when an excess oxazoline donor (donor/acceptor 5:1) was
used. The relatively high-yield transglycosylation suggests
that the Endo-A catalyzed transglycosylation of the sugar
oxazolines was equally efficient for small and large accept-
ors. Finally, we also tested whether Endo-A could hydrolyze
the resulting “ground-state” glycopeptides 28–33 carrying
the N-linked core tri-, tetra-, and pentasaccharide, respec-
tively. It was found that in the presence of large amount of
Endo-A, only glycopeptide 29 that carries the core pentasac-
charide was slowly hydrolyzed by Endo-A to release the tet-
sidering that the corresponding di-, tri-, and tetrasaccharide
oxazolines 1–4 are active for the enzymatic transglycosyla-
tion, the results suggest that the sugar oxazolines as donor
substrates are kinetically more favorable for the transglyco-
sylation than the hydrolysis of the resulting ground-state“
glycopeptides, allowing product accumulation.
Conclusion
An array of oligosaccharide oxazolines was synthesized and
evaluated as donor substrates for the Endo-A catalyzed gly-
copeptide synthesis. It was revealed that the minimum sub-
strate structure required for the Endo-A catalyzed transgly-
cosylation is a Manb1!4GlcNAc oxazoline moiety. Despite
this, the enzyme can tolerate modifications at the 3’- and/or
6’-positions of the disaccharide oxazoline, allowing the trans-
fer of both larger and selectively modified oligosaccharide
moieties to the peptide acceptor. On the other hand, the
enzyme has a great flexibility for the acceptor portion and
could efficiently take both small and large GlcNAc-peptide
as the acceptor substrate. Since this high-yield enzymatic li-
gation allows independent manipulations of the donor
(sugar oxazoline) and the acceptor (GlcNAc-peptide/pro-
tein) portions by well-established oligosaccharide and pep-
rasaccharide
Mana1!6(Mana1!3)Manb1!4GlcNAc
(Man3GlcNAc) and the GlcNAc-peptide moiety. The other
glycopeptides carrying the core tri- and tetrasaccharides 28,
30, 32, as well as those carrying the modified trisaccharide
31 and 33, were resistant to the enzymatic hydrolysis. Con-
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L.-X. Wang et al.
13C NMR(CD3OD, 125 MHz): d
70.9, 70.4, 69.3, 66.8, 65.2, 61.7, 61.1, 13.0; ESI-MS: m/z: calcd for
C14H23NO10: 365.13; found: 366.87 [M+H]+.
=
168.6, 101.2, 99.9, 77.4, 76.4, 72.8,
tide chemistry, it provides a highly convergent approach for
constructing both natural and modified glycopeptides. Stud-
ies directed to the exploration of this chemoenzymatic ap-
proach for constructing large complexglycopeptides and
glycoproteins are underway.[26]
4-Pentenyl O-(2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl)-(1!4)-3,6-di-O-
acetyl-2-acetamido-2-deoxy-b-d-glucopyranoside
(11):
4-Penten-1-ol
(10 mL, 53 mmol) and CSA (300 mg) was added to a solution of 10
(8.85 g, 14.3 mmol) in anhydrous ClCH2CH2Cl (80 mL). The mixture was
stirred under an argon atmosphere at 908C for 2 h. After cooling to RT,
the mixture was neutralized by Et3N and concentrated. The residue was
subjected to flash column chromatography (hexanes/EtOAc 1:3) to
afford 11 (9.1 g, 90%) as an amorphous solid. 1H NMR (500 MHz,
CDCl3): d = 5.83–5.75 (m, 2H, -CH=, NH), 5.18 (t, J=9.0 Hz, 1H),
5.12–4.95 (m, 5H), 4.57–4.54 (m, 2H), 4.45 (d, J=8.0 Hz, 1H, H-1’), 4.39
(dd, J=12.0, 4.5 Hz, 1H), 4.16–4.02 (m, 4H), 3.87–3.69 (m, 6H), 3.64–
3.61 (m, 1H), 3.50–3.45 (m, 3H), 2.14, 2.11, 2.08, 2.06, 2.04, 2.01, 1.98 (s
each, 3H each, 7CH3CO), 1.75–1.64 (m, 2H, -OCH2CH2CH2-); 13C NMR
(CDCl3, 125 MHz): d = 169.9, 169.6, 169.5, 169.3, 169.2, 168.5, 168.4,
137.0, 114.0, 100.1, 99.8, 75.3, 71.9, 71.7, 71.4, 71.0, 70.7, 67.9, 66.9, 61.3,
60.7, 52.5, 29.01, 19.9, 19.8, 19.7, 19.6; ESI-MS: m/z: calcd for
C31H45NO17: 703.27, found: 704.07 [M+H]+.
Experimental Section
General procedures: TLC was performed on aluminum plates coated
with silica gel 60 with detection by charring with 10% (v/v) sulfuric acid
in methanol or by UV detection. Flash column chromatography was per-
formed on silica gel 60 (EM Science, 230–400 mesh). 1H and 13C NMR,
and 2D NMR spectra were recorded on Inova 500 NMR in CDCl3, D2O,
or CD3OD, as specified. Chemical shifts are expressed in ppm downfield
using external Me4Si (0 ppm) as the reference. The ESI-MS spectra were
measured on a micromass ZQ-400 single quadruple mass spectrometer.
Analytic HPLC was carried out with a Waters 626 HPLC instrument on
a Waters NovaPak C18 column (3.9”150 mm) at 408C. The column was
eluted with a linear gradient of 0–90% MeCN containing 0.1% TFA at a
flow rate of 1 mLminꢀ1 over 25 min. Peptide and glycopeptides were de-
tected at two wavelengths (214 and 280 nm). Preparative HPLC was per-
formed with Waters 600 HPLC instrument on a Waters C18 Column
(symmetry 300, 19”300 mm). The column was eluted with a suitable gra-
4-Pentenyl O-(4,6-O-benzylidene-b-d-glucopyranosyl)-(1!4)-2-acetami-
do-2-deoxy-b-d-glucopyranoside (12) and 4-pentenyl O-(2,3-di-O-acetyl-
4,6-O-benzylidene-b-d-glucopyranosyl)-(1!4)-3,6-di-O-acetyl-2-acetami-
do-2-deoxy-b-d-glucopyranoside (13): A solution of the compound 11
(9 g, 13 mmol) in MeOH (100 mL) containing NaOMe (1.3 mmol) was
stirred at RT for 2 h. Then the mixture was neutralized by Dowex W50-
X8 (H+ form), filtered, and the filtrate was concentrated. The residue
was dissolved in DMF (50 mL) and dimethyl acetal benzaldehyde
(6.3 mL, 3.4 equiv) and p-toluenesulfonic acid (1.0 g). The mixture was
stirred at 508C for 10 h. After cooling to RT, the mixture was neutralized
by Et3N and concentrated. The residue was subjected to flash column
chromatography (EtOAc/MeOH 10:1) to give 12 (3.65 g, 53%). ESI-MS:
m/z: calcd for C26H37NO11: 539.24; found: 540.35 [M+H]+.
dient of MeCN containing 0.1% TFA at 12 mLminꢀ1
O-(2,3,4,6-Tetra-O-acetyl-b-d-glucopyranosyl)-(1!4)-2-acetamido-1,3,6-
tri-O-acetyl-2-deoxy-a-d-glucopyranose (9): suspension of (15 g,
.
A
8
22.5 mmol)[19] and anhydrous NaOAc (20 g) in AcOH (250 mL) was
heated to 1008C for 1 h. The reaction was diluted with EtOAc and
washed with NaHCO3 and brine, dried by Na2SO4, and filtered. The fil-
trate was concentrated in vacuo to give a white solid. The solid was dis-
solved in THF (100 mL) and AcOH (50 mL). To the solution were se-
quentially added Ac2O (10 mL) and zinc dust (30 g) in portions. The mix-
ture was stirred at RT for 1 h and filtered through a Celite pad. The fil-
trate was concentrated in vacuo and the residue was subject to flash
column chromatography (hexanes/EtOAc 1:2) to give 9 (9.94 g, 65%).
1H NMR (500 MHz, CDCl3): d = 6.09 (d, J=3.5 Hz, 1H, H-1), 5.72 (d,
J=8.5 Hz, 1H, NH), 5.21 (dd, J=8.8, 10.5 Hz, 1H, H-3), 5.15 (t, J=
9.0 Hz, 1H, H-3’), 5.08 (t, J=9.0 Hz, 1H, H-4’), 4.93 (t, J=9.0 Hz, 1H,
H-2’), 4.55 (d, J=8.4 Hz, 1H, H-1’), 4.44–4.35 (m, 3H), 4.12–4.04 (m,
2H), 3.88–3.82 (m, 2H), 3.70–3.66 (m, 1H), 2.19, 2.12, 2.09, 2.06, 2.04,
2.01, 1.99, 1.95 (s each, 3H each, 8CH3CO); ESI-MS: m/z: calcd for
C28H39NO18: 677.22; found: 678.34 [M+H]+.
The compound was further characterized by transformation to its acety-
lated derivative 13. To a solution of 12 (50 mg) in pyridine (2 mL) was
added Ac2O (0.2 mL). The mixture was stirred at RT for 5 h. The mixture
was then poured into cold NaHCO3 solution and stirred for 2 h at RT.
The mixture was extracted with CH2Cl2 and the organic layer washed
with NaHCO3, HCl (1n) and brine, dried by Na2SO4, and filtered. The
filtrate was concentrated in vacuo and the residue was subjected to flash
column chromatography (hexanes/EtOAc 2:1) to give 13 (72 mg, quanti-
tative) as a white foam. 1H NMR (500 MHz, CDCl3): d = 7.51–7.32 (m,
5H, C6H5), 5.84–5.78 (m, 2H, -CH=, NH), 5.53 (s, 1H, PhCH=), 5.32 (t,
J=9.5 Hz, 1H), 5.12 (t, J=9.2 Hz, 1H), 5.06–4.95 (m, 3H), 4.66 (d, J=
7.5 Hz, 1H, H-1), 4.55–4.39 (m, 3H), 4.16–4.04 (m, 2H), 3.88–3.71 (m,
5H), 3.64–3.63 (m, 2H), 3.55–3.46 (m, 3H), 2.16, 2.10, 2.08, 2.06, 2.00 (s
each, 3H each, 5CH3CO), 1.75–1.66 (m, 2H, -OCH2CH2CH2-); ESI-MS:
m/z: calcd for C34H45NO15: 707.28; found: 708.58 [M+H]+.
O-(2,3,4,6-Tetra-O-acetyl-b-d-glucopyranosyl)-(1!4)-2-acetamido-3,6-di-
O-acetyl-1,2-dideoxy-a-d-glucopyrano)-[2,1-d]-2-oxazoline
(10):
TMSOTf (3.2 mL, 1.1 equiv) was added under argon atmosphere to a sol-
ution of 9 (10.8 g, 16 mmol) in dry ClCH2CH2Cl. The mixture was stirred
at 508C overnight. After cooling to RT, the mixture was neutralized by
Et3N and concentrated. The residue was subjected to a flash column
chromatography (hexanes/EtOAc 1:2) to afford 10 (8.85 g, 90%).
1H NMR (500 MHz, CDCl3): d = 5.90 (d, J=7.3 Hz, 1H, H-1), 5.64 (d,
J=1.8 Hz, 1H, H-3), 5.16 (t, J=9.2 Hz, 1H, H-3’), 5.11 (t, J=9.8 Hz, 1H,
H-4’), 5.00 (t, J=8.4 Hz, 1H, H-2’), 4.70 (d, J=8.4 Hz, 1H, H-1’), 4.37–
4.09 (m, 5H), 3.79–3.76 (m, 1H, H-5), 3.63–3.62 (m, 1H), 3.48–3.45 (m,
1H, H-5’), 2.10, 2.09, 2.08, 2.08, 2.03, 2.00, 1.98 (s each, 3H each, 7CH3);
13C NMR (CDCl3, 125 MHz): d = 169.8, 169.7, 169.4, 168.5, 165.9, 101.2,
98.1, 77.2, 72.1, 71.1, 70.4, 69.2, 67.1, 66.6, 63.9, 62.6, 60.9, 13.0; ESI-MS:
m/z: calcd for C26H35NO16: 617.2, found: 618.43 [M+H]+.
4-Pentenyl O-(3-O-benzoyl-4,6-O-benzylidene-b-d-glucopyranosyl)-(1!
4)-3,6-di-O-benzoyl-2-acetamido-2-deoxy-b-d-glucopyranoside
(14):
BzCN (2.6 mL, 3.3 equiv) in MeCN (25 mL) was added at ꢀ308C in por-
tions over 3 h to a solution of 12 (3.3 g, 61 mmol) in MeCN (50 mL) con-
taining Et3N (3 mL). The reaction was monitored by TLC (hexanes/
EtOAc 2:1). After stirring at ꢀ308C for 5 h, the reaction was quenched
by adding MeOH. The mixture was diluted with EtOAc, washed with
0.1m HCl, NaHCO3 and brine, dried over Na2SO4, and filtered. The fil-
trate was concentrated in vacuo and the residue was subject to flash silica
gel column chromatography (hexanes/EtOAc 2:1) to give 14 (1.77 g,
34%) as a white foam. 1H NMR (500 MHz, CDCl3): d = 8.20–7.26 (m,
20H, C6H5), 6.33 (d, J=8.5 Hz, 1H, NH), 5.75–5.69 (m, 1H, -CH=), 5.64
(dd, J=9.5, 2.0 Hz, 1H, H-3), 5.28 (t, J=9.2 Hz, 1H, H-3’), 5.14 (s, 1H,
PhCH=), 5.01–4.87 (m, 3H), 4.80 (d, J=8.5 Hz, 1H, H-1), 4.63–4.60 (m,
2H), 4.20–4.15 (m, 1H), 4.05–4.03 (m, 2H), 3.95–3.94 (d, J=4.5 Hz, 1H),
3.86–3.81 (m, 2H), 3.69–3.65 (m, 1H), 3.55–3.51 (m, 1H), 3.44 (t, J=
9.0 Hz, 1H, H-4’), 3.35 (dd, J=10.5, 4.8 Hz, 1H), 3.21–3.16 (m, 1H), 2.87
(t, J=10.5 Hz, 1H), 2.06–2.03 (m, 2H, -OCH2CH2CH2-), 1.83 (s, 3H,
CH3CO), 1.68–1.59 (m, 2H, -OCH2CH2CH2-); ESI-MS: m/z: calcd for
C47H49NO14: 851.32; found: 852.42 [M+H]+.
O-(b-d-Glucopyranosyl)-(1!4)-1,2-dideoxy-a-d-glucopyrano)-[2,1-d]-2-
oxazoline (5): A solution of 10 (12 mg, 19 mmol) in MeOH containing
NaOMe (2 mmol) was stirred at RT for 2 h. Then the mixture was con-
centrated in vacuo. The residue was dissolved in water and lyophilized to
give oxazoline 5 (6 mg, quantitative) as a white solid. 1H NMR (CD3OD,
500 MHz): d = 6.03 (d, J=7.0 Hz, 1H, H-1), 4.66 (d, J=7.5 Hz, 1H, H-
1’), 4.32 (s, 1H, H-2), 4.13 (dd, J=7.5, 8.4 Hz, 1H, H-2’), 3.91–3.88 (m,
4H), 3.72–3.50 (m, 12H), 3.55–3.32 (m, 3H), 1.85 (s, 3H, CH3);
3360
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 3355 – 3364

Glycopeptide Synthesis
FULL PAPER
4-Pentenyl O-(2,3-di-O-benzoyl-4,6-O-benzylidene-b-d-mannopyranosyl)-
(1!4)-3,6-di-O-benzoyl-2-acetamido-2-deoxy-b-d-glucopyranoside (15):
Tf2O (690 mL, 2 equiv) at 08C for 1 h was added to a solution of 14
(1.7 g, 19 mmol) in CH2Cl2 (50 mL) containing pyridine (1 mL). The reac-
tion was monitored by TLC. After the completion of the reaction, the
mixture was diluted with CH2Cl2, washed successively with cold HCl
(0.1n), cold saturated NaHCO3 and H2O, dried over Na2SO4, and fil-
tered. The filtrate was concentrated and co-evaporated with toluene. The
residue was then dissolved in toluene (20 mL) and Bu4NOBz (3 g,
76 mmol) was added. The mixture was stirred under reflux for 2 h when
TLC indicated the completion of the reaction. After evaporation, the
mixture was diluted with CH2Cl2, washed successively with saturated
NaHCO3 and H2O, dried over Na2SO4, and filtered. The filtrate was con-
centrated in vacuo and the residue was subject to flash silica gel column
chromatography (hexanes/EtOAc 1:3) to afford 15 (1.25 g, 65%) as a
was concentrated. The residue was dissolved in water and lyophilized to
give the disaccharide oxazoline 1 (34 mg, quantitative) as a white solid.
1H NMR (D2O, 500 MHz): d = 6.03 (d, J=7.5 Hz, 1H, H-1), 4.66 (s, 1H,
H-1’), 4.32 (s, 1H, H-2’), 4.13 (s, 1H, H-2), 3.91–3.88 (m, 4H), 3.72–3.50
(m, 4H), 3.55–3.32 (m, 2H), 1.85 (s, 3H, CH3-); 13C NMR (D2O,
125 MHz): d = 168.6, 101.2, 99.9, 77.4, 76.4, 72.8, 70.9, 70.4, 69.3, 66.8,
65.2, 61.7, 61.1, 13.0 ESI-MS: m/z: calcd for C14H23NO10: 365.13; found:
366.57 [M+H]+.
4-Pentenyl O-(4-O-acetyl-2,3-di-O-benzoyl-6-O-benzyl-b-d-mannopyra-
nosyl)-(1!4)-2-acetamido-3,6-di-O-benzoyl-2-deoxy-b-d-glucopyranoside
(18): Trifluoroacetic anhydride (120 mL, 0.88 mmol) and Et3SiH (240 mL,
0.44 mmol) were added at 08C to a solution of 15 (140 mg, 0.15 mmol) in
dry CH2Cl2 (5 mL). After the reaction mixture was stirred at 08C for
5 min, TFA (112 mL, 0.44 mmol) was added dropwise over 2 min. The re-
action was stirred from 08C to RT overnight. The mixture was diluted
with EtOAc (50 mL), and the solution was washed with NaHCO3 and
brine, dried by Na2SO4, and filtered. The filtrate was dissolved in pyri-
dine (5 mL) containing Ac2O (0.5 mL). The mixture was stirred overnight
at RT and then diluted with CH2Cl2, washed with NaHCO3 and brine.
The organic layer was dried by Na2SO4 and filtered. The filtrate was con-
centrated and the residue was purified by flash silica gel column chroma-
tography (hexanes/EtOAc 2:1) to give 18 (107 mg, 76%) as a white
white foam. 1H NMR (500 MHz, CDCl3):
d = 8.11–7.26 (m, 25H,
5C6H5), 5.87 (d, J=3.5 Hz, 1H, H-2’), 5.77–5.69 (m, 2H), 5.61 (d, J=
9.5 Hz, 1H, NH), 5.45–5.38 (m, 3H), 5.33 (t, J=9.2 Hz, 1H, H-3), 4.97–
4.90 (m, 3H), 4.69 (dd, J=12.0, 3.5 Hz, 1H, H-6a), 4.60 (dd, J=12.0,
3.8 Hz, 1H, H-6b), 4.53–4.48 (m, 2H), 4.19–4.14 (m, 2H), 3.99 (t, J=
10.0 Hz, 1H, H-4’), 3.79–3.68 (m, 3H), 3.48–3.26 (m, 3H), 2.08–2.02 (m,
2H, -OCH2CH2CH2-), 1.83 (s, 3H, CH3CO), 1.68–1.59 (m, 2H, -OCH2
CH2 CH2-); 13C NMR(CDCl3, 125 MHz): d = 170.1, 166.4, 166.3, 165.7,
165.5, 136.0, 133.4, 133.3, 133.1, 130.0, 129.9, 129.8, 128.6, 128.4, 128.2,
126.1, 114.9, 101.7, 101.2, 99.2, 77.4, 77.1, 76.8, 72.8, 70.4, 68.8, 68.0, 67.5,
62.0, 53.7, 30.0, 28.6, 23.3; ESI-MS: m/z: calcd for: C54H53NO15: 955.34;
found: 956.37 [M+H]+.
1
foam. H NMR (500 MHz, CDCl3): d = 8.08–7.26 (m, 25H, 5C6H5), 5.81
(d, J=2.5 Hz, 1H, H-2’), 5.77–5.71 (m, 1H, -CH=), 5.56 (d, J=8.0 Hz,
1H, NH), 5.47 (t, J=10.0 Hz, 1H, H-4’), 5.34 (t, J=9.0 Hz, 1H, H-3),
5.28 (dd, J=7.0, 3.0 Hz, 1H, H-3’), 4.97–4.90 (m, 3H), 4.72 (dd, J=11.5,
2.0 Hz, 1H, H-6a), 4.59 (dd, J=11.5, 4.0 Hz, 1H, H-6b), 4.48 (m, 2H),
4.37 (d, J=12.0 Hz, 1H, PhCH2-), 4.29–4.17 (m, 2H), 3.86–3.81 (m, 2H),
3.49–3.28 (m, 4H), 2.10–2.06 (m, 2H, -OCH2-CH2-CH2-), 1.80, 1.76 (s
each, 3H each, 2CH3CO), 1.72–1.64 (m, 2H, -OCH2-CH2-CH2-); ESI-
MS: m/z: calcd for C56H57NO16: 999.37; found: 1000.16 [M+H]+.
4-Pentenyl O-(2,3,4,6-tetra-O-acetyl-b-d-mannopyranosyl)-(1!4)-3,6-di-
O-acetyl-2-acetamido-2-deoxy-b-d-glucopyranoside (16): A solution of 15
(300 mg, 0.31 mmol) in 80% HOAc (10 mL) was stirred at 508C for 4 h.
The mixture was concentrated in vacuo, and the residue was dissolved in
MeOH (20 mL) containing NaOMe (0.03 mmol). The solution was stirred
4-Pentenyl O-(2,3,4-O-triacetyl-6-O-benzyl-b-d-mannopyranosyl)-(1!4)-
+
at RT for 5 h and then neutralized with DowexW50-X8 (H form). The
2-acetamido-3,6-di-O-acetyl-2-deoxy-b-d-glucopyranoside (19):
A solu-
solution was filtered, and the filtrate was concentrated to dryness. The
residue was then treated with pyridine (5 mL) and Ac2O (1 mL) at RT
for 10 h. The reaction mixture was then poured into cold NaHCO3 solu-
tion and stirred for 2 h. The reaction mixture was extracted with CH2Cl2
and the organic layer was washed with aq. NaHCO3, HCl (1n) and brine,
dried by Na2SO4, and filtered. The filtrate was concentrated in vacuo and
the residue was subject to flash silica gel column chromatography (hex-
anes/EtOAc 1:3) to give 16 (140 mg, 62%). 1H NMR (500 MHz, CDCl3:
d = 5.83–5.75 (m, 1H, -CH=), 5.41 (d, J=9.0 Hz, 1H, NH), 5.39 (d, J=
3.5 Hz, 1H, H-2’), 5.19 (t, J=9.0 Hz, 1H, H-4’), 5.10 (t, J=9.0 Hz, 1H,
H-3), 5.03–4.94 (m, 3H), 4.69 (s, 1H, H-1’), 4.46 (d, J=8.0 Hz, 1H, H-1),
4.40–4.30 (m, 2H), 4.20 (dd, J=12, 4.5 Hz, 1H), 4.13–3.96 (m, 2H), 3.93–
3.81 (m, 3H), 3.64–3.44 (m, 4H), 2.14, 2.11, 2.09, 2.07, 2.04, 2.01, 1.98 (s
each, 3H each, 7CH3CO), 1.75–1.64 (m, 2H, -OCH2CH2CH2-); ESI-MS:
m/z: calcd for C31H45NO17: 703.27; found: 704.47 [M+H]+.
tion of the 18 (100 mg, 0.1 mmol) in MeOH (10 mL) containing NaOMe
(0.1 equiv) was stirred at RT for 2 h. Then the mixture was neutralized
+
by DowexW50-X8 (H form), filtered and concentrated. The foregoing
compound was dissolved in pyridine (5 mL) containing Ac2O (0.5 mL).
The mixture was stirred at RT overnight and then diluted with CH2Cl2,
and washed with NaHCO3 and brine. The organic layer was dried by
Na2SO4 and filtered. The filtrate was concentrated and the residue was
purified by flash silica gel column chromatography (hexanes/EtOAc 1:2)
1
to give 19 (71 mg, 95%). H NMR (500 MHz, CDCl3): d = 7.32–7.26 (m,
5H, C6H5), 5.78–5.76 (m, 1H, -CH=), 5.41 (d, J=9.0 Hz, 1H, NH), 5.37
(d, J=2.3 Hz, 1H, H-2’), 5.22 (t, J=9. 5 Hz, 1H, H-4), 5.14–4.94 (m,
5H), 4.66 (s, 1H, H-1’), 4.55–4.35 (m, 4H), 4.19 (dd, J=12.0, 4.0 Hz, 1H,
H-6), 3.89–3.77 (m, 3H), 3.60–3.54 (m, 4H), 3.45–3.41 (m, 2H), 2.12,
2.09, 2.04, 2.01, 1.97, 1.94, 1.93 (s each, 3H each, 6CH3CO), 1.68–1.58 (m,
2H, -OCH2CH2CH2-); 13C NMR(CDCl3, 125 MHz): d
= 169.6, 169.5,
O-(2,3,4,6-Tetra-O-acetyl-b-d-mannopyranosyl)-(1!4)-2-acetamido-3,6-
di-O-acetyl-1,2-dideoxy-a-d-glucopyrano)-[2,1-d]-2-oxazoline (17): NIS
(15 mg, 1.2 equiv) and TESOTf (25 mL) were added to a solution of 16
(75 mg, 106 mmol) in anhydrous CH2Cl2 (20 mL). The mixture was stirred
at RT for 0.5 h and then diluted with CH2Cl2, and washed with NaHCO3
and brine. The organic layer was dried by Na2SO4 and filtered. The fil-
trate was concentrated and the residue was purified by flash silica gel
column chromatography (hexanes/EtOAc 1:2) to give 17 (57.3 mg, 87%)
as a white solid. 1H NMR (CDCl3, 500 MHz): d = 5.90 (d, J=7.0 Hz,
1H, H-1), 5.54 (t, J=2.5 Hz, 1H, H-3), 5.41 (d, J=3.0 Hz, 1H, H-2’),
5.25 (t, J=10 Hz, 1H, H-4’), 5.05 (dd, J=9.5, 3.0 Hz, 1H, H-3’), 4.81 (s,
1H, H-1’), 4.27 (dd, J=12.5, 5.0 Hz, 1H, H-6’), 4.19–4.09 (m, 4H), 3.70–
3.68 (m, 2H), 3.48–3.46 (m, 1H), 2.20, 2.11, 2.09, 2.09, 2.04, 2.04, (s each,
3H each, 6CH3CO). 1.99 (s, 3H, CH3); 13C NMR (CDCl3, 125 MHz): d
= 170.7, 170.6, 170.5, 170.0, 169.6, 169.3, 166.3, 99.3, 99.0, 76.1, 72.5, 70.8,
70.4, 68.4, 67.8, 66.0, 64.8, 63.6, 62.5, 60.4, 20.1, 20.9, 20.8, 20.7, 20.6, 20.5,
14.2; ESI-MS: m/z: calcd for C26H35NO16: 617.2; found: 618.37 [M+H]+.
169.1, 168.8, 137.0, 136.6, 127.6, 127.1, 126.9, 114.0, 100.3, 96.5, 72.5, 71.5,
71.3, 68.2, 68.0, 61.5, 52.9, 29.0, 27.6, 22.4, 19.9, 19.8, 19.7; ESI-MS: m/z:
calcd for C36H49NO16: 751.77; found: 752.09 [M+H]+.
O-(2,3,4-Tri-O-acetyl-6-O-benzyl-b-d-mannopyranosyl)-(1!4)-3,6-di-O-
acetyl-1,2-dideoxy-a-d-glucopyrano)-[2,1-d]-2-oxazoline
(20):
NIS
(12 mg, 1.3 equiv) and TESOTf (13 mL) were added to a solution of 19
(38 mg, 51 mmol) in anhydrous CH2Cl2 (5 mL), and the mixture was stir-
red at RT for 0.5 h until TLC indicated the completion of the reaction.
The mixture was diluted with CH2Cl2 and washed with NaHCO3 and
brine. The mixture was dried by Na2SO4 and filtered. The filtrate was
concentrated and the residue was purified by flash silica gel column chro-
matography (hexanes/EtOAc/Et3N 1:2:0.01) to give 20 (26 mg, 78%) as a
white solid. 1H NMR (CDCl3, 500 MHz): d = 7.33–7.32 (m, 5H, C6H5),
5.88 (d, J=7.0 Hz, 1H, H-1), 5.54 (dd, J=4.5, 2.5 Hz, 1H, H-3), 5.39 (d,
J=3.5 Hz, 1H, H-2’), 5.35 (t, J=10 Hz, 1H, H-4’), 5.03 (dd, J=10.0,
3.0 Hz, 1H, H-3’), 4.80 (s, 1H, H-1’), 4.52 (d, J=12.0 Hz, 1H, PhCH2-),
4.19 (d, J=12.0 Hz, 1H, PhCH2-), 4.19–4.09 (m, 4H), 3.70–3.68 (m, 2H),
3.73–3.54 (m, 4H), 2.28, 2.10, 2.01, 1.98, 1.90 (s each, 3H each, 5CH3CO-),
1.89 (s, 3H, CH3); 13C NMR(CDCl3, 125 MHz): d = 170.7, 170.1, 169.8,
169.3, 166.2, 138.0, 128.4, 127.9, 127.7, 99.4, 98.8, 73.9, 73.5, 70.9, 70.7,
O-(b-d-Mannopyranosyl)-(1!4)-1,2-dideoxy-a-d-glucopyrano)-[2,1-d]-2-
oxazoline (1): A solution of 17 (57 mg, 100 mmol) in MeOH (2 mL) con-
taining NaOMe (10 mmol) was stirred at RT for 2 h. Then the mixture
Chem. Eur. J. 2006, 12, 3355 – 3364
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3361

L.-X. Wang et al.
69.7, 68.5, 63.6, 21.1, 20.9, 20.8, 20.7, 13.9, 11.4; ESI-MS: m/z: calcd for
C31H39NO15: 665.23; found: 666.16 [M+H]+.
2.06 (m, 2H, -CH2 CH2O), 1.95, 1.77 (s each, 3H each, 2CH3CO), 1.68–
1.58 (m, 2H, -OCH2CH2CH2-); 13C NMR (CDCl3, 125 MHz): d = 170.61,
169.7, 166.3, 166.2, 115.28, 99.0, 98.1, 96.9, 77.4, 77.1, 76.8, 72.7, 72.4, 71.5,
70.0, 68.8, 67.8, 66.9, 63.0, 62.9, 52.44, 30.27, 29.8, 28.3, 23.1, 20.8; ESI-
MS: m/z: calcd for C83H77NO25: 1487.48; found: 1488.61 [M+H]+.
O-(6-Benzyl-b-d-mannopyranosyl)-(1!4)-1,2-dideoxy-a-d-glucopyrano)-
[2,1-d]-2-oxazoline (4): A solution of compound 20 (22 mg, 36 mmol) in
MeOH containing NaOMe (5 mmol) was stirred at RT for 2 h. Then the
mixture was concentrated. The residue was dissolved in water and lyophi-
lized to give the oxazoline 4 (15 mg, quantitative) as a white solid.
1H NMR (CD3OD, 500 MHz): d = 7.39–7.32 (m, 5H, C6H5), 5.99 (d, J=
7.2 Hz, 1H, H-1), 4.64–4.59 (m, 3H, H-1’, 2 PhCH2-), 4.23 (t, J=2.8 Hz,
1H, H-2’), 4.06–4.04 (m, 1H), 3.85–3.29 (m, 11H), 2.13 (s, 3H, CH3-);
4-Pentenyl O-[(2,3,4,6-tetra-O-acetyl-a-d-mannopyranosyl)-(1!6)-(2,3,4-
triacetyl-b-d-mannopyranosyl)]-(1!4)-2-acetamido-3,6-di-O-acetyl-2-
deoxy-b-d-glucopyranoside (25): A solution of 24 (150 mg, 101 mmol) in
MeOH (10 mL) containing NaOMe (10 mmol) was stirred at RT for 2 h.
Then the mixture was neutralized by Dowex W50-X8 (H+ form), filtered
and concentrated. The foregoing compound was dissolved in pyridine
(5 mL) and Ac2O (1 mL). The mixture was stirred at RT overnight. The
mixture was diluted with CH2Cl2 and washed with NaHCO3, brine, and
water. The organic layer was dried by Na2SO4 and filtered. The filtrate
was concentrated and the residue was purified by flash silica gel column
13C NMR (CD3OD, 125 MHz): d
= 168.7, 137.2, 128.8, 128.6, 128.4,
101.3, 99.9, 77.6, 75.1, 73.3, 72.8, 70.9, 70.4, 69.4, 69.3, 13.0; ESI-MS: m/z:
calcd for C21H29NO10: 455.18; found: 456.16 [M+H]+.
4-Pentenyl O-(2,3-di-O-benzoyl-b-d-mannopyranosyl)-(1!4)-2-acetami-
do-3,6-di-O-benzoyl-2-deoxy-b-d-glucopyranoside (21) and 4-pentenyl O-
(4,6-di-O-acetyl-2,3-di-O-benzoyl-b-d-mannopyranosyl)-(1!4)-2-acetami-
1
chromatography (hexanes/EtOAc 1:2) to give 25 (95 mg, 95%). H NMR
do-3,6-di-O-benzoyl-2-deoxy-b-d-glucopyranoside (22):
A solution of
(CDCl3, 500 MHz): d
= 5.87–5.62 (m, 2H, NH, -CH=), 5.43 (d, J=
compound 15 (110 mg, 0.21 mmol) in 80% HOAc (10 mL) was stirred at
508C for 4 h. Concentration of the reaction mixture followed by purifica-
tion on a silica gel column (CH2Cl2/MeOH 30:1) gave compound 21
(95 mg, 92%). ESI-MS: m/z: calcd for C47H49NO15: 867.31; found: 868.28
[M+H]+.
2.8 Hz, 1H, H-2’’), 5.28–5.10 (m, 5H), 5.02–4.94 (m, 3H), 4.77 (s, 1H, H-
1’’), 4.68 (s, 1H, H-1’), 4.67 (d, J=8.8 Hz, 1H, H-1), 4.43–4.10 (m, 6H),
3.81–3.80 (m, 2H), 3.55–3.28 (m, 3H), 2.11, 2.10, 2.08, 2.06, 2.00, 1.99,
1.97, 1.93, 1.92 (9s, 30H, 9CH3); ESI-MS: m/z: calcd for C43H61NO25
991.35; found: 992.15 [M+H]+.
:
The identity of compound 21 was further characterized by conversion to
the 4’,6’-di-O-acetyl derivative 22. To a solution of 21 in pyridine (5 mL)
was added Ac2O (0.5 mL). The mixture was stirred at RT for 4 h, and
then diluted with CH2Cl2. The solution was washed with NaHCO3 and
brine, and the organic layer was dried by Na2SO4 and filtered. The filtrate
was concentrated and the residue was purified by flash silica gel column
chromatography (hexanes/EtOAc 1:1) to give 22 (96 mg, 100%).
1H NMR (500 MHz, CDCl3): d = 8.11–7.26 (m, 20H, 5C6H5), 5.82 (d,
J=3.0 Hz, 1H, H-2’), 5.72–5.69 (m, 1H, -CH=), 5.38 (dd, J=9.0 Hz, 1H,
H-3), 5.27 (dd, J=9.0, 3.5 Hz, 1H, H-3’), 5.01–4.94 (m, 3H, H-1’, =CH2-),
4.60 (m, 2H), 4.51 (d, J=8.0 Hz, 1H, H-1), 4.21–4.16 (m, 2H), 4.06–3.96
(m, 2H), 3.85–3.82 (m, 1H), 3.74–3.69 (m, 3H), 3.48–3.38 (m, 2H), 2.14–
2.10 (m, 2H, -OCH2CH2CH2-), 2.01, 1.85, 1.79 (3s, 9H, 3CH3CO), 1.68–
1.58 (m, 2H, -OCH2CH2CH2-); ESI-MS: m/z: calcd for C51H53NO17:
951.33; found: 952.26 [M+H]+.
O-[(2,3,4,6-Tetra-O-acetyl-a-d-mannopyranosyl)-(1!6)-(2,3,4-tri-O-
acetyl-b-d-mannopyranosyl)]-(1!4)-3,6-di-O-acetyl-1,2-dideoxy-a-d-glu-
copyrano)-[2,1-d]-2-oxazoline (26): NIS (13 mg, 1.3 equiv) and TESOTf
(14 mL) were added to a solution of 25 (50 mg, 50 mmol) in dry CH2Cl2
(5 mL). The mixture was stirred at RT for 0.5 h when TLC indicated the
completion of the reaction. The mixture was diluted with CH2Cl2 and
washed with NaHCO3 and brine. The organic layer was dried by Na2SO4
and filtered. The filtrate was concentrated and the residue was purified
by flash chromatography on silica gel using (EtOAc/hexanes 2:1 contain-
ing 1% Et3N) to give 26 (37 mg, 80%) as a white solid. 1H NMR
(CDCl3, 500 MHz): d = 5.91 (d, J=7.5 Hz, 1H, H-1), 5.61–5.60 (s, 1H,
H-3), 5.40 (d, J=3.5 Hz, 1H, H-2’), 5.31–5.27 (m, 3H), 5.20 (t, J=
10.0 Hz, 1H, H-4’), 5.03 (dd, J=3.0, 10.0 Hz, 1H, H-3’’), 4.85 (s, 1H, H-
1’), 4.84 (s, 1H, H-1’’), 4.34 (dd, J=4.0, 12.0 Hz, 1H, H-6), 4.18–4.08 (m,
5H), 3.92 (dd, J=4.4, 11.0 Hz, 1H), 3.76–3.74 (m, 1H), 3.67–3.59 (m,
2H), 3.45–3.43 (m, 1H), 2.18, 2.15, 2.12, 2.11, 2.10, 2.07, 2.04, 2.01, 1.99,
(s each, 3H each, 10CH3CO), 1.96 (s, 3H, CH3); ESI-MS: m/z: calcd for
C43H61NO25: 905.28; found: 906.53 [M+H]+.
4-Pentenyl O-[(2,3,4,6-tetra-O-benzoyl-a-d-mannopyranosyl)-(1!6)-(2,3-
di-O-benzoyl-b-d-mannopyranosyl)]-(1!4)-2-acetamido-3,6-di-O-benzo-
yl-2-deoxy-b-d-glucopyranoside (23) and 4-pentenyl O-[(2,3,4,6-tetra-O-
benzoyl-a-d-mannopyranosyl)-(1!6)-(4-O-acetyl-2,3-di-O-benzoyl-b-d-
mannopyranosyl)]-(1!4)-2-acetamido-3,6-di-O-benzoyl-2-deoxy-b-d-glu-
copyranoside (24): A suspension of compound 22 (70 mg, 75 mmol) and
2,3,4,6-tetra-O-benzoyl-a-d-mannopyranosyl trichloroacetimidate (60 mg,
81 mmol) in dry CH2Cl2 (4 mL) containing activated 4 ꢁ molecular sieves
(100 mg) was stirred under an atmosphere of argon at RT for 30 min.
After cooling to ꢀ208C, a solution of TMSOTf in CH2Cl2 (0.1m, 40 mL,
4 mmol) was added and the resulting mixture was stirred from ꢀ208C to
RT for 3 h. The reaction was quenched with Et3N and diluted with
CH2Cl2 (10 mL). The solution was washed with brine and the organic
layer was dried over Na2SO4 and filtered. The filtrate was concentrated
in vacuo and the residue was purified by flash silica gel column chroma-
tography (hexanes/EtOAc 2:1) to give compound 23 (82 mg, 76%) as a
white foam. ESI-MS: m/z: calcd for C81H75NO24: 1445.47; found: 1446.52
[M+H]+.
O-[(a-d-Mannopyranosyl)-(1!6)-(b-d-mannopyranosyl)]-(1!4)-1,2-di-
deoxy-a-d-glucopyrano)-[2,1-d]-2-oxazoline (3): A solution of 26 (22 mg,
24 mmol) in MeOH containing NaOMe (3 mmol) was stirred at RT for
2 h. Then the mixture was concentrated. The residue was dissolved in
water and lyophilized to give 3 (13 mg, quantitative) as a white solid.
1H NMR (CD3OD, 500 MHz): d = 6.00 (d, J=7.2 Hz, 1H, H-1), 4.79 (s,
1H, H-1’), 4.62 (s, 1H, H-2’), 4.59 (s, 2H, H-1’’, H-2’’), 4.20 (s, 1H, H-2),
4.04–4.02 (m, 1H), 3.9–3.26 (m, 14H), 2.01 (s, 3H, CH3-); 13C NMR
(CD3OD, 125 MHz): d = 168.6, 100.0, 99.7, 99.6, 77.8, 74.5, 73.0, 72.7,
70.4, 70.1, 69.9, 69.4, 66.8, 66.3, 65.1, 61.7, 60.8, 13.0; ESI-MS: m/z: calcd
for C20H33NO15: 527.19; found: 528.29 [M+H]+.
Acceptor GlcNAc-peptide 27: GlcNAc-tripeptide 27 was synthesized on
a Pioneer automatic peptide synthesizer (Applied Biosystems) using
Fmoc chemistry on a PAL-PEG-PS resin (on 0.2 mmol scale), following
the previously described procedure.[7] After the solid-phase synthesis, the
peptide was retrieved from the resin with 95% TFA, followed by the
treatment with 5% hydrazine to remove the O-acetyl groups. The result-
ing crude GlcNAc-peptide was purified by preparative RP-HPLC to pro-
vide the GlcNAc-tripeptide 27 (104 mg, 88%). 1H NMR (D2O,
500 MHz): d = 4.98 (d, J=9.8 Hz, 1H, NH-Ac), 4.65 (d, J=7.0 Hz, 1H,
H-1), 4.25 (d, J=4.4 Hz, 1H, Thr-Ha), 4.17 (d, J=7.5 Hz, 1H, Leu-Ha),
4.16 (dd, J=4.5, 6.5 Hz, 1H,Asn-Ha), 3.81 (dd, J=12.4, 1.8 Hz, 1H, H-3),
3.74 (t, J=10.0 Hz, 1H, H-2), 3.68 (dd, J=12.3, 4.8 Hz, 1H,H-6), 3.53 (t,
J=8.4 Hz, 1H,H-4), 3.45–3.38 (m, 3H, Leu-Hb, H-5, H-6’), 2.77 (dd, J=
5.8, 16.0 Hz, 1H, Asn-Hb), 2.65 (dd, J=16.0, 7.8 Hz, 1H, Asn-Hb), 1.95
(s, 3H, NHCOCH3), 1.94 (s, 3H, NHCOCH3), 1.87–1.90 (m, 1H, Leu-
Hb), 1.45–1.48 (m, 2H, Leu-CH2), 1.14 (d, J=6.6 Hz, 3H, Thr-CH3), 0.85
Compound 23 was further characterized by its conversion to the 4’-O-
acetyl derivative 24. Compound 23 (40 mg, 27 mmol) was dissolved in pyr-
idine (2 mL) containing Ac2O (0.5 mL). The mixture was stirred at RT
overnight. The mixture was diluted with CH2Cl2 and washed with
NaHCO3, brine, dried by Na2SO4 and filtered. The filtrate was concen-
trated and the residue was purified by flash silica gel column chromatog-
raphy (hexanes/EtOAc 2:1) to give 24 (42 mg, quantitative) as a white
1
foam. H NMR (500 MHz, CDCl3): d = 8.09–7.32 (m, 40H, 8C6H5), 6.11
(d, J=10.0 Hz, 1H, H-4’’), 5.92–5.87 (m, 3H, NH, H-4’, H-3’’), 5.78–5.76
(m, 1H), 5.72–5.67 (m, 1H, =CH-), 5.65–5.60 (m, 2H), 5.37 (dd, J=10.0,
3.5 Hz, 1H, H-3’), 5.05 (s, 1H, H-1’’), 4.96–4.89 (m, 3H), 4.82 (d, J=
8.5 Hz, 1H), 4.70–4.60 (m, 4H), 4.49–4.42 (m, 2H), 4.28–4.26 (m, 2H),
4.19 (t, J=9.5 Hz, 1H, H-4), 3.71–3.62 (m, 3H), 3.46–3.30 (m, 3H), 2.10–
3362
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 3355 – 3364

Glycopeptide Synthesis
FULL PAPER
(d, J=7 Hz, 3H, Leu-CH3), 0.80 (t, J=7 Hz, 3H, Leu-CH3); ESI-MS:
m/z: calcd for C24H42N6O11: 590.29; found: 591.29 [M+H]+.
Glycopeptide 33: Yield: 5.0 mg, 68% (based on the reaction with
1.5 mmol of GlcNAc-C34); ESI-MS: m/z: calcd for: 4946.44; found:
1649.68 [M+3H]3+, 1237.50 [M+4H]4+, 990.20 [M+5H]5+
.
General procedure for the ENGase-catalyzed transglycosylation: A mix-
ture of the respective oligosaccharide oxazoline (10–30 mmol) and the
GlcNAc-tripeptide 27 (3 molar equivalents of the donor) or the GlcNAc-
C34[7] (5 molar equivalents of the donor) in a phosphate buffer (50 mm,
pH 6.5, 0.5–1 mL) was incubated at 258C with the enzyme Endo-A (100
mU). The oxazoline derivative was added in portions during the reaction.
The reaction was monitored by analytical HPLC by taking aliquots at in-
tervals. It was observed that the transglycosylation reaction with the dis-
accharide oxazoline derivatives 1 and 4 proceeded slowly and took more
than 48 h for reaching the plateau, while the transglycosylation with the
tetrasaccharide oxazoline 2 proceeded quickly and would be complete
within 2 h. The reaction was stopped by heating in a boiling water bath
for 3 min when the peak of the transglycosylation product reached the
maximum. The product was purified by preparative HPLC on a Waters
preparative column (Symmetry 300, 19x300 mm) to afford the respective
glycopeptide, which was characterized by NMR and ESI-MS).
Pronase digestion of glycopeptides 32 and 33: Glycopeptide 32 (0.5 mg)
was digested with pronase (30 mg, Sigma) in a phosphate buffer (pH 8.2,
100 mL) at 378C for 4 h. The reaction mixture was subjected to Sephadex
G-10 gel filtration and the carbohydrate positive fractions (detected by
anthrone assay) were pooled and subjected to ESI-MS analysis. ESI-MS:
m/z: calcd for Man2GlcNAc2Asn: 862.78; found: 863.62 [M+H]+. Simi-
larly the Bn-tagged glycopeptide 33 (0.3 mg) was digested with pronase
as described above, and the digestion product was isolated and character-
ized. ESI-MS: m/z: calcd for Bn-Manb1!4GlcNAcb1!4GlcNAcb1!
Asn: 790.31; found: 791.40 [M+H]+.
Acknowledgements
We thank Professor K. Takegawa for kindly providing the pGEX-2T/
Endo-A plasmid that was used to overexpress Endo-A. The work was
supported by the National Institutes of Health (AI054354 and
AI051235).
Glycopeptide 28: yield: 3.0 mg, 63% (based on the reaction with 5 mmol
of 27); H NMR (D2O, 500 MHz): d = 4.97 (d, J=10.0 Hz, 1H, NH-Ac),
1
4.66 (d, J=7.0 Hz, 1H, H-1), 4.65 (s, 1H, H-1’’), 4.53 (d, J=7.5 Hz, 1H,
H-1’), 4.24 (d, J=4.4 Hz, 1H, Thr-Ha), 4.20 (d, J=7.5 Hz, 1H, Leu-Ha),
4.15 (dd, J=6.2, 4.4 Hz, 1H, Asn-Ha), 4.00 (d, J=3.2 Hz, 1H, H-2’’),
3.86–3.32 (m, 18H), 2.77 (dd, J=5.8, 16.0 Hz, 1H, Asn-Hb), 2.65 (dd, J=
7.8, 16.0 Hz, 1H, Asn-Hb), 1.99 (s, 3H, NHCOCH3), 1.95 (s, 3H,
NHCOCH3), 1.93 (s, 3H, NHCOCH3), 1.92–1.82 (m, 1H, Leu-Hb), 1.42–
1.34 (m, 2H, Leu-CH2), 1.14 (d, J=6.6 Hz, 3H, Thr-CH3), 0.85 (d, J=
7.0 Hz, 3H, Leu-CH3), 0.80 (t, J=7 Hz, 3H, Leu-CH3); ESI-MS: m/z:
calcd for C38H65N7O21: 955.42; found: 956.39 [M+H]+.
[1] a) A. Varki, Glycobiology 1993, 3, 97–130; b) R. A. Dwek, Chem.
Rev. 1996, 96, 683–720; c) P. M. Rudd, T. Elliott, P. Cresswell, I. A.
Wilson, R. A. Dwek, Science 2001, 291, 2370–2376.
[2] a) G. Arsequell, G. Valencia, Tetrahedron: Asymmetry 1999, 10,
3045–3094; b) K. M. Koeller, C. H. Wong, Nat. Biotechnol. 2000, 18,
835–841; c) O. Seitz, ChemBioChem 2000, 1, 214–246; d) H. Herz-
ner, T. Reipen, M. Schultz, H. Kunz, Chem. Rev. 2000, 100, 4495–
4538; e) B. G. Davis, Chem. Rev. 2002, 102, 579–601; f) M. J.
Grogan, M. R. Pratt, L. A. Marcaurelle, C. R. Bertozzi, Annu. Rev.
Biochem. 2002, 71, 593–634; g) P. H. Seeberger, Chem. Commun.
2003, 1115–1121; h) S. Hanson, M. Best, M. C. Bryan, C. H. Wong,
Trends Biochem. Sci. 2004, 29, 656–663; i) B. G. Davis, Science 2004,
303, 480–482; j) C. H. Wong, J. Org. Chem. 2005, 70, 4219–4225.
[3] a) D. Macmillan, C. R. Bertozzi, Angew. Chem. 2004, 116, 1379–
1383; Angew. Chem. Int. Ed. 2004, 43, 1355–1359; b) T. J. Tolbert,
C. H. Wong, Methods Mol. Biol. 2004, 283, 255–266; c) J. D.
Warren, J. S. Miller, S. J. Keding, S. J. Danishefsky, J. Am. Chem.
Soc. 2004, 126, 6576–6578; d) X. Geng, V. Y. Dudkin, M. Mandal,
S. J. Danishefsky, Angew. Chem. 2004, 116, 2616–2619; Angew.
Chem. Int. Ed. 2004, 43, 2562–2565; e) B. G. Davis, R. C. Lloyd,
J. B. Jones, J. Org. Chem. 1998, 63, 9614–9615; f) D. Macmillan,
R. M. Bill, K. A. Sage, D. Fern, S. L. Flitsch, Chem. Biol. 2001, 8,
133–145; g) G. M. Watt, J. Lund, M. Levens, V. S. Kolli, R. Jefferis,
G. J. Boons, Chem. Biol. 2003, 10, 807–814; h) J. Ni, S. Singh, L. X.
Wang, Bioconjugate Chem. 2003, 14, 232–238.
[4] a) S. Mezzato, M. Schaffrath, C. Unverzagt, Angew. Chem. 2005,
117, 1677–1681; Angew. Chem. Int. Ed. 2005, 44, 1650–1654; b) M.
Fumoto, H. Hinou, T. Matsushita, M. Kurogochi, T. Ohta, T. Ito, K.
Yamada, A. Takimoto, H. Kondo, T. Inazu, S. Nishimura, Angew.
Chem. 2005, 117, 2590–2593; Angew. Chem. Int. Ed. 2005, 44, 2534–
2537; c) T. Matsushita, H. Hinou, M. Kurogochi, H. Shimizu, S.
Nishimura, Org. Lett. 2005, 7, 877–880; d) Y. Kajihara, N. Yamamo-
to, T. Miyazaki, H. Sato, Curr. Med. Chem. 2005, 12, 527–550.
[5] a) K. Yamamoto, J. Biosci. Bioeng. 2001, 92, 493–501; b) L. X.
Wang, S. Singh, J. Ni, in Synthesis of Carbohydrates through Biotech-
nology (Eds.: P. G. Wang, Y. Ichikawa), American Chemical Society,
Washington, DC, 2004, pp. 73–92.
Glycopeptide 29: yield: 5.5 mg, 86% (based on the reaction with 5 mmol
of 27); 1H NMR (D2O, 500 MHz): d = 5.02 (s, 1H, H-1’’’’), 4.97 (d, J=
10.0 Hz, 1H, NH-Ac), 4.84 (d, J=1.40 Hz, 1H, H-1’’’), 4.65 (d, J=7.1 Hz,
1H, H-1), 4.55 (d, J=7.8 Hz, 1H, H-1’’), 4.53 (d, J=7.5 Hz, 1H, H-1’),
4.24 (d, J=4.4 Hz, 1H, Thr-Ha), 4.18 (d, J=7.5 Hz, 1H, Leu-Ha), 4.15
(dd, J=4.4, 6.2 Hz, 1H, Asn-Ha), 3.99 (dd, J=2.0, 3.5 Hz, 1H,H-2’’), 3.90
(dd, J=1.5, 3.5 Hz, 1H, H-2’’’’), 3.86–3.46 (m, 29H), 2.77 (dd, J=5.8,
16.0 Hz, 1H, Asn-Hb), 2.65 (dd, J=7.8, 16.0 Hz, 1H, Asn-Hb), 1.99 (s,
3H, NHCOCH3), 1.95 (s, 3H, NHCOCH3), 1.93 (s, 3H, NHCOCH3),
1.92–1.82 (m, 1H, Leu-Hb), 1.42–1.34 (m, 2H, Leu-CH2), 1.14 (d, J=
6.6 Hz, 3H, Thr-CH3), 0.85 (d, J=7 Hz, 3H, Leu-CH3), 0.80 (t, J=7 Hz,
3H, Leu-CH3); ESI-MS: m/z: calcd for C50H89N7O31: 1279.53; found:
1280.62 [M+H]+.
Glycopeptide 30: yield: 4 mg, 72% (based on the reaction with 5 mmol of
27); 1H NMR (D2O, 500 MHz): d = 4.97 (d, J=8.7 Hz, 1H, NH-Ac),
4.84 (s, 1H, H-1’’’), 4.72 (d, J=7.5 Hz, 1H, H-1), 4.65 (s, 1H, H-1’’), 4.53
(d, J=7.5 Hz, 1H, H-1’), 4.24 (d, J=4.5 Hz, 1H, Thr-Ha), 4.20 (d, J=
7.5 Hz, 1H, Leu-Ha), 4.15 (dd, J=4.4, 6.2 Hz, 1H, Asn-Ha), 4.00 (d, J=
3.2 Hz, 1H, H-2’’), 3.88–3.32 (m, 23H), 2.75 (dd, J=5.8, 16.0 Hz, 1H,
Asn-Hb), 2.61 (dd, J=7.8, 16.0 Hz, 1H, Asn-Hb), 2.00 (s, 3H,
NHCOCH3), 1.94 (s, 3H, NHCOCH3), 1.93 (s, 3H, NHCOCH3), 1.92–
1.82 (m, 1H, Leu-Hb), 1.42–1.34 (m, 2H, Leu-CH2), 1.14 (d, J=6.6 Hz,
3H, Thr-CH3), 0.85 (d, J=7 Hz, 3H, Leu-CH3), 0.80 (t, J=7 Hz, 3H,
Leu-CH3); ESI-MS: m/z: calcd for C44H75N7O26: 1117.48; found: 1118.60
[M+H]+.
Glycopeptide 31: yield: 2.87 mg, 55% (based on the reaction with 5 mmol
of 27); 1H NMR (D2O, 500 MHz): d = 7.38–7.32 (m, 5H, C6H5), 4.97 (d,
J=8.5 Hz, 1H, NH-Ac), 4.66 (d, J=7.2 Hz, 1H, H-1), 4.53 (d, J=7.5 Hz,
1H, H-1’), 4.24 (d, J=4.4 Hz, 1H, Thr-Ha), 4.19 (d, J=7.5 Hz, 1H, Leu-
Ha), 4.15 (dd, J=4.4, 6.2 Hz, 1H, Asn-Ha), 3.98 (d, J=3.8 Hz, 1H, H-2’’),
3.86–3.40 (m, 21H), 2.77 (dd, J=5.8, 16.0 Hz, 1H, Asn-Hb), 2.65 (dd, J=
7.8, 16.0 Hz, 1H, Asn-Hb), 1.99 (s, 3H, NHCOCH3), 1.95 (s, 3H,
NHCOCH3), 1.93 (s, 3H, NHCOCH3), 1.90–1.82 (m, 1H, Leu-Hb), 1.42–
1.34 (m, 2H, Leu-CH2), 1.14 (d, J=6.6 Hz, 3H, Thr-CH3), 0.85 (d, J=
7 Hz, 3H, Leu-CH3), 0.80 (t, J=7 Hz, 3H, Leu-CH3); ESI-MS: m/z:
calcd for C45H71N7O21: 1045.47, found: 1046.40 [M+H]+.
[6] a) K. Takegawa, M. Tabuchi, S. Yamaguchi, A. Kondo, I. Kato, S.
Iwahara, J. Biol. Chem. 1995, 270, 3094–3099; b) K. Haneda, T.
Inazu, K. Yamamoto, H. Kumagai, Y. Nakahara, A. Kobata, Carbo-
hydr. Res. 1996, 292, 61–70; c) L. X. Wang, J. Q. Fan, Y. C. Lee, Tet-
rahedron Lett. 1996, 37, 1975–1978; d) L. X. Wang, M. Tang, T.
Suzuki, K. Kitajima, Y. Inoue, S. Inoue, J. Q. Fan, Y. C. Lee, J. Am.
Chem. Soc. 1997, 119, 11137–11146; e) M. Mizuno, K. Haneda, R.
Iguchi, I. Muramoto, T. Kawakami, S. Aimoto, K. Yamamoto, T.
Glycopeptide 32: Yield: 5.6 mg, 75% (based on the reaction with
1.5 mmol of GlcNAc-C34); ESI-MS: m/z: calcd for: 5018.22; found:
1674.04 [M+3H]3+, 1255.64 [M+4H]4+, 1004.78 [M+5H]5+
.
Chem. Eur. J. 2006, 12, 3355 – 3364
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3363

L.-X. Wang et al.
Inazu, J. Am. Chem. Soc. 1999, 121, 284–290; f) K. Haneda, M. Ta-
gashira, E. Yoshino, M. Takeuchi, T. Inazu, K. Toma, H. Iijima, Y.
Isogai, M. Hori, S. Takamatsu, Y. Fujibayashi, K. Kobayashi, K. Ya-
mamoto, Glycoconjugate J. 2004, 21, 377–386; g) K. Fujita, K. Take-
gawa, Biochem. Biophys. Res. Commun. 2001, 282, 678–682; h) S.
Singh, J. Ni, L. X. Wang, Bioorg. Med. Chem. Lett. 2003, 13, 327–
330; i) H. Li, S. Singh, Y. Zeng, H. Song, L. X. Wang, Bioorg. Med.
Chem. Lett. 2005, 15, 895–898.
[16] S. Shoda, T. Kiyosada, H. Mori, S. Kobayashi, Heterocycles 2000, 52,
599–602.
[17] a) S. Kobayashi, H. Morii, R. Itoh, S. Kimura, M. Ohmae, J. Am.
Chem. Soc. 2001, 123, 11825–11826; b) S. Kobayashi, S. Fujikawa,
M. Ohmae, J. Am. Chem. Soc. 2003, 125, 14357–14369; c) H.
Ochiai, M. Ohmae, S. Kobayashi, Carbohydr. Res. 2004, 339, 2769–
2788.
[18] V. Y. Dudkin, D. Crich, Tetrahedron Lett. 2003, 44, 1787–1789.
[19] T. K. M. Shing, A. S. Perlin, Carbohydr. Res. 1984, 130, 65–72.
[20] R. U. Lemieux, R. M. Ratcliffe, Can. J. Chem. 1979, 57, 1244–1251.
[21] S. Nakabayashi, C. D. Warren, R. W. Jeanloz, Carbohydr. Res. 1986,
150, C7–C10.
[7] L. X. Wang, H. Song, S. Liu, H. Lu, S. Jiang, J. Ni, H. Li, ChemBio-
Chem 2005, 6, 1068–1074.
[8] K. Takegawa, S. Yamaguchi, A. Kondo, H. Iwamoto, M. Nakoshi, I.
Kato, S. Iwahara, Biochem. Int. 1991, 24, 849–855.
[9] K. Yamamoto, S. Kadowaki, J. Watanabe, H. Kumagai, Biochem. Bi-
ophys. Res. Commun. 1994, 203, 244–252.
[22] G. W. J. Twaddle, D. Y. Yashunsky, A. V. Nikolaev, Org. Biomol.
Chem. 2003, 1, 623–628.
[10] D. H. Crout, G. Vic, Curr. Opin. Chem. Biol. 1998, 2, 98–111.
[11] B. Li, Y. Zeng, S. Hauser, H. Song, L. X. Wang, J. Am. Chem. Soc.
2005, 127, 9692–9693.
[12] a) K. A. Brameld, W. D. Shrader, B. Imperiali, W. A. Goddard, 3rd,
J. Mol. Biol. 1998, 280, 913–923; b) A. C. Terwisscha van Scheltinga,
S. Armand, K. H. Kalk, A. Isogai, B. Henrissat, B. W. Dijkstra, Bio-
chemistry 1995, 34, 15619–15623; c) I. Tews, A. C. Terwisscha van
Scheltinga, A. Perrakis, K. S. Wilson, B. W. Dijkstra, J. Am. Chem.
Soc. 1997, 119, 7954–7959.
[13] a) B. L. Mark, D. J. Vocadlo, S. Knapp, B. L. Triggs-Raine, S. G.
Withers, M. N. James, J. Biol. Chem. 2001, 276, 10330–10337;
b) S. J. Williams, B. L. Mark, D. J. Vocadlo, M. N. James, S. G. With-
ers, J. Biol. Chem. 2002, 277, 40055–40065.
[23] a) S. David, A. Malleron, C. Dini, Carbohydr. Res. 1989, 188, 193–
200; b) W. Gꢂnther, H. Kunz, Carbohydr. Res. 1992, 228, 217–241;
c) W. Gꢂnther, H. Kunz, Angew. Chem. 1990, 102, 1068–1069;
Angew. Chem. Int. Ed. Engl. 1990, 29, 1050–1051.
[24] M. P. DeNinno, J. B. Etienne, K. C. Duplantier, Tetrahedron Lett.
1995, 36, 669–672.
[25] a) R. S. Rush, P. L. Derby, D. M. Smith, C. Merry, G. Rogers, M. F.
Rohde, V. Katta, Anal. Chem. 1995, 67, 1442–1452; b) S. Elliott, T.
Lorenzini, S. Asher, K. Aoki, D. Brankow, L. Buck, L. Busse, D.
Chang, J. Fuller, J. Grant, N. Hernday, M. Hokum, S. Hu, A. Knudt-
en, N. Levin, R. Komorowski, F. Martin, R. Navarro, T. Osslund, G.
Rogers, N. Rogers, G. Trail, J. Egrie, Nat. Biotechnol. 2003, 21, 414–
421.
[14] M. Fujita, S. Shoda, K. Haneda, T. Inazu, K. Takegawa, K. Yama-
moto, Biochim. Biophys. Acta 2001, 1528, 9–14.
[26] H. Li, B. Li, H. Song, L. Breydo, I. V. Baskakov, L. X. Wang, J. Org.
Chem. 2005, 70, 9990–9996.
[15] S. Kobayashi, T. Kiyosada, S. Shoda, J. Am. Chem. Soc. 1996, 118,
13113–13114.
Received: September 28, 2005
Published online: February 7, 2006
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