A convergent strategy for the preparation of N-glycan core di-, tri-, and pentasaccharide thioaldoses for the site-specific glycosylation of peptides and proteins bearing free cysteines

Article abstract of DOI:10.1016/j.carres.2003.10.029

Mammalian glycoprotein biosynthesis produces heterogeneous ranges of proteins that possess the same peptide backbone but differ in the nature and site of glycosylation. This feature has frustrated efforts to develop therapeutic glycoproteins as well as the elucidation of biological functions of individual glycoforms. We have developed an attractive approach to well-defined glycoforms of glycoproteins by oxidative coupling of thioaldoses to cysteine-containing peptides and proteins to give disulfide-linked neoglycoconjugates. To this end, the chemical synthesis di-, tri-, and pentasaccharide N-glycan thioaldoses was undertaken. A convergent approach was used for the preparation of the pentasaccharide containing a 'synthetically difficult' β-mannoside linkage. This linkage was installed by forming initially the corresponding β-glucoside-containing pentasaccharide, followed by inversion of configuration at C-2. This approach exploited a levulonyl ester at C-2 of a glucosyl donor, which directed the coupling to give the β-glucoside exclusively and could be removed selectively using hydrazine acetate without affecting other base-labile functionalities. The resulting alcohol was converted into a triflate, which was displaced by tri-n-butylammonium acetate to give a β-mannosidic linkage. The trisaccharide N-glycan was prepared in a similar manner. Thioaldoses were prepared by displacing the peracetylated α-glycosyl chlorides with thioacetate to give the peracetylated β-thioacetates, which upon saponification gave the desired compounds. The incubation of molar excesses of chitobiose thioaldose with cysteine-containing glutathione and BSA resulted in the site-specific formation of a disulfide-linked neoglycopeptide and neoglycoprotein, respectively.

Full text of DOI:10.1016/j.carres.2003.10.029

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 181–193
A convergent strategy for the preparation of N-glycan core di-, tri-,
and pentasaccharide thioaldoses for the site-specific glycosylation of
peptides and proteins bearing free cysteines
Gregory M. Watt and Geert-Jan Boons*
Complex Carbohydrate Research Center, University of Georgia, 220 Riverbend Road, Athens, GA 30602, USA
Received 4 September 2003; accepted 31 October 2003
Abstract—Mammalian glycoprotein biosynthesis produces heterogeneous ranges of proteins that possess the same peptide backbone
but differ in the nature and site of glycosylation. This feature has frustrated efforts to develop therapeutic glycoproteins as well as the
elucidation of biological functions of individual glycoforms. We have developed an attractive approach to well-defined glycoforms
of glycoproteins by oxidative coupling of thioaldoses to cysteine-containing peptides and proteins to give disulfide-linked neogly-
coconjugates. To this end, the chemical synthesis di-, tri-, and pentasaccharide N-glycan thioaldoses was undertaken. A convergent
approach was used for the preparation of the pentasaccharide containing a Ôsynthetically difficultÕ b-mannoside linkage. This linkage
was installed by forming initially the corresponding b-glucoside-containing pentasaccharide, followed by inversion of configuration
at C-2. This approach exploited a levulonyl ester at C-2 of a glucosyl donor, which directed the coupling to give the b-glucoside
exclusively and could be removed selectively using hydrazine acetate without affecting other base-labile functionalities. The resulting
alcohol was converted into a triflate, which was displaced by tri-n-butylammonium acetate to give a b-mannosidic linkage. The
trisaccharide N-glycan was prepared in a similar manner. Thioaldoses were prepared by displacing the peracetylated a-glycosyl
chlorides with thioacetate to give the peracetylated b-thioacetates, which upon saponification gave the desired compounds. The
incubation of molar excesses of chitobiose thioaldose with cysteine-containing glutathione and BSA resulted in the site-specific
formation of a disulfide-linked neoglycopeptide and neoglycoprotein, respectively.
ꢀ 2003 Elsevier Ltd. All rights reserved.
Keywords: Carbohydrates; Glycosylation; Glycoprotein; Glycopeptide; Ligation
1. Introduction
hormone action, arthritis, and viral and bacterial
infections.7–9 The cell- and tissue-specific changes in cell-
surface oligosaccharides during development have indi-
cated that these structures may be involved in cell
adhesion and migration events during embryogenesis.
Alterations in the branching and extension of N-glycans
have also been found on the surfaces of cells that have
undergone oncogenic transformation, and these changes
correlate with alterations in cell adhesion, invasiveness,
and metastasis of transformed cells.10–12
Protein glycosylation is an important co-translational
modification, which ranges from monosaccharides on
nuclear and cytoplasmic proteins to enormously com-
plex structures on extracellular N- or O-linked glyco-
proteins and proteoglycans.1–6 At the cellular level,
N- and O-glycans have been shown to contribute to a
myriad of functions, including cell-adhesion events
during immune surveillance, inflammatory reactions,
Protein-bound oligosaccharides are assembled by a
large number of glycosyltransferases that catalyze the
transfer of monosaccharide residues from nucleoside
mono- or diphosphate sugars to growing oligosaccha-
ride chains. Protein glycosylation is not under direct
* Corresponding author. Tel.: +1-706-542-9196; fax: +1-706-564-4412;
e-mail: gjboons@ccrc.uga.edu
0008-6215/$ - see front matter ꢀ 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2003.10.029

182
G. M. Watt, G.-J. Boons / Carbohydrate Research 339 (2004) 181–193
genetic control, and therefore results in the formation of
a number of glycoforms that possess the same peptide
backbone but differ in the nature and site of glycosyla-
tion.13 It has been suggested that each glycoform possess
a unique set of biological activities. The few studies that
have compared single glycoforms have required abun-
dant sources, extensive enzymatic processing, and diffi-
cult chromatographic separations. Obviously, there is an
urgent need for more efficient approaches for preparing
homogeneous forms of glycoproteins. Such preparations
will not only be important to study biological properties
of individual glycoforms, but will also be of critical
importance for the development of therapeutic glyco-
proteins.
The de novo synthesis of glycoproteins offers a
promising approach to well-defined glycoforms, and
several different ways have been explored each repre-
senting unique and difficult challenges.14 The chemical
synthesis of glycoproteins using standard peptide syn-
thesis is only feasible for peptides under 30 amino acids
in length. Small glycoproteins can be obtained by Ôthe
state of the artÕ native ligation techniques,15 which allow
efficient combining of peptide fragments.16–20 A more
direct approach to well-defined glycoproteins involves
the use of synthetic oligosaccharides that are function-
alized in such a way that they react in a site-specific
manner with a unique functionality within the protein
structure. One innovative idea involves the coupling of a
sugar bearing an iodoacetamide group with a thiol of a
free cysteine moiety.21;22 This method was, for example,
applied to the in vitro glycosylation of a human eryth-
ropoietin cysteine mutant.23
We have developed a general approach for site-
selective glycosylation of proteins,24 whereby an elec-
trophilic sulfur-containing nitropyridinesulfenyl thio-
glycoside is reacted with the thiol group of a free
cysteine residue of a peptide or protein. This approach is
advantageous in that the resulting disulfide linkage be-
tween the oligosaccharide and the protein can adopt a
conformation similar to that of the amide linkage found
in natural N-linked glycoproteins, a feature which may
be important for reproducing the contact points between
the glycan and the peptide chain.
Here we report a highly convergent approach for the
synthesis of N-glycan core di-, tri-, and pentasaccharide
thioaldoses and the glycosylation of cysteine-containing
peptide, glutathione, and protein, bovine serum albumin
(BSA). Difficulties were encountered in synthesizing ni-
tropyridinesulfenyl thioglycosides of oligosaccharides;
however, disulfide-linked neoglycopeptides and proteins
could be obtained conveniently by coupling of thioal-
doses directly with the thiol group of free cysteine-
containing peptides or proteins. As a cysteine can be
introduced at any point of a protein by site-directed
mutagenesis, a protein can be glycosylated at a specific
site.14;25–27
2. Results and discussion
It was envisaged that thioaldoses 1, 2, and 3 (Fig. 1),
which are derived from the core region of N-linked ol-
igosaccharides, would be ideally suited for site-specific
glycosylation of proteins by disulfide formation. The
preparation of compounds 2 and 3 is complicated by the
presence of a b-linked mannoside and the necessity to
introduce a b-thioaldose. b-Mannosides are difficult to
synthesize because the axial C-2 hydroxyl group steri-
cally blocks the b-face and exerts a strong a-directing
anomeric effect. As a result, attempts to introduce these
linkages by standard chemical glycosylation methods
usually produce mixtures of anomers. Despite these
difficulties, several elegant strategies to prepare b-
mannosides have reported including the following: the
use of insoluble silver salt promoters,28 intramolecular
glycosylations,29–34 epimerization of a C-2 hydroxyl of a
b-glucoside,35–38 and the use of an a-mannosyl triflate
for the introduction of a b-mannosidic linkage.39 Ele-
gant chemical preparations of the core N-glycan pen-
tasaccharide or larger structures have been reported40–49
employing these strategies. Glycosidases50–52 and glyco-
syltransferases53 have also been used for the synthesis of
the b-mannosyl-linked core trisaccharide.
We have designed a synthetic route for thioaldoses 2
and 3 in which the b-mannosidic linkage is installed by
the initial formation of a b-glucoside, followed by
inversion of configuration of the C-2 hydroxyl of the
latter moiety. This strategy is attractive since b-gluco-
sides can be stereospecifically obtained by exploiting
neighboring-group participation of a C-2 ester protect-
ing group. Thus, coupling of trisaccharide 15, which has
OH
O
NHAc
HO
HO
O
SH
HO
O
NHAc
OH
1
OH
OH
O
NHAc
HO
HO
HO
O
HO
O
O
SH
O
HO
NHAc
OH
2
HO
HO
HO
HO
O
O
OH
OH
NHAc
O
O
HO
O
O
HO
SH
O
HO
O
NHAc
HO
HO
OH
O
OH
OH
3
Figure 1. Target compounds.

G. M. Watt, G.-J. Boons / Carbohydrate Research 339 (2004) 181–193
183
a levulonyl (Lev) ester at C-2, with disaccharide 8 should
provide key pentasaccharide 19 (Scheme 3) in a con-
vergent manner. An attractive feature of the Lev ester is
that after the glycosylation, it can be removed under
mild conditions using hydrazine acetate without affect-
ing other ester functionalities. The resulting alcohol can
then be converted to a triflate leaving group, which upon
SN2 displacement with acetate should yield the desired
b-mannosidic linkage.
Glycosyl acceptor 8 was easily obtained by a two-
directional glycosylation approach54–57 in which
thioglycosyl donor 4 was coupled with 2-trimethyl-
silylethanol using trimethylsilyl triflate (TMSOTf)/
N-iodosuccinamide (NIS) as the promoter58 to give 6,
which was immediately used in the next NIS/TMSOTf-
mediated glycosylation and coupled with thioglycosyl
donor 5 to give disaccharide 7 (Scheme 1). Deacetyla-
tion of 7 using NaOMe in methanol afforded the
required glycosyl acceptor 8 in a yield of 82%.
BnO
BnO
OAc
O
O
Ph
O
O
BnO
SEt
HO
+
NH
CCl3
O
OH
9
10
TMSOTf
61%
R2O
R2O
O
SEt
O
OR1
BnO
BnO
O
LevOH
DCC
11. R1 = H, R2:R2 = CHPh
OAc
12. R1 = Lev, R2:R2 = CHPh
13. R1 = Lev, R = H
BnO
AcOH/H2O
9, TMSOTf
70%
BnO
BnO
OAc
O
BnO
O
O
RO
SEt
O
The trisaccharide 15 was efficiently prepared by a
chemoselective glycosylation strategy, which exploits the
fact that trichloroacetimidates can be activated with
TMSOTf59 without affecting thioglycosides (Scheme 2).
However, at a later stage of the synthesis a thioglycoside
can act as a glycosyl donor in the presence of a thiophilic
promoter, minimizing protecting-group manipulations
at the anomeric center. TMSOTf-promoted glycosyla-
tion of mannosyl trichloroacetimidate 9 with thiogly-
cosyl acceptor 10 gave, after selective crystallization, the
(1fi3)-linked disaccharide 11 in a yield of 61%. Com-
pound 11 was the major regioisomer formed due to the
higher reactivity of the C-3 hydroxyl of 10. The
remaining free hydroxyl of 11 was protected with a Lev
group60 by reaction with levulonic acid activated by
DCC and 4-(N,N-dimethylamino)pyridine (DMAP) to
OLev
BnO
BnO
O
OAc
Ac2O
Py
14. R = H
15. R = Ac
BnO
Scheme 2.
give fully protected 12. Next, the benzylidene acetal of
12 was removed by hydrolysis using a mixture of acetic
acid and water to give 13 in an overall yield of 95% (two
steps). The resulting 4,6-diol acceptor 13 was coupled
with mannosyl donor 9 in the presence of a catalytic
amount of TMSOTf to give regioselectively the 3,6-O-
branched trisaccharide 14 in 70% yield. This reaction
exploits the higher reactivity of the primary sugar hy-
droxyls over secondary ones. Finally, glycosyl acceptor
15 was obtained by protection of the C-4 hydroxyl of 14
as an acetyl ester by reaction with acetic anhydride and
pyridine.
OBn
Having glycosyl acceptor 8 and glycosyl donor 15 at
hand, attention was focused on the preparation of
pentasaccharide 19 (Scheme 3). Thus, coupling of 8 with
15 in the presence of NIS and TMSOTf afforded the b-
glucosyl-linked pentasaccharide 16 in a yield of 65%.
Next, selective removal of the levulonyl ester of 16 using
hydrazine acetate gave compound 17, with its central
glucosyl residue C-2 hydroxyl unmasked, in 94% yield.
The C-2-triflate 18, which was obtained upon reaction of
the foregoing compound with triflic anhydride in pyri-
dine, was subjected to a nucleophilic substitution with
tri-n-butylammonium acetate in toluene with sonica-
tion48 to give the corresponding b-mannoside 19 in an
overall yield of 89%. Analysis of the 1H NMR spectrum
of 19 revealed a singlet at 4.87 ppm corresponding to the
b-mannosyl anomeric proton. The following protecting
group manipulations were performed in good yields to
give the peracetylated TMSEt glycoside 20: treatment
O
HO
SEt
NPhth
BnO
4
HO(CH2)2TMS
NIS/TMSOTf
Ac2O
Py
OBn
OBn
O
O
AcO
BnO
HO
SEt
NPhth
OSE
NPhth
BnO
5
6
NIS/TMSOTf
OBn
O
NPhth
BnO
RO
O
OSE
NPhth
BnO
O
OBn
7. R = Ac
8. R = H
NaOMe
Scheme 1.

184
G. M. Watt, G.-J. Boons / Carbohydrate Research 339 (2004) 181–193
15
+
8
BnO
BnO
O
+
8
SEt
BnO
BnO
BnO
OAc
O
BnO
NIS/TMSOTf (65%)
OR
24
O
OBn
O
OSE
NPhth
NIS/TMSOTf (65%)
NPhth
NPhth
O
BnO
O
AcO
OBn
O
O
BnO
O
BnO
OR
O
O
OBn
BnO
BnO
BnO
BnO
BnO
O
OSE
NPhth
O
BnO
O
O
16. R = Lev
17. R = H
18. R = Tf
(H2N)2.HOAc
Tf2O, pyridine
OAc
OBn
OR
OBn
25. R = Lev
26. R = H
27. R = Tf
(H2N)2.HOAc
OAc
O
BnO
BnO
BnO
Bu4NOAc (89%, tree-steps)
Tf2O, pyridine
O
OBn
OAc
O
NPhth
Bu4NOAc (80%)
O
BnO
O
AcO
O
OSE
NPhth
O
BnO
O
O
OBn
BnO
BnO
OAc
O
NPhth
O
OBn
O
BnO
BnO
19
BnO
OSE
NPhth
O
BnO
O
BnO
OAc
i) NH2(CH2)2NH2
ii)Ac2O, pyridine
OBn
OBn
28
iii) Pd/C, H2, EtOH/H2O
iv) Ac2O, pyrdine
AcO
AcO
i) NH2(CH2)2NH2
ii) Ac2O, pyridine
OAc
O
AcO
iii) Pd/C, H2, EtOH/H2O
iv) Ac2O, pyrdine
O
OAc
OAc
O
NHAc
O
O
AcO
O
OAc
AcO
R
OAc
O
AcO
AcO
O
AcO
O
NHAc
O
NHAc
AcO
O
OAc
O
AcO
AcO
R
AcO
AcO
O
O
NHAc
20. R = OSE (α)
21. R = OH (α/β)
22. R = Cl (α)
OAc
OAc
10% TFA
SOCl2
OAc
29. R = OSE (α)
30. R = OH (α/β)
21. R = Cl (α)
10% TFA
SO2Cl2
KSAc
KSAc
23. R = SAc (β)
NaOMe
(87%)
32 R = SAc (β)
3
NaOMe(87% from 29)
2
Scheme 3.
Scheme 4.
with ethylenediamine removed the phthalamido groups,61
and the resulting amines were acetylated with acetic
anhydride in pyridine to give an N-acetylated com-
pound. Next, debenzylation was accomplished by cata-
lytic hydrogenation over 10% palladium-on-charcoal,
and the resulting hydroxyl groups were acetylated using
acetic anhydride and pyridine. The aglycone of 20 was
cleaved by 10% trifluoroacetic acid in dichloromethane
to afford hemiacetal 21, which was treated with thionyl
chloride to give the corresponding a-glycosyl chloride
22, which, in turn, was reacted with potassium thioac-
etate in acetone to give the peracetylated thioaldose 23,
in 40% yield. The relatively low yield of this step was due
to a side reaction, which led to the formation of an
oxazoline-containing product. The core-pentasaccharide
thioaldose 3 was obtained by deacetylation of 23 using
sodium methoxide in methanol. The 1H NMR spectrum
of 3 showed the reducing-end anomeric proton at
4.52 ppm, which had a coupling constant of 11.0 Hz,
demonstrating that the anomeric configuration of the
thiol group was exclusively in the b-configuration.
Trisaccharide 2 was prepared by a similar strategy,
but in this case chitobiose acceptor 8 was coupled with
glucoside 24 using NIS/TMSOTf as the promoter to give
trisaccharide 25 in 81% yield (Scheme 4). Selective re-
moval of the Lev ester of 25 using standard conditions
gave 26, containing a C-2 hydroxyl, which was con-
verted to a triflate-leaving group and subsequently
subjected to nucleophilic displacement with tributylam-
monium acetate to give b-mannoside 28 in 82% yield.
Using a similar procedure as described for 3, compound
28 was deblocked and the thioaldose moiety installed to
give target compound 2.
Peracetylated chitobiose was converted into its ano-
meric chloride with thionyl chloride (details not shown),
and subsequent treatment with potassium thioacetate
was followed by base-catalyzed deacetylation, which
gave thioaldose 1.
Having successfully prepared thioaldoses 1–3, atten-
tion was focused on the site-specific glycosylation of
peptides and proteins that have a cysteine moiety.
Although the nitropyridinesulfenyl thioglycoside of
GlcNAc could easily be obtained by reaction of 1 with
2,20-dithiobis(5-nitropyridine) (DTNP) in acetic acid/

G. M. Watt, G.-J. Boons / Carbohydrate Research 339 (2004) 181–193
185
water,24 the preparation of similar derivatives of the
more complex thioaldoses 1–3 proved problematic, and
mainly disulfide-linked dimers were obtained. It was
anticipated that oxidative coupling of a thiol moiety of a
cysteine residue with a thioaldose should offer an alter-
native approach to disulfide-linked neoglycoconjugates.
It is well known that thiolates can easily be oxidized to
disulfides, whereas analogous thiols are very slow to
react. Thus, treatment of a cysteine-containing peptide
or protein with an excess of a thioaldose at relatively
high pH should lead to the formation of a mixed
disulfide. Indeed, the combination of equimolar
amounts of thioaldose 1 and glutathione in aqueous
ammonium acetate at pH 8.5 yielded the disulfide-linked
glycopeptide 33, the thioaldose homodisulfide and glu-
tathione disulfide in a statistical 2:1:1 ratio as deter-
mined by 1H NMR spectroscopy (Scheme 5). The use of
2 equiv of the thioaldose afforded the products in ratio
of 1:0.9:0.2, thus increasing the amount of the hetero-
disulfide compared to glutathione disulfide. When a
larger excess of the thioaldose (5–20 equiv) was used
only the sugar homodimer and glycopeptide were pres-
ent, and the oxidized glutathione disulfide was not de-
tected.
Bovine serum albumin (BSA) was chosen as a model
protein on the merit of its availability and the conve-
nience of having a free cysteine residue at Cys-58 of its
sequence. After treatment of BSA with excess of 1 in
aqueous ammonium acetate, the BSA-glycoconjugate 34
was isolated free from the excess amount of 1 and its
homodimer by ultrafiltration using Centricon concen-
tration devices fitted with 10-kDa membranes. Ellman
type analysis, using dithiodipyridine,62 of the isolated
conjugate against a blank sample of nonreacted BSA
gave a zero absorbance reading at 340 nm, showing the
absence of free thiols, and indicating that the conjuga-
tion had gone to completion. Further spectroscopic
corroboration of the conjugate came from MALDI-
TOF mass spectrometry, which showed a mass increase
for the conjugate over BSA sample of 477 Da (while the
theoretical difference is 438 Da, indicating the incorpo-
ration of the thiochitobiose moiety). The attachment of
the synthetic oligosaccharides to IgG fragments, engi-
neered to have Cys-297 in place of glycan-linked Asn,
has been achieved with thioaldoses, where peptide
mapping indicated selective glycosylation of the free
cysteine, and biological assays demonstrated improved
effector functions. The results of these studies will be
reported elsewhere.
3. Conclusion
Mammalian glycoprotein biosynthesis produces heter-
ogeneous ranges of proteins that possess the same pep-
tide backbone but differ in the nature and site of
glycosylation. This feature has frustrated efforts to elu-
cidate biological functions of glycoproteins, in particu-
lar in those cases in which the precise structure of a
glycan determines biological activity. Furthermore, the
inability to control N-glycan formation in cell culture is
a major obstacle in the development of therapeutic
glycoproteins. We have developed an attractive ap-
proach to well-defined glycoforms of glycoproteins by
the chemical synthesis of a range of N-glycan thioal-
doses, which can be attached to cysteine-containing
peptides and proteins by an oxidative coupling to give
disulfide-linked neoglycoconjugates. The synthetic oli-
gosaccharides 2 and 3 were prepared by a strategy,
whereby the b-mannosidic linkages were installed by the
initial formation of the corresponding b-glucosides,
followed by inversion of configuration at C-2. The
anomeric thiols of 1, 2, and 3 were prepared by
displacing the peracetylated a-glycosyl chlorides
NH2
NH2
H
H
COOH
COOH
OH
O
NHAc
O
O
H
HO
HO
1, aq NH4OAc, pH 8.5
O
HS
NH
H
S
HO
S
NH
O
NHAc
OH
HN
O
HN
O
COOH
COOH
33
34
OH
NHAc
O
1, aq NH4OAc, pH 8.5
HO
HO
O
S
HO
BSA
HS
BSA
O
S
NHAc
OH
Scheme 5.

186
G. M. Watt, G.-J. Boons / Carbohydrate Research 339 (2004) 181–193
with thioacetate to give the peracetylated b-thioacetates,
whereupon saponification of the O- and S-acetates gave
the desired compounds. Coupling studies involving the
incubation of chitobiose thioaldose 1 in molar excess
and the cysteine-containing peptide glutathione found
that the disulfide-linked glycopeptide was formed along
with the homodimer of 1. Free cysteine-containing
protein BSA, was incubated with 1 also to give a neo-
glycoprotein product, where MALDI-TOF mass spec-
trometry indicated the addition of thiochitobiose, and
the free thiol concentration, determined using dithiodi-
pyridine, was found to be 0. In addition, to providing
well-defined glycoforms, in vitro glycosylation technol-
ogy offers several unique advantages such as the quan-
titative incorporation of synthetic oligosaccharides that
are of low natural abundance, but which are known to
display exceptional bioactivities. Furthermore, unnatu-
ral oligosaccharides can be used that will give neogly-
coproteins with improved biostabilities. Also, toxins or
other reagents can be incorporated to give conjugates
with novel effector functions.
passed through a sintered glass filter, and the eluent was
washed with 15% aq sodium thiosulfate (2 · 200 mL),
then with satd aq NaHCO3 (200 mL), followed by dry-
ing (MgSO4), filtration and concentration in vacuo.
Flash silica gel column chromatography of the residue
using 9:1 toluene–EtOAc as the eluent afforded com-
pound 6 (6.06 g, 78%) as a white foam. The H NMR
spectrum was identical to a preparation of 6 reported
previously.64
1
4.3. Trimethylsilylethyl 4-O-acetyl-3,6-di-O-benzyl-2-
deoxy-2-phthalamido-b-
D
-glucopyranosyl-(1!4)-3,6-di-
O-benzyl-2-deoxy-2-phthalamido-b-
D
-glucopyranoside (7)
A suspension of compound 565 (8.00 g, 13.9 mmol) and 6
ꢀ
(6.06 g, 10.3 mmol) containing activated 4 A molecular
sieves (10 g) in dry CH2Cl2 (30 mL) was stirred under an
atmosphere of argon at room temperature for 30 min.
After cooling to 0 ꢁC, successively N-iodosuccinamide
(3.75 g, 16.68 mmol) and trimethylsilyl triflate (252 lL,
1.28 mmol) were added and stirring was continued at
0 ꢁC for 15 min. The reaction mixture was diluted with
CH2Cl2 (200 mL), passed through a sintered glass filter,
and the eluent was washed with 15% sodium thiosulfate
(2 · 200 mL), then with satd aq NaHCO3 (200 mL) fol-
lowed by drying (MgSO4), filtration and concentration
in vacuo. Flash silica gel column chromatography of the
residue using 9:1 toluene–EtOAc as the eluent afforded
4. Experimental
4.1. General methods
Chemicals were purchased from Acros or Aldrich, Sig-
ma, and Fluka. Protein concentrations were determined
using a Lowry protein concentration kit from Sigma,
where UV absorption was recorded at 690 nm using a
Finstruments 96 well ELISA-plate reader. Thiol con-
centrations were determined using purified S,S-dithio-
dipyridine as described by Grassetti and Murray,62 and
the absorption was recorded at 340 nm using a Fin-
struments ELISA-plate reader. Mass spectra were ob-
tained using a Micromass Q-Tof-2 mass spectrometer
and a Hewlett–Packard G2025A MALDI-TOF mass
spectrometer in positive-ion mode using 2,5-dihydroxy-
benzoic acid as the matrix, unless stated otherwise.
High-resolution mass spectra were obtained using a
Voyager delayed extraction STR with 2,5-dihydroxy-
benzoic acid as an internal calibration matrix.
1
compound 7 (10.02 g, 88%) as white foam. H NMR
(CDCl3, 300 MHz): d 8.00–7.00 (m, 28H, Ar), 5.54 (d,
1H, J1 –2 8.2 Hz, H-10), 5.34 (dd, 1H, J3 –4 9.1, J4 –5
0
0
0
0
0
0
9.5 Hz, H-40), 5.28 (d, 1H, J1–2 7.4 Hz, H-1), 5.04 (d, 1H,
JAB 12.4 Hz, CH2Ph), 4.83–4.61 (m, 7H, H-30 and
6 · CH2Ph), 4.53 (d, 1H, JAB 12.1 Hz, CH2Ph), 4.50 (dd,
1H, J2 –3 10.7 Hz, H-20), 4.36 (m, 3H, H-2, H-3, and H-
4), 3.99 (m, 1H, CH2CH2TMS), 3.80–3.45 (m, 7H,
CH2CH2TMS, H-5, H-50, H-6b, H-6a, H-6b0, and H-
6a0), 2.11 (s, 3H, COCH3), 0.90 (m, 2H, CH2CH2TMS),
and 0.00 (s, 9H, TMS); MALDI-TOF MS: m=z 1124.9
(M+Naþ) and 1141.0 (M+Kþ); Anal. Calcd for
C63H66N2O14Si (1103.3): C, 68.58; H, 6.03; N, 2.54.
Found: C, 68.32; H, 6.13; N, 2.49.
0
0
4.4. Trimethylsilylethyl 3,6-di-O-benzyl-2-deoxy-2-
D
4.2. Trimethylsilylethyl 3,6-di-O-benzyl-2-deoxy-2-
phthalamido-b-
phthalamido-b-
-glucopyranosyl-(1!4)-3,6-di-O-benzyl-
D
-glucopyranoside (6)
2-deoxy-2-phthalamido-b-
D
-glucopyranoside (8)
A suspension of compound 463 (7.03 g, 13.18 mmol) and
trimethylsilylethanol (28.2 mL, 19.77 mmol) containing
ꢀ
activated 4 A molecular sieves (10 g) in dry CH2Cl2
(30 mL) was stirred under an atmosphere of argon at
room temperature for 30 min. After cooling to 0 ꢁC,
successively N-iodosuccinamide (3.85 g, 16.68 mmol)
and trimethylsilyl triflate (260 lL, 1.32 mmol) were ad-
ded, and the stirring was continued at 0 ꢁC for 30 min.
The reaction mixture was diluted with CH2Cl2 (200 mL),
To compound 7 (1.38 g, 1.26 mmol) in a mixture of 1:1
dry THF–dry MeOH (20 mL, 1/1, v/v) was added a
freshly prepared solution of NaOMe in MeOH until the
pH was 11, and the reaction mixture left for 6 h at room
temperature. Neutralization of the reaction with Am-
berlite 650 cation-exchange resin [Hþ-form] was fol-
lowed by filtration and concentration of the filtrate in
vacuo. Flash silica gel column chromatography of the
residue (eluent: 9:1 toluene–EtOAc) afforded compound

G. M. Watt, G.-J. Boons / Carbohydrate Research 339 (2004) 181–193
187
8 (1.09 g, 82%) as white foam. 1H NMR (CDCl3,
trated in vacuo. The crude residual 12 was treated with
80% HOAc (15 mL) at room temperature for 20 h, at the
end of which time the reaction mixture was concentrated
in vacuo. Flash silica gel column chromatography of the
residue using 4:1 toluene–EtOAc as the eluent afforded
13 (368 mg, 95%) as a syrup. 1H NMR (CDCl3,
300 MHz) d 7.38–7.10 (m, 15H, Ph), 5.28 (s, 1H, H-10),
0
0
300 MHz) d 7.95–7.00 (m, 28H, Ar), 5.55 (d, 1H, J1 –2
8.2 Hz, H-10), 5.18 (d, 1H, J1–2 8.0 Hz, H-1), 5.00 (m, 2H,
2 · CH2Ph), 4.68 (m, 6H, 6 · CH2Ph), 4.44 (dd, 1H, J2–3
10.7 Hz, H-2), 4.37 (m, 4H, H-20, H-3, H-4, and H-30),
4.02 (m, 1H, H-40), 4.00 (m, 1H, CH2CH2TMS), 3.90
(dd, 1H, J50–6b 4.4, J6a0–6b 10.2 Hz, H-6b0), 3.74 (m, 2H,
H-6b, H-6a0), 3.66–3.38 (m, 4H, CH2CH2TMS, H-5, H-
50, and H-6a), 0.90 (m, 2H, CH2CH2TMS), and 0.00 (s,
9H, TMS); MALDI-TOF MS: m=z 1083.1 (M+Naþ)
and 1099.0 (M+Kþ). Anal. Calcd for C61H64N2O13Si
(1061.2): C, 69.04; H, 6.08; N, 2.64. Found: C, 68.28; H,
6.14; N, 2.55.
0
0
5.23 (dd, 1H, J1 –2 1.9, J2 –3 3.0 Hz, H-20), 4.95 (dd, 1H,
J1–2 9.6, J2–3 9.3 Hz, H-2), 4.84 (d, 1H, JAB 11.3 Hz,
CH2Ph), 4.74 (d, 1H, JAB 11.0 Hz, CH2Ph), 4.54 (m, 4H,
4 · CH2Ph), 4.40 (d, 1H, H-1), 4.19 (d, 1H, J4-OH 5.2 Hz,
0
0
0
0
4-OH), 4.08 (m, 1H, H-50), 3.93 (dd, 1H, J3 –4 9.1 Hz,
H-30), 3.79 (m, 3H, H-3, H-4, and H-6b0), 3.68 (m, 1H,
H-6b), 3.65–3.55 (m, 3H, H-6a, H-6a0, and H-40), 3.25
(m, 1H, H-5), 2.62 (m, 6H, CH2CH2COCH3 and
SCH2CH3), 2.10 (m, 1H, 6-OH), 2.10 and 2.08 (2 · s,
2 · 3H, CH2CH2COCH3 and COCH3), and 1.22 (t, 3H,
SCH2CH3); MALDI-TOF MS: m=z 819.7 (M+Naþ) and
835.5 (M+Kþ). Anal. Calcd for C42H52O13S (796.3):
C, 63.30; H, 6.58. Found: C, 63.27; H, 6.61.
0
0
4.5. Ethyl 2-O-acetyl-3,4,6-tri-O-benzyl-a-D-mannopyr-
anosyl-(1!3)-4,6-O-p-methoxybenzylidene-1-thio-b-
D-
glucopyranoside (11)
To a cooled ()60 ꢁC) and stirred solution of trichloro-
acetimidate 9 (781 mg, 1.23 mmol) and compound 10
(350 mg, 1.03 mmol) in dry CH2Cl2 (5 mL) under an
argon atmosphere was added trimethylsilyl triflate
(11 lL, 62 lmol). The temperature was allowed to rise to
0 ꢁC over a period of 2 h, whereafter, TLC (4:1 toluene–
ethyl acetate) showed that a major and minor product
had been formed. Et3N (20 lL) was added to quench the
reaction and after dilution with CH2Cl2 (20 mL) and
washing with a satd aq NaHCO3 (25 mL) the CH2Cl2
phase was dried (MgSO4), filtered and concentrated in
vacuo. Precipitation of the residue from hexane and
HOAc afforded 11 (494 mg, 61%) as an amorphous
4.7. Ethyl 2-O-acetyl-3,4,6-tri-O-benzyl-a-
D
-mannopyr-
-manno-
D-glucopyrano-
anosyl-(1!6)-[2-O-acetyl-3,4,6-tri-O-benzyl-a-
D
pyranosyl-(1!3)-]-2-O-levulonyl-1-thio-b-
side (14)
A cooled ()60 ꢁC) and stirred solution of 9 (172 mg,
0.27 mmol) and disaccharide 13 (165 mg, 0.20 mmol) in
dry CH2Cl2 (2 mL) under argon was treated with trim-
ethylsilyl triflate (5 lL, 27 lmol). After stirring for 2 h,
the reaction was quenched with Et3N (8 lL) and after
aqueous workup, the residue was subjected to flash silica
gel column chromatography (eluent: 4:1 toluene–
EtOAc) affording 14 (172 mg, 70%) as a syrup. 1H NMR
(CDCl3, 500 MHz): d 7.38–7.05 (m, 30H, Ar), 5.36 (dd,
1
material. H NMR (CDCl3, 300 MHz) d 7.32–6.77 (m,
19H, Ph), 5.43 (m, 2H, CHPhOCH3 and H-20), 5.17 (d,
1H, J1 –2 1.4 Hz, H-10), 4.76 (d, 1H, JAB 10.7 Hz,
CH2Ph), 4.59 (d, 1H, JAB 12.8 Hz, CH2Ph), 4.49 (m, 4H,
4 · CH2Ph), 4.31 (d, 1H, J1–2 9.9 Hz, H-1), 4.25 (dd, 1H,
J5–6b 5.0, J6a–6b 10.4 Hz, H-6b), 4.15 (m, 1H, H-50), 3.93
0
0
1H, J1
1.5, J2
2.9 Hz, H-200), 5.19 (s, 1H, H-10),
00 –300
00 –200
5.17 (dd, 1H, J1 –2 2.0, J2 –3 3.4 Hz, H-20), 4.92 (dd, 1H,
J1–2 9.1, J2–3 9.3 Hz, H-2), 4.84 (d, 1H, JAB 11.2 Hz,
CH2Ph), 4.82 (d, 1H, JAB 11.6 Hz, CH2Ph), 4.81 (d, 1H,
H-100), 4.73 (d, 1H, JAB 11.2 Hz, CH2Ph), 4.70 (d, 1H,
JAB 11.2 Hz, CH2Ph), 4.68 (d, 1H, JAB 12.2 Hz, CH2Ph),
4.53 (m, 3H, 3 · CH2Ph), 4.47 (m, 4H, 4 · CH2Ph), 4.34
(d, 1H, H-1), 4.21 (d, 1H, J4-OH 5.4 Hz, 4-OH), 4.03
00
0
0
0
0
(dd, 1H, J2 –3 3.3, J3 –4 9.1 Hz, H-30), 3.71 (m, 2H, H-3
and H-40), 3.68 (s, 3H, PhOCH3), 3.64 (m, 3H, H-6b0, H-
6a0, and H-6a), 3.54 (t, 1H, J 9.3 Hz, H-4), 3.38 (m, 2H,
H-2 and H-5), 2.96 (d, 1H, J2–2OH 3.3 Hz, 2-OH), 2.61
(m, 2H, SCH2CH3), 2.02 (s, 3H, COCH3), and 1.22 (t,
3H, SCH2CH3); MALDI-TOF MS: m=z 837.7 (M+Naþ)
and 853.6 (M+Kþ).
0
0
0
0
10.3 Hz, H-500),
00 –500
00 –6a00
(ddd, 1H, J500
1.5, J5
6.8, J4
–6b
3.92 (m, 2H, H-30 and H-300), 3.84 (m, 2H, H-40 and H-
50), 3.76 (dd, 1H, J6a00 10.3 Hz, H-6b00), 3.74 (m, 4H,
–6b00
4.6. Ethyl 2-O-acetyl-3,4,6-tri-O-benzyl-a-
D
-mannopyr-
-glucopyranoside
anosyl-(1!3)-2-O-levulinoyl-1-thio-b-
D
H-3, H-4, H-6b, and H-6b0), 3.67 (m, 1H, H-6a0), 3.64
(dd, 1H, J3
9.7 Hz, H-400), 3.60 (dd, 1H, H-6a00), 3.51
00 –400
(13)
(dd, 1H, J5–6a 1.9, J6aꢀ6b 11.2 Hz, H-6a), 3.30 (ddd, 1H,
J5–6b 5.4, J4–5 9.4 Hz, H-5), 2.60 (m, 6H,
CH2CH2COCH3 and SCH2CH3), 2.04, 2.10, and 2.08
(3 · s, 3 · 3H, CH2CH2COCH3 and 2 · COCH3) and
1.15 (t, 3H, SCH2CH3); MALDI-TOF MS: m=z 1295.3
(M+Naþ) and 1311.0 (M+Kþ). Anal. Calcd for
C71H82O19S (1271.5): C, 67.07; H, 6.50. Found: C, 67.28;
H, 6.44.
Compound 11 (400 mg, 0.49 mmol) was dissolved in dry
CH2Cl2 (4 mL) and levulinic acid (100 lL, 0.98 mmol),
DCC (202 mg, 0.98 mmol), and DMAP (6 mg, 50 lmol)
were added, and the reaction was stirred at room tem-
perature for 2 h. Dilution with DCM (20 mL) was fol-
lowed by washing with a satd aq NaHCO3 (25 mL), the
CH2Cl2 phase was dried (MgSO4), filtered and concen-

188
G. M. Watt, G.-J. Boons / Carbohydrate Research 339 (2004) 181–193
0
0
4.8. Ethyl 2-O-acetyl-3,4,6-tri-O-benzyl-a-
D
-mannopyr-
-manno-
8.3 Hz, H-300), 4.38 (dd, 1H, J2 –3 10.7 Hz, H-20), 4.31 (m,
4H, H-2, H-3, H-4, and H-40), 4.1100(00m, 3H, H-3000, H-5000,
and H-30000 ), 4.00 (m, 7H, H-4000, H-4 , H-6b000, H-6b0000 , H-
6b0, H-6a0, and CH2CH2TMS), 3.85 (m, 4H, H-300, H-
00
anosyl-(1!6)-[2-O-acetyl-3,4,6-tri-O-benzyl-a-
D
pyranosyl-(1!3)-]-4-O-acetyl-2-O-levulonyl-1-thio-b-
D-
glucopyranoside (15)
50000 , H-6a000, and H-6a0000 ), 3.82 (dd, 1H, J500
3.9, J6a00
–6b
–6b00
Compound 14 (172 mg, 0.14 mmol) was treated with
pyridine (1 mL) and Ac2O (0.5 mL) for 18 h, after which
time the reaction was concentrated in vacuo and the
residue was subjected to flash silica gel column chro-
matography (eluent: 4:1 toluene–EtOAc) to afford 15
11.2 Hz, H-6b00), 3.77 (m, 1H, H-5), 3.72 (dd, 1H, J5
00 –6a00
3.4 Hz, H-6a00), 3.67 (m, 1H, H-6b), 3.52 (m, 3H, H-6a,
H-50 and CH2CH2TMS), 3.46 (m, 1H, H-500), 2.96 (m,
1H, CH2CH2COCH3), 2.72 (m, 2H, CH2CH2COCH3),
2.60 (m, 1H, CH2CH2COCH3), 2.38, 2.35, and 2.18(·2)
(4 · s, 4 · 3H, CH2CH2COCH3 and 3 · COCH3), 0.90
(m, 2H, CH2CH2TMS), and 0.00 (s, 9H, TMS); MAL-
DI-TOF MS: m=z 2335.1 (M+Naþ) and 2351.9
(M+Kþ).
1
(173 mg, 98%) as syrup. H NMR (CDCl3, 500 MHz): d
00 –200
00 –300
2.1, J2
7.40–7.04 (m, 30H, Ar), 5.33 (dd, 1H, J1
2.9 Hz, H-200), 5.11 (dd, 1H, J1 –2 1.5, J2 –3 2.9 Hz, H-20),
5.02 (d, 1H, H-10), 5.01 (dd, 1H, J3ꢀ4 9.3, J4ꢀ5 9.8 Hz, H-
4), 4.94 (dd, 1H, J1–2 9.7, J2–3 9.3 Hz, H-2), 4.84 (d, 1H,
JAB 10.7 Hz, CH2Ph), 4.82 (d, 1H, H-100), 4.80 (d, 1H,
JAB 11.2 Hz, CH2Ph), 4.79 (d, 1H, JAB 11.2 Hz, CH2Ph),
4.70–4.60 (m, 4H, 4 · CH2Ph), 4.54 (d, 1H, JAB 11.2 Hz,
CH2Ph), 4.49 (m, 3H, 3 · CH2Ph), 4.41 (d, 1H, JAB
0
0
0
0
4.10. Trimethylsilylethyl 2-O-acetyl-3,4,6-tri-O-benzyl-a-
D
-mannopyranosyl-(1!6)-[2-O-acetyl-3,4,6-tri-O-benzyl-
a-
-mannopyranosyl-(1!3)-]-2,4-di-O-acetyl-b-
mannopyranosyl-(1!4)-3,6-di-O-benzyl-2-deoxy-2-
phthalamido-b-
-glucopyranosyl-(1!4)-3,6-di-O-benzyl-
2-deoxy-2-phthalamido-b- -glucopyranoside (19)
D
D-
00 –400
10.7 Hz, CH2Ph), 4.33 (d, 1H, H-1), 3.93 (dd, 1H, J3
D
9.3 Hz, H-300), 3.88 (m, 4H, H-3, H-30, H-40, and H-400),
D
3.80 (m, 1H, H-500), 3.75 (m, 4H, H-50, H-6b0, H-6b00, and
00
00 –6a00
H-6b), 3.68 (dd, 1H, J5
1.5 and J6a00
10.3 Hz, H-
A solution of compound 16 (121 mg, 52 lmol) in MeOH
(2 mL) containing hydrazine acetate (12 mg, 124 lmol)
was stirred at room temperature for 1 h, then at 55 ꢁC
for 20 h. The reaction was concentrated in vacuo and
partitioned between CH2Cl2 (20 mL) and water (20 mL),
and the organic phase was dried (MgSO4), filtered and
concentrated in vacuo. Flash silica gel column chro-
matography (eluent: 4:1 toluene–EtOAc) of the residue
–6b
6a00), 3.62 (m, 1H, H-6a0), 3.51 (m, 2H, H-5 and H-6a),
2.59 (m, 6H, CH2CH2COCH3 and SCH2CH3), 2.13,
2.12, 2.11, and 1.97 (4 · s, 4 · 3H, CH2CH2COCH3 and
3 · COCH3) and 1.14 (t, 3H, SCH2CH3); MALDI-TOF
MS: m=z 1335.9 (M+Naþ) and 1352.1 (M+Kþ). Anal.
Calcd for C73H84O19S (1312.5): C, 66.75; H, 6.45.
Found: C, 66.48; H, 6.54.
1
afforded 17 (108 mg, 94%) as a white foam. H NMR
(CDCl3, 500 MHz): d 8.00–6.90 (m, 58H, Ar), 5.46 (d,
4.9. Trimethylsilylethyl 2-O-acetyl-3,4,6-tri-O-benzyl-a-
0000
1H, J1 –2 8.8 Hz, H-10), 5.44 (m, 1H, H-2 ), 5.34 (m, 1H,
0
0
D
-mannopyranosyl-(1!6)-[2-O-acetyl-3,4,6-tri-O-benzyl-
H-2000), 5.22 (dd, 1H, J3
9.8 and J4
9.3 Hz, H-400),
00 –400
00 –500
a-
b-
D
-mannopyranosyl-(1!3)-]-4-O-acetyl-2-O-levulonyl-
-glucopyranosyl-(1!4)-3,6-di-O-benzyl-2-deoxy-2-
D
5.15 (d, 1H, J1–2 7.8 Hz, H-1), 5.11 (d, 1H, H-1000), 5.06
(m, 3H, 3 · CH2Ph), 5.06 (m, 1H, CH2Ph), 5.00 (m, 1H,
CH2Ph), 4.97 (m, 1H, CH2Ph), 4.96 (s, 1H, H-10000 ), 4.89–
4.62 (m, 14H, 14 · CH2Ph), 4.77 (m, 1H, H-100), 4.55 (dd,
phthalamido-b-
D
-glucopyranosyl-(1!4)-3,6-di-O-benzyl-
D
2-deoxy-2-phthalamido-b- -glucopyranoside (16)
1H, J2 –3 10.3, J3 –4 8.8 Hz, H-30), 4.48 (d, 1H, JAB
9.7 Hz, CH2Ph), 4.31 (m, 6H, H-2, H-3, H-4, H-20, H-40,
0
0
0
0
A cooled (0 ꢁC) and stirred solution of 15 (160 mg,
0.12 mmol) and acceptor 8 (155 mg, 0.15 mmol) in dry
CH2Cl2 (2 mL) containing 4 A molecular sieves (0.1 g)
and H-500), 4.20 (dd, 1H, J2000 2.9 and J3
ꢀ
000 –4000
9.3 Hz, H-
-3000
was treated with N-iodosuccinamide (31 mg, 0.13 mmol)
and trimethylsilyl triflate (3 lL, 12 lmol). Stirring was
continued at 0 ꢁC for 15 min, after which time, the
reaction was subjected to the same workup as described
for the preparation of compound 7 to afford 16 (171 mg,
3000), 4.15 (s, 1H, 200-OH), 4.11 (m, 1H, H-6b0), 4.07 (m,
2H, H-4000 and H-30000 ), 4.00 (t, 1H, J 9.3 Hz, H-40000 ), 3.87
(m, 7H, H-50000 , H-6b000, H-6b0000 , H-6b00, H-6a0,
CH2CH2TMS, and H-6a0000 ), 3.72 (m, 3H, H-6b, H-6a00,
and H-6a000), 3.64 (m, 2H, H-200 and H-300), 3.52 (m, 5H,
H-5, H-6a, H-50, H-500, and CH2CH2TMS), 2.38 (·2)
and 2.18 (3 · s, 3 · 3H, 3 · COCH3), 0.90 (m, 2H,
CH2CH2TMS), and 0.00 (s, 9H, TMS); MALDI-TOF
MS: m=z 2237.2 (M+Naþ) and 2253.1 (M+Kþ). The
foregoing compound 17 (108 mg, 49 lmol) in CH2Cl2
(2 mL) was treated with pyridine (130 lL) and triflic
anhydride (110 lL) at 0 ꢁC. The reaction was stirred at
this temperature for 2 h, after which time it was diluted
with CH2Cl2 (50 mL), washed with satd aq NaHCO3
(50 mL), dried (MgSO4), filtered and concentrated in
1
65%) as a white foam. H NMR (CDCl3, 500 MHz): d
8.00–6.90 (m, 58H, Ar), 5.49 (d, 1H, J1 –2 7.8 Hz, H-10),
0
0
5.43 (dd, 1H, J1
2.0, J2
4.9 Hz, H-20000 ), 5.33 (dd,
0000 –20000
0000 –30000
1H, J1
9.8 and J4
2.0, J2
00 –500
2.4 Hz, H-2000), 5.30 (dd, 1H, J3
000 –3000 00 –400
000 –2000
9.3 Hz, H-400), 5.18 (d, 1H, H-1000), 5.17 (m,
2H, H-200 and H-1), 5.11 (d, 1H, JAB 13.2 Hz, CH2Ph),
5.04 (d, 1H, JAB 10.7 Hz, CH2Ph), 5.02 (d, 1H, JAB
11.2 Hz, CH2Ph), 4.96 (m, 1H, CH2Ph), 4.94 (s, 1H, H-
10000 ), 4.83 (m, 3H, 3 · CH2Ph), 4.75–4.56 (m, 13H,
13 · CH2Ph), 4.72 (m, 1H, H-100), 4.45 (dd, 1H, J2
00 –300

G. M. Watt, G.-J. Boons / Carbohydrate Research 339 (2004) 181–193
189
9.7, J3 –4 10.1 Hz, H-30), 5.07 (t, 1H, J 8.8 Hz, H-400), 5.05
(dd, 1H, J2–3 8.8, J3ꢀ4 8.4 Hz, H-3), 4.99 (m, 2H, H-1000
and H-2000), 4.83 (s, 1H, H-10000 ), 4.65 (s, 1H, H-100), 4.47
0
0
vacuo. The residual 18 was taken up in dry toluene
(4 mL) and treated with Bu4NOAc (100 mg, 0.33 mmol)
and the resulting suspension was subjected to sonication
at room temperature under argon for 20 h. The reaction
was subjected directly to flash-column chromatography
(eluent: 9:1 toluene–EtOAc) affording 19 (98 mg, 89%)
(d, 1H, J1 –2 8.4 Hz, H-10), 4.44 (d, 1H, J1–2 7.5 Hz, H-1),
0
0
4.39 (dd, 1H, J50–6b 3.5, J6a0–6b 12.9 Hz, H-6b0), 4.34 (dd,
0
0
000
1H, J5000
1.9, J6a000
11.0 Hz, H-6b000), 4.31 (m, 1H,
000
–6b
–6b
H-6b0000 ), 4.29 (m, 2H, H-6b and H-6a000), 4.24 (m, 1H, H-
1
as a glass-like foam. H NMR (CDCl3, 500 MHz): d
8.00–6.90 (m, 58H, Ar), 5.54 (d, 1H, J2
2.9 Hz, H-200),
6a0), 4.20 (dd, 1H, J5
11.5 Hz, H-6a0000 ),
00 –300
0000 –6a0000
2.0, J6a0000
0000
–6b
5.45 (m, 1H, H-20000 ), 5.36 (m, 2H, H-10 and H-400), 5.27
(m, 1H, H-2000), 5.09 (d, 1H, J1–2 7.8 Hz, H-1), 5.08 (d,
1H, H-1000), 5.03–4.94 (m, 3H, 3 · CH2Ph), 4.96 (s, 1H,
H-10000 ), 4.87 (s, 1H, H-100), 4.84 (d, 1H, JAB 12.2 Hz,
CH2Ph), 4.79–4.58 (m, 15H, 15 · CH2Ph), 4.54 (d, 1H,
4.16 (dd, 1H, J5–6a 2.2, J6a–6b 12.3 Hz, H-6a), 4.09 (m, 1H,
H-5000), 4.03 (m, 1H, H-50000 ), 3.99 (ddd, 1H, H-2), 3.90 (m,
2H, H-20 and CH2CH2TMS), 3.89 (m, 1H, H-300), 3.87
(dd, 1H, J4 –5 9.7 Hz, H-40), 3.83 (m, 1H, H-6b00), 3.77
0
0
(dd, 1H, J4–5 7.9 Hz, H-4), 3.63 (m, 2H, H-5 and H-50),
00
00 –6a00
0
0
0
0
JAB 10.7 Hz, CH2Ph), 4.37 (dd, 1H, J2 –3 10.4, J3 –4
3.60 (dd, 1H, J5
3.1, J6a00
11.0 Hz, H-6a00), 3.53
–6b
8.8 Hz, H-30), 4.27 (m, 5H, H-2, H-3, H-4, H-20, and 0H000 -
40), 4.09 (m, 2H, H-4000 and H-5000), 4.05 (dd, 1H, H-3 ),
3.95 (m, 4H, H-3000, H-40000 , H-6b000, and CH2CH2TMS),
(m, 2H, H-500 and CH2CH2TMS), 2.40–180 (16 · s,
16 · 3H, 16 · COCH3), 0.90 (m, 2H, CH2CH2TMS), and
0.00 (s, 9H, TMS); MALDI-TOF MS: m=z 1622.6
(M+Naþ) and 1638.8 (M+Kþ).
3.92 (dd, 1H, J50000
4.4, J6a0000
10.8 Hz, H-6b0000 ), 3.86
0000
0000
–6b
–6b
(m, 2H, H-50000 and H-6a000), 3.77 (m, 4H, H-300, H-6b0, H-
6b00, and H-6a0000 ), 3.72 (dd, 1H, J5
4.1, J6a00
–6b
00 –6a00
00
11.2 Hz, H-6a00), 3.65 (m, 2H, H-6b and H-6a0), 3.59 (m,
2H, H-6a and CH2CH2TMS), 3.39 (m, 3H, H-5, H-50,
H-500), 2.30, 2.27, 2.26, and 2.07 (4 · s, 4 · 3H,
4 · COCH3), 0.90 (m, 2H, CH2CH2TMS), and 0.00 (s,
9H, TMS); MALDI-TOF MS: m=z 2278.8 (M+Naþ)
and 2295.0 (M+Kþ).
4.12. 2,3,4,6-Tetra-O-acetyl-a-
(1!6)-[2,3,4,6-tetra-O-acetyl-a-
(1!3)]-2,4-di-O-acetyl-b- -mannopyranosyl-(1!4)-2-
acetamido-3,6-di-O-acetyl-2-deoxy-b- -glucopyranosyl-
(1!4)-2-acetamido-1-S-acetyl-3,6-di-O-acetyl-2-deoxy-
1-thio- -glucopyranose (23)
D
-mannopyranosyl-
D
-mannopyranosyl-
D
D
D
Compound 20 (41 mg, 26 lmol) in CH2Cl2 (0.5 mL) was
treated with CF3CO2H (1 mL) for 45 min at room tem-
perature. The mixture was concentrated in vacuo, and
the crude hemiacetal 21 was taken up in 1:1 anhyd
CH2Cl2–toluene (0.5 mL) and treated with thionyl
chloride (1 mL) at room temperature for 2 h. After re-
moval of the solvent and reagent in vacuo, the resulting
a-glycosyl chloride 22 was treated with dry acetone
(3 mL) containing potassium thioacetate (5.8 mg,
51 lmol). The resulting mixture was stirred at room
temperature under argon for 18 h, and the solvent was
removed under reduced pressure. Flash silica gel column
chromatography of the residue using a 1–6% gradient of
MeOH in CH2Cl2 afforded 23 (16.3 mg, 41%). 1H NMR
(CDCl3, 500 MHz): d 5.94 (d, 1H, J2-NH 8.8 Hz, 2-NH),
4.11. Trimethylsilylethyl 2,3,4,6-tetra-O-acetyl-a-
mannopyranosyl-(1!6)-[2,3,4,6-tetra-O-acetyl-a-
-mannopyr-
D-
D
-
mannopyranosyl-(1!3)-]-2,4-di-O-acetyl-b-
D
anosyl-(1!4)-2-acetamido-3,6-di-O-acetyl-2-deoxy-b-
D-
glucopyranosyl-(1!4)-2-acetamido-3,6-di-O-acetyl-2-
deoxy-D-glucopyranose (20)
A stirred solution of compound 19 (98 mg, 43 lmol) in
1-butanol (12 mL) under argon was treated with ethy-
lenediamine (2.40 mL) at 80 ꢁC for 15 h. After 1 h at
room temperature, the reaction mixture was concen-
trated in vacuo and co-evaporated with toluene three
times. The resulting residue was taken up in pyridine
(8 mL) and Ac2O (4 mL) and after 48 h at room tem-
perature the reaction was concentrated in vacuo. To the
residue in 95% EtOH (25 mL) was added 10% palla-
dium-on-charcoal (200 mg) and the resulting suspension
stirred was vigorously under a slight over pressure of
hydrogen gas for 48 h. After filtration through a plug of
Celite, the filtrate was concentrated in vacuo and treated
with pyridine (8 mL) containing Ac2O (4 mL). After
48 h, the reaction was concentrated in vacuo and flash silica
gel column chromatography (eluent: MeOH gradient in
CH2Cl2) of the residue gave 20 (50 mg, 73%) as a syrup.
1H NMR (CDCl3, 500 MHz): d 6.05 (d, 1H, J2-NH
5.64 (d, 1H, J2 -NH 9.8 Hz, 20-NH), 5.31 (t, 1H, J 9.8 Hz,
0
H-40000 ), 5.30 (m, 1H, H-200), 5.25 (t, 1H, J 10.3 Hz, H-4000),
5.20 (m, 1H, H-20000 ) 5.15 (dd, 1H, J2
2.9 Hz, H-30000 ),
0000 –30000
5.06 (m, 2H, H-30 and H-3000), 4.99 (t, 1H, J3
00 –400
00 –500
9.3, J4
11.2 Hz, H-400), 4.97 (d, 1H, J1–2 8.8 Hz, H-1), 4.91 (m,
3H, H-3, H-1000, and H-2000), 4.75 (s, 1H, H-10000 ), 4.57 (s,
1H, H-100), 4.39 (d, 1H, J1 –2 8.3 Hz, H-10), 4.32 (dd, 1H,
0
0
J50–6b 3.9, J6a0–6b 12.20H000 z, H-6b0), 4.20 (m, 4H, H-2, H-
0
0
6b, H-6b000, and H-6b ), 4.16 (m, 1H, H-6a0), 4.12 (dd,
1H, J5
12.7 Hz, 6a000), 4.08 (dd, 1H,
000
000 –6a000
2.4, J6a000
–6b
9.8 Hz, 2-NH), 5.65 (d, 1H, J2 -NH 8.8 Hz, 20-NH), 5.38
0000
0000 –6a0000
0
J5
2.0, J6a0000
12.2 Hz, H-6a0000 ), 4.01 (m, 2H, H-6a
–6b
4.4 Hz, H-50000 ), 3.81
(m, 1H, H-40000 ), 5.35 (m, 1H, H-200), 5.33 (t, 1H, J
and H-5000), 3.95 (ddd, 1H, J5
0000 –6a0000
10.1 Hz, H-4000), 5.27 (m, 2H, H-20000 and H-30000 ), 5.15 (dd,
(m, 2H, H-20 and H-300), 3.76 (m, 2H, H-40 and H-6b00),
3.71 (m, 1H, H-4), 3.64 (m, 1H, H-5), 3.52 (m, 2H, H-50
10.1 Hz, H-3000), 5.11 (dd, 1H, J2 –3
000 –3000
000 –4000
0
0
1H, J2
3.1, J3

190
G. M. Watt, G.-J. Boons / Carbohydrate Research 339 (2004) 181–193
and H-6a00), 3.47 (m, 1H, H-500), 2.28 (s, 3H, COCH3),
and 2.10–1.85 (m, 16 · 3H, 16 · COCH3); MALDI-TOF
MS: m=z 1580.5 (M+Naþ) and 1596.5 (M+Kþ).
(M+Naþ) and 1629.4 (M+Kþ). Anal. Calcd for
C93H98N2O20Si (1591.8): C, 70.17; H, 6.21; N, 1.76.
Found: C, 70.02; H, 6.17; N, 1.75.
4.13. a-
(1!3)]-b-
deoxy-b-
b-1-thio-
D
-Mannopyranosyl-(1!6)-[a-
D-mannopyranosyl-
-mannopyranosyl-(1!4)-2-acetamido-2-
4.15. Trimethylsilylethyl 2-O-acetyl-3,4,6-tri-O-benzyl-b-
D
D
-mannopyranosyl-(1!4)-3,6-di-O-benzyl-2-deoxy-2-
phthalamido-b-
-glucopyranosyl-(1!4)-3,6-di-O-benzyl-
2-deoxy-2-phthalamido-b- -glucopyranoside (28)
D
-glucopyranosyl-(1!4)-2-acetamido-2-deoxy-
D
D
-glucopyranose (3)
D
Compound 23 (3.0 mg, 1.9 lmol) was treated with a 1 M
solution NaOMe in MeOH (2 mL) at 0 ꢁC for 30 min.
After neutralization of the reaction mixture with Am-
berlite 650C ion-exchange resin, the solvent was re-
moved in vacuo to give 3 (1.7 mg) in 96% yield. 1H
NMR (D2O, 500 MHz): d 4.92 (s, 1H, H-1000), 4.72 (s,
1H, H-10000 ), 4.61 (s, 1H, H-100), 4.52 (d, 1H, J1–2 11.0 Hz,
A solution of compound 25 (1.20 g, 0.754 mmol) in
MeOH (20 mL) was treated with hydrazine acetate
(83 mg, 905 mmol) at room temperature for 2 h. The
reaction was concentrated in vacuo and partitioned be-
tween CH2Cl2 (100 mL) and H2O (100 mL), and the
organic phase was dried (MgSO4), filtered and concen-
trated in vacuo. Flash silica gel column chromatography
(eluent: 9:1 toluene–EtOAc) of the residue afforded 26
(900 mg, 80%) as a white foam. 1H NMR (CDCl3,
H-1), 4.44 (d, 1H, J1 –2 7.7 Hz, H-10), 4.08 (d, 1H, J2
00 –300
0
0
3.4 Hz, H-200), 3.89 (m, 1H, H-2000), 3.79 (m, 1H, H-20000 ),
3.78–3.66 (m, 8H), 3.66–3.51 (m, 10H), 3.51–3.42 (m,
6H), 3.42–3.30 (m, 3H), 1.92 and 1.89 (2 · s, 6H,
2 · COCH3); MALDI-TOF MS: m=z 949.9 (M+Naþ)
and m=z 965.8 (M+Kþ); HRMS (m=z): Calcd for
C34H58N2NaO25S 949.2898; found m=z 949.2947.
0
0
600 MHz): d 8.02–6.90 (m, 43H, Ar), 5.45 (d, 1H, J1 –2
8.4 Hz, H-10), 5.15 (d, 1H, J1–2 7.9 Hz, H-1), 5.12 (d, 1H,
JAB 11.0 Hz, CH2Ph), 5.12 (d, 1H, JAB 13.2 Hz, CH2Ph),
5.04 (d, 1H, JAB 12.8 Hz, CH2Ph), 5.00 (d, 1H, JAB
11.0 Hz, CH2Ph), 4.99 (d, 1H, JAB 11.0 Hz, CH2Ph), 4.80
7.7 Hz, H-100), 4.78 (d, 1H, JAB 12.4 Hz,
00 –200
(d, 1H, J1
CH2Ph), 4.75 (d, 1H, JAB 12.1 Hz, CH2Ph), 4.71 (d, 1H,
JAB 11.3 Hz, CH2Ph), 4.71 (d, 1H, JAB 11.3 Hz, CH2Ph),
4.70 (d, 1H, JAB 12.5 Hz, CH2Ph), 4.65 (d, 1H, JAB
12.8 Hz, CH2Ph), 4.65 (d, 1H, JAB 12.8 Hz, CH2Ph), 4.62
4.14. Trimethylsilylethyl 3,4,6-tri-O-benzyl-2-O-levulin-
oyl-b-
2-phthalamido-b-
zyl-2-deoxy-2-phthalamido-b-
D
-glucopyranosyl-(1!4)-3,6-di-O-benzyl-2-deoxy-
-glucopyranosyl-(1!4)-3,6-di-O-ben-
-glucopyranoside (25)
D
D
0
0
(d, 1H, JAB 11.0 Hz, CH2Ph), 4.61 (dd, 1H, J2 –3 10.6,
J3 –4 8.4 Hz, H-30), 4.60 (d, 1H, JAB 12.5 Hz, CH2Ph),
0
0
To a solution of compound 24 (700 mg, 1.18 mmol) and
disaccharide acceptor 8 (1.04 g, 0.98 mmol) in dry
ꢀ
CH2Cl2 (9 mL) was added activated 4 A molecular sieves
4.41 (m, 2H, H-20 and H-3), 4.25 (dd, 1H, J3 –4 8.4, J4 –5
0
0
0
0
9.9 Hz, H-40), 4.12 (m, 2H, H-2 and H-4), 4.02 (dd, 1H,
00
00
J500
1.9, J6a00
–6b
11.7 Hz, H-6b00), 3.97 (m, 1H,
1.8 Hz, H-6a00), 3.82
(1 g). After stirring at room temperature for 30 min un-
der argon, the mixture was cooled (0 ꢁC) and N-iodo-
succinamide (3.75 g, 16.68 mmol) and trimethylsilyl
triflate (252 lL, 1.28 mmol) were added. Stirring was
continued at 0 ꢁC for 15 min, at the end of which time
the reaction was subjected to the same workup as de-
scribed for the preparation of compound 7 to afford 25
(1.27 g, 81%) as a white foam. 1H NMR (CDCl3,
–6b
00 –6a00
CH2CH2TMS), 3.86 (dd, 1H, J5
(m, 1H, H-6b0), 3.80 (m, 1H, H-400), 3.76 (dd, 1H, J5 –6a
0
0
4.4, J6a0–6b 10.6 Hz, H-6a0), 3.74 (dd, 1H, J5–6b 1.1, J6a–6b
0
11.0 Hz, H-6b), 3.71 (m, 1H, H-200), 3.68 (dd, 1H, J2
00 –300
00 –400
8.4, J3
9.2 Hz, H-300), 3.60 (dd, 1H, J5–6a 3.7 Hz, H-
6a), 3.52 (m, 3H, CH2CH2TMS, H-50, and H-500), 3.48
(m, 1H, H-5), 0.85 (m, 2H, CH2CH2TMS), and 0.00 (s,
9H, TMS); MALDI-TOF MS: m=z 1516.1 (M+Naþ)
and 1532.2 (M+Kþ). The foregoing compound 26
(565 mg, 0.38 mmol) was taken up in CH2Cl2 (8 mL) and
cooled to 0 ꢁC before pyridine (91 lL, 1.13 mmol) and
triflic anhydride (95 lL, 0.57 mmol) were added as de-
scribed for compound 19. The reaction was stirred at
this temperature for 2 h, after which time it was diluted
with CH2Cl2 (50 mL), washed with satd aq NaHCO3
(50 mL), dried (MgSO4), filtered and concentrated in
vacuo. The residual 27 was taken up in dry toluene
(18 mL) and Bu4NOAc (684 mg, 2.27 mmol) was added,
and the resulting suspension was subjected to sonication
at room temperature under argon for 20 h. The reaction
was subjected directly to flash column chromatography
(eluent: 9:1 toluene–EtOAc) affording 28 (477 mg, 82%)
0
0
300 MHz): d 8.08–6.90 (m, 43H, Ar), 5.48 (d, 1H, J1 –2
9.2 Hz, H-
7.9 Hz, H-10), 5.22 (dd, 1H, J1
00 –200
00 –300
8.4, J2
300), 5.15 (d, 1H, J1–2 7.9 Hz, H-1), 5.09 (m, 2H,
2 · CH2Ph), 4.96 (m, 2H, 2 · CH2Ph), 4.89 (m, 2H,
2 · CH2Ph), 4.78 (m, 2H, H-100 and CH2Ph), 4.75–4.60
0
0
0
0
(m, 7H, 7 · CH2Ph), 4.48 (dd, 1H, J2 –3 10.6, J3 –4 7.9 Hz,
H-30), 4.41 (dd, 1H, H-20), 4.31 (m, 4H, H-2, H-3, H-4,
and H-40), 3.93 (m, 5H, CH2CH2TMS, H-400, H-6b0, H-
6b00, and H-6a00), 3.81 (dd, 1H, J5 –6a 4.0, J6a0–6b 11.0 Hz,
0
0
0
H-6a0), 3.76 (dd, 1H, J3 8.8 Hz, H-200), 3.70 (m, 1H,
00 –400
H-6b), 3.64–3.49 (m, 5H, CH2CH2TMS, H-5, H-50,
H-500, and H-6a), 2.90 (m, 2H, COCH2CH2COCH3),
2.60 (m, 2H, COCH2CH2COCH3), 2.35 (s, 3H,
COCH2CH2COCH3), 0.90 (m, 2H, CH2CH2TMS), and
0.00 (s, 9H, TMS); MALDI-TOF MS: m=z 1613.3

G. M. Watt, G.-J. Boons / Carbohydrate Research 339 (2004) 181–193
191
1
as a white foam. H NMR (CDCl3, 600 MHz): d 8.02–
6.90 (m, 43H, Ar), 5.68 (d, 1H, J2
3.0 Hz, H-200), 5.45
4.17. 2,3,4,6-Tetra-O-acetyl-b-
2-acetamido-3,6-di-O-acetyl-2-deoxy-b-D-glucopyrano-
D
-mannopyranosyl-(1!4)-
00 –300
(d, 1H, J1 –2 8.8 Hz, H-10), 5.19 (d, 1H, JAB 12.7 Hz,
CH2Ph), 5.17 (d, 1H, J1–2 8.3 Hz, H-1), 5.04 (d, 1H, JAB
13.7 Hz, CH2Ph), 5.02 (d, 1H, JAB 11.2 Hz, CH2Ph), 4.90
(s, 1H, H-100), 4.84 (d, 1H, JAB 11.2 Hz, CH2Ph), 4.84 (d,
1H, JAB 12.2 Hz, CH2Ph), 4.73–4.64 (m, 8H, 8 · CH2Ph),
syl-(1!4)-2-acetamido-1-S-acetyl-3,6-di-O-acetyl-2-
0
0
deoxy-1-thio-D-glucopyranose (32)
Compound 29 (55 mg, 54 lmol) in CH2Cl2 (0.5 mL) was
treated with CF3CO2H (1 mL) for 45 min at room
temperature. After concentration in vacuo, crude 30
was dissolved in 1:1 anhyd CH2Cl2–anhyd toluene
(0.6 mL) and treated with thionyl chloride (0.8 mL)
as described for 23. Removal of the solvent and reagent
under reduced pressure gave the a-glycosyl chloride 31,
which was subsequently dissolved in dry acetone (2 mL)
containing potassium thioacetate (8.6 mg, 76 lmol) as
0
0
4.56 (d, 1H, JAB 11.2 Hz, CH2Ph), 4.50 (dd, 1H, J2 –3
10.7, J3 –4 8.3 Hz, H-30), 4.40 (dd, 1H, H-20), 4.37 (m,
0
0
1H, H-3), 4.35 (dd, 1H, J3 –4 8.8, J4 –5 9.8 Hz, H-40), 4.10
(m, 2H, H-2 and H-4), 3.97 (m, 1H, CH2CH2TMS), 3.95
0
0
0
0
9.8 Hz, H-400), 3.90–3.80 (m,
00 –500
00 –400
(dd, 1H, J3
9.3, J4
4H, H-6b00, H-6b0, H-6a0, and H-6b), 3.74 (m, 1H, H-6a),
3.65 (dd, 1H, H-300), 3.60 (dd, 1H, J5
3.9, J6a00
–6b
00
00 –6a00
1
10.7 Hz, H-6a00), 3.56–3.47 (m, 4H, CH2CH2TMS, H-500,
H-50, and H-5), 2.39 (s, 3H, COCH3), 0.90 (m, 2H,
CH2CH2TMS), and 0.00 (s, 9H, TMS); MALDI-TOF
MS: m=z 1558.0 (M+Naþ) and 1574.2 (M+Kþ). Anal.
Calcd for C90H94N2O19Si (1535.8): C, 70.38; H, 6.17; N,
1.82. Found: C, 70.05; H, 6.19; N, 1.70.
described for 23 to afford 32 (22 mg, 41%). H NMR
0
(CDCl3, 500 MHz): d 5.88 (d, 1H, J20-NH 8.8 Hz,
NHAc0), 5.75 (d, 1H, J2-NH 9.8 Hz, NHAc), 5.32 (d, 1H,
3.4 Hz, H-200), 5.13 (t, 1H, J 9.8 Hz, H-400), 4.98 (m,
00 –300
J2
3H, H-3, H-30, and H-1), 4.92 (dd, 1H, H-300), 4.60 (s,
1H, H-100), 4.33 (d, 1H, J1 –2 7.8 Hz, H-10), 4.27–4.17 (m,
0
0
6H, H-2, H-6b00, H-6b0, H-6b, H-6a0, and H-6a), 4.04
00
00 –6a00
(dd, 1H, J5
2.4, J6a00
12.2 Hz, H-6a00), 3.87 (ddd,
–6b
1H, J2 –3 8.8 Hz, H-20), 3.75 (t, 1H, J 9.3 Hz, H-40), 3.67
4.16. Trimethylsilylethyl 2,3,4,6-tetra-O-acetyl-b-
mannopyranosyl-(1!4)-2-acetamido-3,6-di-O-acetyl-2-
deoxy-b-
-glucopyranosyl-(1!4)-2-acetamido-3,6-di-O-
acetyl-2-deoxy- -glucopyranose (29)
D
-
0
0
00
(m, 2H, H-4 and H-5), 3.55 (ddd, 1H, J500
4.9 Hz, H-
–6b
500), 3.49 (dt, 1H, J 3.4 Hz, H-50), 2.28, 2.08, 2.06, 2.06,
2.03, 2.02, 1.97, 1.95, 1.91, 1.88, and 1.86 (11 · s, 33H,
11 · COCH3); MALDI-TOF MS: m=z 1002.2 (M+Naþ)
and 1018.1 (M+Kþ).
D
D
A stirred solution of compound 28 (107 mg, 70 lmol) in
1-butanol (14 mL) under argon was treated with ethy-
lenediamine (2.85 mL) as described for the synthesis of
compound 20. The syrupy residue was taken up in 95%
EtOH (18 mL) and 10% palladium-on-charcoal (120 mg)
added. The suspension was stirred vigorously under a
slight over pressure of hydrogen for 48 h, at the end of
which time the mixture was filtered through a plug of
Celite. The filtrate was concentrated in vacuo and dis-
solved in pyridine (5 mL) containing Ac2O (2 mL) and
left to stand at room temperature for 48 h. The reaction
mixture was concentrated in vacuo, and flash silica gel
column chromatography (eluent: MeOH gradient in
4.18. b-
b-
-glucopyranosyl-(1!4)-2-acetamido-2-deoxy-b-1-
thio- -glucopyranose (2)
D
-Mannopyranosyl-(1!4)-2-acetamido-2-deoxy-
D
D
Compound 32 (5.0 mg, 5.1 lmol) was treated with a
0.5 M solution NaOMe in MeOH (1.5 mL) as described
for compound 3 to give 2 (3.0 mg, 100%). 1H NMR
(D2O, 500 MHz): d 4.60 (s, 1H, H-100), 4.50 (d, 1H, J1–2
10.3 Hz, H-1), 4.42 (d, 1H, J1 –2 7.6 Hz, H-10), 3.88 (d,
0
0
1H, J2
3.4 Hz, H-200), 3.81 (dd, 1H, J500
2.0, J6a00
0
–6b00
–6b
00 –300
00
12.2 Hz, H-6b00), 3.77 (dd, 1H, J50–6b 2.0, J6a0–6b 12.3 Hz,
H-6b0), 3.71 (dd, 1H, J5–6b 2.0, J6a–6b 12.3 Hz, H-6b),
3.69–362 (m, 5H, H-2, H-20, H-30, H-40, and H-6a0), 3.61
0
1
CH2Cl2) gave 29 (55 mg, 77%) as a syrup. H NMR
0
(CDCl3, 300 MHz): d 6.20 (d, 1H, J20-NH 9.3 Hz,
NHAc0), 5.94 (d, 1H, J2-NH 9.3 Hz, NHAc), 5.37 (d, 1H,
(dd, 1H, J5
6.8 Hz, H-6a00), 3.55–3.50 (m, 4H, H-3,
00 –6a00
3.0 Hz, H-200), 5.18 (t, 1H, J 9.9 Hz, H-400), 5.12 (m,
H-4, H-6a, and H-300), 3.50 (m, 1H, H-50), 3.45 (dd, 1H,
9.8 Hz, H-400), 3.34 (m, 1H, H-5), 3.30
00 –300
J2
2H, H-3 and H-30), 5.00 (dd, 1H, H-300), 4.68 (s, 1H, H-
00 –400
J3
00 –500
9.3, J4
100), 4.53 (d, 1H, J1–2 7.7 Hz, H-1), 4.47 (d, 1H, J1 –2
(m, 1H, H-500), 1.94 and 1.93 (2 · s, 6H, 2 · COCH3);
MALDI-TOF MS: m=z 624.7 (M+Naþ) and 640.7
(M+Kþ); HRMS (m=z): Calcd for C22H38N2NaO15S
625.1855; found m=z 625.1891.
0
0
8.2 Hz, H-10), 4.30 (m, 5H, H-6b00, H-6b0, H-6b, H-6a0,
00
00 –6a00
and H-6a), 4.10 (dd, 1H, J5
2.5, J6a00
12.4 Hz, H-
–6b
6a00), 3.98–3.78 (m, 4H, CH2CH2TMS, H-2, H-20, and
H-4), 3.71 (dd, 1H, J3–4 7.7, J4–5 8.5 Hz, H-4), 3.68–3.48
(m, 4H, CH2CH2TMS, H-5, H-50, and H-500), 2.14, 2.12,
2.11, 2.09, 2.08, 2.03, 2.02, 1.97, 1.94 and 1.93 (10 · s,
30H, 10 · COCH3), 0.90 (m, 2H, CH2CH2TMS), and
0.00 (s, 9H, TMS); MALDI-TOF MS: m=z 1044.9
(M+Naþ) and 1060.8 (M+Kþ); HRMS (m=z): Calcd for
C43H66N2O24 1022.3775; found m=z 1022.3864.
4.19. 2-Acetamido-2-deoxy-b-
D
-glucopyranosyl-(1!4)-2-
acetamido-2-deoxy-1-thio-b-
thione disulfide (33)
D
-glucopyranose-S0-S-gluta-
A solution (20 mL) of 1 (5 mg, 11.0 lM) and glutathione
(1.7 mg, 5.7 lM) in aq 25 mM NH4OAc at pH 8.5 was

192
G. M. Watt, G.-J. Boons / Carbohydrate Research 339 (2004) 181–193
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stirred at room temperature for 18 h. The reaction
mixture was freeze-dried, and the disulfide-linked gly-
copeptide 33 (3.2 mg, 76%) was isolated by P-2 gel fil-
tration (eluent: H2O). 1H NMR (D2O, 500 MHz): d 4.80
(dd, 1H, J 3.9 and 9.8 Hz, CH-Cys), 4.62 (d, 1H, J1;2
10.7 Hz, H-1), 4.58 (d, 1H, J1 ;2 8.9 Hz, H-10), 4.04 (dd,
0
0
0
0
1H, J2;3 9.8 Hz, H-2), 3.91 (dd, 1H, J50;6b 1.9, J6a0;6b
11.7 Hz, H-6b0), 3.86 (dd, 1H, J5;6b 1.9, J6a;6b 11.7, H-6b),
3.69–3.81 (m, 6H, CH2-Gly, CH-Glu, H-20, H-3, and H-
6a0), 3.66 (dd, 1H, J5;6a 5.9 Hz, H-6a), 3.65 (dd, 1H, J3;4
0
0
0
0
8.8, J4;5 9.8 Hz, H-4), 3.55 (dd, 1H, J2 ;3 10.7, J3 ;4 8.8 Hz,
H-30), 3.54 (m, 1H, H-5), 3.50 (m, 1H, H-50), 3.46 (dd,
1H, J4 ;5 9.7 Hz, H-40), 3.36 (dd, 1H, J 3.9 and 14.6 Hz,
CH2-Cys), 2.99 (dd, 1H, J 9.8 and 14.6 Hz, CH2-Cys),
2.52 and 2.15 (2 · m, 4H, 2 · CH2-Glu), 2.06 and 2.01
(2 · s, 6H, 2 · CH3CO); MALDI-TOF MS: m=z 746.2
(M+Hþ), 768.2 (M+Naþ), 784.2 (M+Kþ), and 789.2
(M)Hþ+2Naþ).
0
0
24. Macindoe, W. M.; van Oijen, A.; Boons, G. J. Chem.
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4.20. 2-Acetamido-2-deoxy-b-
D
-glucopyranosyl-(1!4)-2-
acetamido-2-deoxy-1-thio-b-
disulfide (34)
D
-glucopyranose-S0,S-BSA
28. Paulsen, H. Angew. Chem., Int. Ed. Engl. 1982, 21, 155–
173.
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A solution of BSA (15 lM) and 2-acetamido-2-deoxy-b-
D
-glucopyranosyl-(1!4)-2-acetamido-2-deoxy-1-thio-b-
D
-glucopyranose (1, 750 lM) in 0.25 M NH4OAc at
pH 8.5 (1 mL) was stirred at 30 ꢁC. After 24 h the free
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and subjected to ultrafiltration using Millipore Centri-
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and dissolved in water (300 lL) to give neoglycoprotein
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sinapinic acid as matrix); 34: m=z 67,089 (BSA: m=z
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