Pre-activation-based one-pot synthesis of an α-(2,3)-sialylated core-fucosylated complex type bi-antennary N-glycan dodecasaccharide

Article abstract of DOI:10.1002/chem.200800757

Synthesis of N-glycans is of high current interests due to their important biological properties. A highly efficient convergent strategy based on the pre-activation method for assembly of the complex type core fucosylated bi-antennary N-glycan dodecasaccharide has been developed. Retrosynthetically, this extremely challenging target is broken down to three modules: a sialyl disaccharide, a glucosamine building block and a hexasaccharide diol acceptor. The sialyl disaccharide was easily obtained by selective activation of a new 5-N-trichloroacetyl protected sialyl donor in the presence of a thiogalactoside acceptor. The hexasaccharide diol module was produced by double mannosylation of a fucosylated tetrasaccharide acceptor, which in turn was generated by glycosylation of a α-fucosylated disaccharide with a β-man-nose containing disaccharide donor. The union of the three modules was performed in one-pot giving the fully protected dodecasaccharide in high yield. This synthesis is characterized by minimum protective group and aglycon adjustment on oligosaccharide intermediates, thus greatly enhancing the overall synthetic efficiency. The modular feature of this strategy suggests that this method can be readily adapted to the synthesis of a wide variety of N-glycan structures.

Full text of DOI:10.1002/chem.200800757

DOI: 10.1002/chem.200800757
Pre-Activation-Based One-Pot Synthesis ofan a-(2,3)-Sialylated Core-
Fucosylated Complex Type Bi-Antennary N-Glycan Dodecasaccharide
Bin Sun,[a, b] Balasubramanian Srinivasan,[a] and Xuefei Huang*[a, b]
Dedicated to Professor Chi-Huey Wong on the occasion of his 60th birthday
Abstract: Synthesis of N-glycans is of
high current interests due to their im-
portant biological properties. A highly
efficient convergent strategy based on
the pre-activation method for assembly
of the complex type core fucosylated
bi-antennary N-glycan dodecasacchar-
ide has been developed. Retrosyntheti-
cally, this extremely challenging target
is broken down to three modules: a
was easily obtained by selective activa-
tion of a new 5-N-trichloroacetyl pro-
tected sialyl donor in the presence of a
thiogalactoside acceptor. The hexasac-
charide diol module was produced by
double mannosylation of a fucosylated
tetrasaccharide acceptor, which in turn
was generated by glycosylation of a a-
fucosylated disaccharide with a b-man-
nose containing disaccharide donor.
The union of the three modules was
performed in one-pot giving the fully
protected dodecasaccharide in high
yield. This synthesis is characterized by
minimum protective group and aglycon
adjustment on oligosaccharide inter-
mediates, thus greatly enhancing the
overall synthetic efficiency. The modu-
lar feature of this strategy suggests that
this method can be readily adapted to
the synthesis of a wide variety of N-
glycan structures.
sialyl disaccharide,
a
glucosamine
Keywords: carbohydrates · glycosy-
lation · N-glycans · synthesis design
building block and a hexasaccharide
diol acceptor. The sialyl disaccharide
Introduction
despite intensive studies, detailed structure-function rela-
tionships have not been established in many cases, mainly
due to the difficulties in accessing sufficient quantities of
these glycan structures.
Glycosylation is one of the major types of postsynthetic
modification of mammalian proteins, which include the at-
tachment of oligosaccharides to asparagine (N-glycan) and
to serine or threorine (O-glycan).[1] Unlike protein synthesis,
the addition of carbohydrates to proteins is not under direct
genetic control, which often leads to heterogeneous glycosy-
lation patterns on the same protein backbone. The identities
of carbohydrate moieties can have a profound effect on bio-
logical functions of glycoproteins such as their immunoge-
nicities, stabilities, and affinities to receptors.[1–4] However,
We became interested in the synthesis of a-(2,3)-sialylated
core-fucosylated complex type bi-antennary N-Glycan do-
decasaccharide 1, which is one of the prototypical structures
of mammalian N-glycans.[5] The dodecasaccharide 1 has
been found on alpha fetoprotein (AFP) isolated from pa-
tients having hepatocellular carcinoma, a form of liver
cancer. It is proposed that the structures of N-glycans can
be a much more reliable marker to differentiate benign and
malignant liver diseases.[6,7] Similarly, studies on N-glycans
linked to prostate specific antigen (PSA) suggest that a-
(2,3) linked sialylated N-glycan on PSA can be potentially
used to identify prostate cancer.[8] Dodecasaccharide 1 has
also been found on the surface of erythropoietin.[9] The pres-
ence of fucose and sialic acid moieties in attached carbohy-
drates is believed to be important for the in vivo activity of
erythropoietin.[10] There are intense interests in remodeling
glycoproteins such as erythropoietin with homogeneous car-
bohydrate structures.[11] Thus, the ready availability of do-
decasaccharide 1 can greatly facilitate the exploration of its
glyco-biological functions and biomedical applications.
[a] Dr. B. Sun, Dr. B. Srinivasan, Prof. Dr. X. Huang
Department of Chemistry, The University of Toledo
2801 W. Bancroft Street, MS 602
Toledo, OH 43606 (USA)
[b] Dr. B. Sun, Prof. Dr. X. Huang
Current address:
Department of Chemistry, Michigan State University
East Lansing, MI 48824 (USA)
Fax : (+1)517-353-1793
E-mail: xuefei@chemistry.msu.edu
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200800757.
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FULL PAPER
Although N-glycan struc-
tures can be assembled via sev-
eral approaches,[12–23] total syn-
thesis of dodecasaccharide 1
via chemical methods still
presents
a
daunting task,
which requires the construc-
tion of many difficult glycosyl
linkages such as b-mannose, a-
sialic acid, a-fucose as well as
branching sequences. More-
over, the acid lability of the fu-
cosyl linkage limits the scope
of reagents that can be used.
To date the only chemical total
synthesis of the dodesacchar-
ide in its fully protected form
was accomplished by the Dani-
shefsky group using a variety
of glycosyl donors including
glycals, thioglycosides, glycosyl
sulfoxide, glycosyl fluoride and
glycosyl phosphite. The tour-
de-force total synthesis took
20 steps starting from protect-
ed monosaccharide building
blocks with 13 steps and 3.1%
overall yield for the longest
linear sequence.[11,24] Herein,
we report our studies on an ef-
ficient chemical synthesis of
dodecasaccharide
1 via the
pre-activation based one-pot
method predominantly using
p-tolyl thioglycosides to simpli-
fy building block design.
Results and Discussion
In order to enhance the overall
efficiency, our synthetic se-
quence was designed to mini-
mize protective group manipu-
lations and aglycon adjust-
ments on intermediate oligo-
saccharides while achieving
high stereoselectivities and
yields. Previously, we discov-
Scheme 1. Retrosynthetic analysis of dodecasaccharide 1.
ered that the reaction of a
mannosyl diol acceptor with a
thiomannosyl donor in the absence of a participating protec-
tive group on its 2-O position led to products as an anome-
ric mixture.[26] Therefore, the strategic disconnections be-
tween glycan rings C/D and C’/D’ in dodecasaccharide 2
were chosen, leading to ABC trisaccharide fragment 3 and
the core DD’EFGH hexasaccharide 4 (Scheme 1).
For the installation of DD’ units in 4, to minimize the
steric hindrance to the 3-O position of the E mannosyl unit,
a popular strategy is to introduce the D’ unit first followed
by protective group adjustment and glycosylation on the 6-
O position.[12,27] For higher efficiency, we decided to explore
the possibility of double mannosylation[17,28] of tetrasacchar-
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7073

X. Huang et al.
ide 6 by the 2-O acetylated thi-
omannoside 5. In tetrasacchar-
ide 6, the formation of b-man-
nosyl linkage between ring E
and F is crucial. b-Mannosides
can be accessed via a variety
of methods, including SN2 in-
version of b glucosides[15,18,29]
intramolecular aglycon deliv-
ery,[27,30,31] insoluble silver salt
promoted mannosyl halide re-
action[32] and glycosylation by
benzylidene bearing mannosyl
donors.[33–35] The ground break-
ing work by Crich and co-
workers has demonstrated
high b selectivity can be ob-
tained using benzylidene bear-
ing mannosyl donors,[33–36] al-
though the aglycon of the
product typically needs to be
further modified to convert it
into a suitable donor for subse-
quent glycosylation to extend
the chain.[20]
Our journey commenced
from the preparation of the re-
ducing end disaccharide
through fucosylation of diol
acceptor 8[37] by donor
9
Scheme 2. a) CuBr2, Bu4NBr, CH2Cl2, DMF, 08C ! RT; b) p-TolSCl, CH2Cl2, ꢀ788C; then TTBP, 11;
c) PhBCl2, Et3SiH, CH2Cl2, ꢀ788C; then levulinic acid, EDC, DMAP; d) AgOTf, p-TolSCl, TTBP, 9, CH2Cl2,
ꢀ78 ! 08C; e) hydrazine acetate, CH3OH, CH2Cl2; f) AgOTf, p-TolSCl, CH2Cl2, ꢀ78 ! 08C, then TMSOTf,
08C; g) NaOCH3, CH3OH, RT.
7
(Scheme 2a).[38] Although ex-
clusive a selectivity was ob-
tained when donor 7 reacted
with acceptors containing sec-
ondary hydroxyl groups using
N-iodosuccinimide/triflic acid
or p-TolSCl/AgOTf promoter
systems,[39–41] a:b mixtures
were generated with acceptors
bearing
primary
hydroxyl
groups under these conditions.
In order to enhance the a se-
lectivity, we applied the in situ anomerization protocol
(CuBr2, tetrabutylammonium bromide)[27,28,42] first devel-
oped by Lemieux and co-workers.[43] Satisfactory yield
(75%) was obtained for the desired disaccharide 9, with its
regio- and stereo-chemistry confirmed by NMR analysis.
The formation of the key b mannosyl linkage using the
benzylidene bearing thiomannosyl donor 10 was examined
next. Activation of thiomannosyl donor 10a in the absence
of any acceptor (pre-activation)[44] by p-TolSOTf, formed in
situ through the reaction of p-TolSCl and AgOTf,[44,45] was
followed by addition of thioglycosyl acceptor 11,[46] which
formed disaccharide 12 in 57% yield (82% based on the
amount of donor consumed) with an b/a ratio of 6:1
(Scheme 2b). The aglycon stereochemistry of the donor did
not affect the reaction as donor 10b gave identical result as
10a. The newly formed glycosyl linkage in 12b was con-
firmed by the one bond coupling constant between the
anomeric carbon and proton of the mannose moiety
(1J(C1,H1)=157 Hz).[47] Disaccharide 12b was then directly
A
used as a donor without any aglycon manipulations.
The reaction of disaccharide 12b and 9 promoted by p-
TolSCl/AgOTf produced the desired tetrasaccharide 13 de-
spite the presence of the bulky fucosyl group on the 6-O po-
sition of acceptor 9. However, treatment of 13 with PhBCl2
and Et3SiH for opening the benzylidene group[48] cleaved off
the fucose unit. After several alternative reaction routes in-
cluding postponing the introduction of fucose were ex-
plored, the most efficient way was determined to be adjust-
ing the protective groups on disaccharide 12b. The p-me-
thoxybenzyl (PMB) group in 12b can be oxidatively re-
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Oligosaccharides
FULL PAPER
moved first followed by benzylidene opening.[19,49] We
discovered that mixing PhBCl2 and Et3SiH with 12b
not only regioselectively converted the benzylidene
group to 4-O benzyl but also removed the 3-O-PMB
group simultaneously, leading to a diol which was sub-
sequently protected as levulinoyl ester 14 (Scheme 2c,
77%). Reaction of the levulinoyl disaccharide donor
14 with fucosylated acceptor 9 proceeded smoothly in
86% yield. The two Lev moieties in the tetrasacchar-
ide product 15 were selectively deprotected using hy-
drazine acetate giving diol 6 (Scheme 2c). Double
mannosylation of 6 by donor 5 was explored next with
our p-TolSCl/AgOTf promoter. A product with the
same molecular weight as the desired hexasaccharide
was isolated, which was found to be extremely labile
to acid. NMR studies suggested that it was an or-
thoester. To overcome this problem, TMSOTf[50] was
added at the end of the glycosylation reaction at 08C
to rearrange the orthoester in situ. Gratifyingly, the
desired hexasaccharide 16 was obtained in 77% yield
Table 1. Sialylation results using sialyl donor 17.
Entry
1
Acceptor
Product
Yield [%] (a/b)[a]
68 (a only)
2
3
75 (14:1)
91 (33:1)
4
85 (13:1)
following
TMSOTf
promoted
rearrangement
(Scheme 2d). Hexasaccharide 16 was then de-acetylat-
ed resulting in the core hexasaccharide diol acceptor
4.
Next we focused our attention on the assembly of
the branching sequence. Sialylation is known to be no-
toriously difficult due to the low reactivity of the
sialyl donor and the challenge in stereochemical con-
trol.[51] The presence of the carboxylic acid moiety in
sialic acid also necessitates additional consideration of
5
6
85 (a only)
77 (a only)
1
[a] Anomeric ratios were determined by H NMR analysis.
protective group compatibility in synthetic design.[22] Re-
cently, it was discovered that the replacement of the 5-N-
acetyl group on a sialyl donor with an electron withdrawing
protective group[52–61] significantly enhanced the reaction
yield and stereoselectivity. Thus, we designed sialyl donor 17
bearing the 5-N trichloroacetyl (TCA) moiety, as NH-TCA
can be converted to acetamide under a variety of reaction
conditions including hydrogenolysis, radical reduction and
basic cleavage followed by acetylation.[62–64] The trifluoroa-
cetimidate aglycon leaving group[58,65,66] was chosen due to
the possibility for its selective activation over a thioglyco-
side. This was realized by treating a mixture of donor 17 and
thiogalactoside acceptor 18[62–64] with a catalytic amount of
TMSOTf leading to disaccharide 24 in 68% yield (Table 1,
entry 1), which was benzoylated to give disaccharide 30. The
a sialyl linkage was confirmed by the three bond coupling
lylated thioglycoside product can be used as a donor for fur-
ther glycosylation without additional aglycon leaving group
adjustment.
With all building blocks in hand, one-pot glycosylation
was performed using the pre-activation based protocol
(Scheme 3).[39,44,46,68,69] Pre-activation of disaccharide 30 by
p-TolSCl/AgOTf,[44,45] was rapidly achieved at ꢀ788C. Addi-
tion of the thioglycosyl acceptor 31[39] to the reaction mix-
ture produced trisaccharide 3,[38] which underwent double
glycosylation of hexasaccharide 4 in the same reaction flask
leading to dodecasaccharide 2. It took only four hours for
this three-component one-pot assembly process and the de-
constant between C1 and H3ax (3J
(C1,H3ax)=5.8 Hz) of the
A
sialic acid.[67] The scope of this sialylation reaction was ex-
amined next. A variety of acceptors including primary alkyl
alcohol, carbohydrate hydroxyl groups of galactose, galac-
tosamine and glucosamine were sialylated in good yields
and stereoselectivities, which represented some of the
common natural sialyl linkages (Table 1). Acid labile benzyl-
idene and isopropylidene groups were stable under the reac-
tion condition. In addition to acceptor 18, thioglycoside 23
also served as an excellent substrate for the reaction. This
selective activation protocol is attractive as the resulting sia-
Scheme 3. a) p-TolSCl, AgOTf, CH2Cl2, ꢀ788C; then TTBP, 21;
b) AgOTf, p-TolSCl, 4, CH2Cl2, ꢀ78 ! 0 8C.
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7075

X. Huang et al.
sired dodecasaccharide 2 was easily isolated from the reac-
tion by flash column chromatography in an excellent 65%
yield from disaccharide 30.
saccharide 2 was produced in a total of 10 steps with 7 steps
and 11% overall yield for the longest linear sequence. The
successful completion and high efficiency of this synthesis
highlight the power of the pre-activation based approach.
Due to the convergent modular approach taken, this strat-
egy can be readily adapted towards the assembly of a library
of N glycan sequences including bisecting GlcNAc and
multi-antennary structures as well as un-natural N glycan
analogs. We are currently synthesizing other N-glycan se-
quences and investigating the usage of these molecules as
disease markers for early diagnosis.
Deprotection in complex oligosaccharide synthesis can be
very challenging. Previously, Ito and co-workers reported
that the removal of dichlorophthalimide (DCPhth) group in
the presence of protected sialic acids did not lead to the de-
sired product presumably due to cross reactivities of the
methyl ester of sialic acids with reagents used to remove
DCPhth and vice versa.[22] They designed an elegant ap-
proach by first converting all DCPhth groups into azide
prior to the attachment of sialic acid, thus bypassing the
cross reactivity problem. As an alternative, we explored the
possibility of deprotecting sialic esters in the presence of
phthalimide (Phth) groups. Treatment of the fully protected
dodecasaccharide 2 with LiI in pyridine[54,70] at 1108C clean-
ly cleaved the two methyl esters without affecting the Phth
groups (Scheme 4). The resulting dicarboxylic acid was then
added to hydrazine in refluxing ethanol, which was followed
Experimental Section
General experimental procedures: All reactions were carried out under
nitrogen with anhydrous solvents in flame-dried glassware, unless other-
wise noted. All glycosylation reactions were performed in the presence
of molecular sieves, which were flame-dried right before the reaction
under high vacuum. Glycosylation
solvents were dried using a solvent
purification system and used directly
without further drying. Chemicals
used were reagent grade as supplied
except where noted. Analytical thin-
layer chromatography was performed
using silica gel 60 F254 glass plates;
Compound spots were visualized by
UV light (254 nm) and by staining
with a yellow solution containing Ce-
A
(NO3)6
(0.5 g)
and
(NH4)6Mo7O24·4H2O (24.0 g) in 6%
H2SO4 (500 mL). Flash column chro-
matography was performed on silica
gel 60 (230–400 Mesh). NMR spectra
were referenced using Me4Si (0 ppm),
1
residual CHCl3 (d H NMR 7.26 ppm,
13C NMR 77.0 ppm). Peak and cou-
pling constant assignments are based
on 1H NMR, 1H,1H gCOSY and (or)
1H,13C gHMQC and 1H,13C gHMBC
experiments. All optical rotations
were measured using the sodium D
Scheme 4. a) LiI, pyridine, 1108C; b) hydrazine, ethanol, reflux; c) Ac2O, Et3N, MeOH; d) H2, Pd(OH)2/C,
MeOH, H2O.
by selective acetylation in methanol to yield compound 32.
line. ESI mass spectra were recorded using ESQUIRE Ion Trap LC/MS.
High-resolution mass spectra were recorded on a Micromass electrospray
mass spectrometer equipped with an orthogonal electrospray source (Z-
spray) operated in positive ion mode.
The usage of ethylene diamine instead of hydrazine to
remove the Phth groups led to a lower yield. Finally, the
fully deprotected dodecasaccharide 1 was produced by cata-
lytic hydrogenolysis of 32 with Pd(OH)2/C (49% overall
yield from 2). The NMR and MS data of 1 are consistent
with its structure.
Characterization ofanomeric stereochemistry : The stereochemistries of
the newly formed glycosidic linkages in the oligosaccharides (except
sialyl and mannosyl linkages) were determined by 3J
ACHTREUNG
1H NMR and/or 1J
coupling). Smaller coupling constants of JH1,H2 (around 3 Hz) indicate
1,2-cis a linkages and larger coupling constants 3J
(H1,H2) (7.2 Hz or
ACHTREUNG
3
AHCTREUNG
larger) indicate 1,2-trans b linkages. This can be further confirmed by
Conclusion
1JC1,H1 (ꢁ170 Hz) for a linkages and 1J
(C1,H1) (ꢁ160 Hz) for b linkag-
A
es.[47] For the mannosyl linkages, one bond coupling constants between C1
We have achieved a stereocontrolled synthesis of the fucosy-
lated complex type N glycan dodecasaccharide 2 via the
pre-activation based chemoselective glycosylation method.
This is the most complex oligosaccharide assembled by any
one-pot methods to date, which contains twelve monosac-
charide units and several synthetically challenging linkages
such as a-sialic acids, b-mannose, a-fucose as well as branch-
ing. Starting from monosaccharide building blocks, dodeca-
and H1 were measured by gHMQC 2-D NMR. 1J
cates a-mannosyl linkages and 1J
(C1,H1) ꢁ160 Hz suggests b linkages.
For the sialyl linkages, three bond coupling constants between C1 and
A
AHCTREUNG
H3ax of the sialic acid was measured by gHMBC 2D NMR. 1J
A
)
ꢁ5.8 Hz indicates a-sialyl linkage and 1J
ACHTREUNG
age.
Benzyl 2,3,4-tri-O-benzyl-a-l-fucopyranosyl-(1!6)-3-O-benzyl-2-deoxy-
2-phthalimido-b-d-glucopyranoside (9): mixture of CuBr2 (0.9 g,
6.27 mmol), Bu4NBr (1.31 g, 4.06 mmol), and molecular sieves 4 A (1.2 g)
A
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Chem. Eur. J. 2008, 14, 7072 – 7081

Oligosaccharides
FULL PAPER
in CH2Cl2/DMF 2:1 (18 mL) was stirred and cooled over an ice-water
on the flask wall. The characteristic yellow color of p-TolSCl in the reac-
tion solution dissipated rapidly within a few seconds indicating depletion
of p-TolSCl. After the donor was completely consumed according to
TLC analysis (about 5 min at ꢀ788C), a solution of acceptor 11 (345 mg,
579 mmol) in CH2Cl2 (6 mL) was added slowly along the flask wall. The
reaction mixture was allowed to warm up to ꢀ208C and quenched by
using triethylamine. The reaction mixture was diluted with CH2Cl2 and
filtered through Celite, which was washed extensively with CH2Cl2 until
TLC indicated no more organic compounds in the filtrate. The organic
layer was then extracted with satd. NaHCO3 solution, dried over anhy-
drous sodium sulfate and concentrated under reduced pressure. After
flash column chromatography (hexanes/EtOAc 7:3), the a and b mixture
(231 mg, 82% based on consumption donor, recover donor 70 mg) was
obtained. The pure 12a (33 mg) and 12b (198 mg) (a/b 1:6) were isolated
by column chromatography using 3% acetone in toluene. [a]2D0 =+19.4
(c=1.0 in CHCl3). Data for 12b: 1H NMR (600 MHz, CDCl3): d=7.83–
7.80 (m, 1H), 7.71–7.64 (m, 2H), 7.63–7.60 (m, 1H), 7.48–7.42 (m, 4H),
7.38–7.30 (m, 7H), 7.29–7.21 (m, 8H), 7.01–6.98 (m, 2H), 6.92–6.89 (m,
bath.
A solution of compound 7 (1.07 g, 1.98 mmol) and 8 (0.9 g,
1.84 mmol) in CH2Cl2 (12 mL) was added dropwise and the mixture was
stirred for 17 h from 08C to room temperature. The resulting mixture
was quenched with aq. NaHCO3, diluted with EtOAc, and filtered
through Celite. The filtrate was washed successively with aq. NaHCO3
and brine, and dried over Na2SO4. The organic layer was then evaporated
in vacuo. The residue was purified by flush column chromatography (hex-
anes/EtOAc 2:1) to afford
9
(1.25 g, 75%). [a]2D0 =ꢀ78.1 (c=1.0 in
CHCl3); 1H NMR (600 Hz, CDCl3): d=7.79 (brs, 1H), 7.67 (brs, 2H),
7.52 (brs, 1H), 7.42–7.40 (m, 2H), 7.38–7.22 (m, 14H), 7.08–6.98 (m,
6H), 6.89–6.86 (m, 3H), 5.12–5.08 (m, 1H), 4.98 (d, 3J
ACHTREUNG
3
3
ACHTREUNG
1H), 4.87 (d, J
(d, 3J
A
ACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
3
ACHTREUNG
AHCTREUNG
2H), 6.86–6.80 (m, 5H), 5.51 (s, 1H), 5.44 (d, 3J
4.90–4.82 (m, 3H), 4.65 (d, 3J(H,H)=12 Hz, 1H), 4.58 (d, 3J
12 Hz, 1H), 4.55 (s, 1H), 4.51 (d, 3J
(H,H)=12 Hz, 1H), 4.40 (t, 2H, J=
12.6 Hz), 4.25–4.22 (m, 1H), 4.19–4.15 (m, 2H), 4.05 (t, 1H, J=9.6 Hz),
3.96 (t, 1H, J=9.6 Hz), 3.78 (s, 3H), 3.75 (d, 3J
(H,H)=3 Hz, 1H), 3.68
(d, 3J
(H,H)=12 Hz, 1H), 3.59–3.51 (m, 3H), 3.43 (dd, 1H, J=3.0,
ACHTREUNG
AHCTREUNG
G
ACHTREUNG
(150 Hz, CDCl3): d=138.80, 138.79, 138.7, 138.3, 137.3, 133.8, 131.9,
AHCTREUNG
128.72, 128.66, 128.6, 128.52, 128.47, 128.34, 128.25, 128.24, 128.18, 128.1,
127.9, 127.83, 127.76, 127.5, 123.3, 98.9 (C1 fucose, 1J
U
1
AHCTREUNG
97.6 (C1 glucosamine, J
(C1,H1)=161 Hz), 79.5, 77.8, 77.7, 76.6, 75.1, 74.4,
AHCTREUNG
74.4, 74.1, 73.2, 73.2, 60.6, 55.8, 16.7 ppm. Correlations between C1 of
fucose and H6 and H6’ of the glucosamine unit were observed in gHMBC
2-D NMR spectrum confirming the fucose is linked to 6-O of the glucos-
amine. HRMS: m/z: calcd for C55H55NO11Na: 928.3673 [M+Na]+, found:
928.3654.
10.2 Hz), 3.16–3.12 (m, 1H), 2.27 ppm (s, 3H); 13C NMR (100 MHz,
CDCl3): d=168.4, 167.5, 159.4, 138.9, 138.8, 138.5, 138.2, 137.9, 134.1,
134.1, 133.9, 133.7, 131.9, 131.8, 130.8, 129.8, 129.4, 129.3, 129.1, 128.7,
128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6,
127.5, 127.2, 126.3, 123.7, 123.5, 113.9, 102.3 (C1 mannose, 1J
A
p-Tolyl 2-O-benzyl-4,6-O-benzylidene-3-O-p-methoxybenzyl-1-thio-a-d-
mannopyranoside (10a): nBu2SnO (3.72 g, 14.6 mmol) was added to a so-
lution of p-tolyl 4,6-O-benzylidene-1-thio-a-d-mannopyranoside[71] (4.9 g,
13.6 mmol) in toluene (65 mL) and the resulting mixture was heated
under reflux using a Dean-Stark apparatus for 3 h. The reaction mixture
was cooled to room temperature and p-methoxybenzyl chloride (2.8 mL,
20.4 mmol) and nBu4NI (1.01 g, 2.7 mmol) were added. The reaction mix-
ture was heated under reflux again for 2 h followed by addition of H2O
(2 mL) to quench the reaction. After removing the solvents, the residue
was purified by column chromatography (silica gel, hexanes/EtOAc 7:3)
to give p-tolyl 4,6-O-benzylidene-3-O-p-methoxybenzyl-1-thio-a-d-man-
nopyranoside (5.96 g, 92%).
1
154 Hz), 101.6, 83.4 (C1 glucosamine, J
A
78.2, 77.3, 75.2, 75.1, 73.8, 72.6, 68.9, 68.8, 67.6, 55.5, 55.0, 21.4 ppm;
HRMS: m/z: calcd for C63H61NNaO12S: 1078.3812 [M+Na]+, found:
1078.3807.
p-Tolyl 2,4-di-O-benzyl-4,6-di-O-levulinoyl-b-d-mannopyranosyl-(1!4)-
3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyranoside (14): A solu-
tion of 12b (210 mg, 0.20 mmol) in CH2Cl2 (6 mL) was stirred with acti-
vated molecular sieves (MS 3 Š) (600 mg) and cooled to ꢀ788C. Triethyl-
silane (130 mL, 0.20 mmol) was added followed by dichlorophenyl borane
(105 mL, 0.20 mmol). The reaction was stirred for 6 h and quenched by
adding triethyl amine. The reaction mass was diluted with CH2Cl2 and ex-
tracted with a saturated aqueous NaHCO3 solution. The organic layer
was dried over anhydrous sodium sulfate and concentrated under re-
duced pressure. Diol p-tolyl 2,4-di-O-benzyl-b-d-mannopyranosyl-(1!4)-
This product was dissolved in anhydrous DMF (50 mL) and NaH (95%,
457 mg, 18 mmol) was added to the mixture. After 1 h at room tempera-
ture, benzyl bromide (2.47 g 18 mmol) was added to the mixture and
stirred overnight. The reaction mixture was diluted with EtOAc
(300 mL), washed with water, 10% NH4Cl and brine and dried over an-
hydrous Na2SO4. The organic phase was evaporated and the residue was
purified by flash column chromatography (silica gel, hexanes/EtOAc 4:1)
to give compound 10a (6.3 g, 90%). 1H NMR (600 MHz, CDCl3): d=
7.58–7.53 (m, 2H), 7.44–7.27 (m, 12H), 7.15–7.11 (m, 2H), 6.93–6.87 (m,
3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyranoside
(167 mg)
was obtained from flash column chromatography (EtOAc/hexanes 1:1) in
1
89% yield. [a]2D0 =+40.6 (c=1.0 in CHCl3); H NMR (600 MHz, CDCl3):
d=7.83–7.80 (m, 1H), 7.71–7.61 (m, 3H), 7.40–7.25 (m, 16H), 7.04–7.01
(m, 2H), 6.92–6.88 (m, 5H), 5.43 (d, 3J
ACHTREUNG
3J
3J
A
ACHTREUNG
T
ACHTREUNG
2H), 5.67 (s, 1H), 5.46 (d, 3J
(H,H)=1.8 Hz, 1H), 4.76 (d, 3J
1H), 4.52–4.48 (d, 3J
ACHTREUNG
12 Hz, 1H), 4.73 (s, 2H), 4.62 (d, 3J
ACHTREUNG
3J
ACHTREUNG
3
2H), 4.27–4.22 (dd, J
(H,H)=10.8, 4.2 Hz, 1H), 4.40–4.10 (m, 1H), 4.10–
(m, 1H), 3.44–3.39 (m, 2H), 3.14–3.11 (m, 1H), 2.28 (s, 3H), 1.86–
1.83 ppm (m, 1H); 13C NMR (100 MHz, CDCl3): d=168.5, 167.9, 138.8,
138.6, 137.8, 137.5, 136.6, 136.2, 134.3, 134.2, 134.1, 133.9, 133.2, 132.1,
131.9, 131.6, 129.9, 128.8, 128.6, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9,
127.8, 127.7, 127.6, 127.3, 123.9, 123.5, 123.3, 96.8, 96.7, 78.776.3, 75.5,
74.7, 74.6, 74.4, 74.0, 73.6, 70.9, 69.9, 62.5, 60.7, 56.5, 56.1, 27.2 ppm; ESI-
MS: m/z: calcd for C55H55NO11SNa: 960.35 [M+Na]+, found: 960.36.
3.97 (m, 1H), 3.94- 3.88 (m, 1H), 3.82 (s, 3H), 2.35ppm (s, 3H);
13C NMR (150 MHz, CDCl3): d=159.5, 138.2, 138.1, 137.9, 132.6, 130.7,
130.2, 130.16, 129.6, 129.1, 128.7, 128.5, 128.4, 128.1, 126.4, 114.0, 101.7,
87.7, 79.3, 78.2, 76.0, 73.2, 73.0, 68.8, 65.8, 21.4 ppm; HRMS: m/z: calcd
for C35H36O6SNa: 607.2130 [M+Na]+, found 607.2089.
p-Tolyl 2-O-benzyl-4,6-O-benzylidene-3-O-p-methoxybenzyl-b-d-manno-
pyranosyl-(1!4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyra-
noside (12b): A mixture of donor 10a (226 mg, 387 mmol) and 2,4,6-tri-
tert-butylpyrimidine (TTBP; 180 mg, 726 mmol) in dry CH2Cl2 (20 mL)
was stirred with activated molecular sieves 4 Š (800 mg) for 30 min at
room temperature and then cooled to ꢀ758C. AgOTf (118 mg,
0.46 mmol) in acetonitrile (300 mL) was added directly to the solution
without touching the flask wall. After 10 minutes, orange colored p-
TolSCl (55 mL, 387 mmol) was added via a microsyringe. Since the reac-
tion temperature was lower than the freezing point of p-TolSCl, p-TolSCl
was added directly into the reaction mixture to prevent it from freezing
To a solution of p-tolyl 2,4-di-O-benzyl-b-d-mannopyranosyl-(1!4)-3,6-
di-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyranoside
(167 mg,
0.178 mmol) in CH2Cl2 (5 mL), levulinic acid (73 mL, 0.712 mmol), N-
ethyl N,N-dimethylaminopropyl carbodiimide hydrochloride (EDC)
(136.5 mg, 0.712 mmol) and N,N-dimethylamino pyridine (4.4 mg,
0.036 mmol) were added and the reaction was stirred overnight at room
temperature. The reaction mixture was diluted with CH2Cl2 (50 mL) and
extracted with satd. NaHCO3 solution. The organic layer was evaporated
to dryness and 14 (173 mg) was obtained in 86% yield after flash column
chromatographic purification (hexanes/EtOAc 2:1). [a]2D0 =+17.8 (c=1.0
Chem. Eur. J. 2008, 14, 7072 – 7081
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7077

X. Huang et al.
in CHCl3); 1H NMR (600 Hz, CDCl3): d=7.78–7.74 (m, 1H), 7.68–7.62
(m, 2H), 7.64–7.61 (m, 1H), 7.42–7.24 (m, 17H), 6.99–6.97 (m, 1H),
1H), 3.18–3.13 (m, 1H), 2.34 (d, 3J
3J(H,H)=6.6 Hz, 3H); 13C NMR (150 MHz, CDCl3): d=168.7, 168.1,
A
ACHTREUNG
6.83–6.81 (m, 2H), 6.73–6.69 (m, 3H), 5.39 (d, 3J
A
168.0, 167.9, 139.2, 139.1, 139.0, 138.9, 138.8, 138.5, 138.3, 1378.0, 137.3,
134.3, 134.1, 133.9, 132.1, 132.0, 131.7, 128.9, 128.74, 128.69, 128.66,
128.64, 128.61, 128.4, 128.38, 128.3, 128.26, 128.2, 128.1, 128.08, 128.0,
127.94, 127.81, 127.8, 127.77, 127.7, 127.64, 127.6, 127.4, 127.2, 123.9,
123.5, 101.4, 97.0, 96.99, 96.9, 79.8, 79.2, 79.0, 77.8, 77.4, 76.8, 76.2, 75.4,
75.37, 75.36, 75.3, 75.0, 74.9, 74.7, 74.67, 74.4, 73.7, 73.69, 73.4, 72.7, 70.2,
68.1, 66.3, 64.0, 62.6, 60.7, 56.7, 56.1, 16.6 ppm; HRMS: m/z: calcd for
C103H102N2O22Na: 1742.6855 [M+Na]+, found: 1742.6771.
4.85 (d, 3J
3J(H,H)=3.0, 9.6 Hz, 1H), 4.70 (d, 3J
4.54 (d, 3J
3J
A
ACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
A
ACHTREUNG
1H), 2.67–2.33 (m, 8H), 2.25 (s, 3H), 2.12 (s, 3H), 2.01 ppm (s, 3H);
13C NMR (100 MHz, CDCl3): d=206.7, 206.2, 172.3, 172.0, 167.9, 167.2,
138.7, 138.6, 138.2, 137.89, 137.85, 133.8, 133.6, 129.5, 128.5, 128.4, 128.2,
127.94, 127.89, 127.78, 127.75, 127.71, 127.66, 127.51, 126.8, 123.3, 123.2,
100.7, 83.18, 79.0, 78.9, 77.9, 76.2, 76.1, 74.8, 74.7, 74.6, 73.4, 73.07, 73.05,
68.5, 63.1, 54.7, 37.74, 37.69, 29.8, 29.7, 27.88, 27.85, 21.1 ppm; MALDI-
MS: m/z: calcd for C65H67NO15SNa: 1156.42 [M+Na]+, found: 1156.61.
Benzyl 2-O-acetyl-3,4,6-tri-O-benzyl-a-d-mannopyranosyl-(1!6)-(2-O-
acetyl-3,4,6-tri-O-benzyl-a-d-mannopyranosyl-(1!3))-2,4-di-O-benzyl-b-
d-mannopyranosyl-(1!4)-O-3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-
glucopyranosyl-(1!4)-(2,3,4-tri-O-benzyl-a-l-fucopyranosyl-(1!6))-3-O-
benzyl-2-deoxy-2-phthalimido-b-d-glucopyranoside (16): The mixture of
donor 5 (26 mg, 45 mmol), acceptor 6 (17 mg, 15.7 mmol) and freshly acti-
vated MS 4 Š (600 mg) in anhydrous CH2Cl2 (6 mL) was stirred for 30
minutes at room temperature and cooled down to ꢀ788C followed by
the addition of AgOTf (34 mg, 133 mmol) in anhydrous acetonitrile
(0.1 mL) directly to the solution without touching the wall of reaction
flask. After 5 min, p-TolSCl (6.8 mL, 45 mmol) was added via a microsyr-
inge. The reaction mixture was stirred for 1.5 h until the temperature
reached 08C, which was followed by addition of TMSOTf (2 mL). The re-
action was stirred further for 1.5 h from 08C to room temperature and
triethylamine (25 mL) was added. The mixture was diluted with CH2Cl2
(50 mL) and filtered through Celite. The Celite was washed with CH2Cl2
until no organic product was present in the filtrate by TLC. The filtrate
was combined, concentrated and purified by flash column chromatogra-
Benzyl 2,4-di-O-benzyl-3,6-di-O-levulinoyl-b-d-mannopyranosyl-(1!4)-
3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl-(1!4)-(2,3,4-
tri-O-benzyl-a-l-fucopyranosyl-(1!6))-3-O-benzyl-2-deoxy-2-phthalimi-
do-b-d-glucopyranoside (15): The mixture of donor 14 (100 mg, 88 mmol),
acceptor 9 (64 mg, 70.4 mmol) and freshly activated MS 4 Š (600 mg) in
dry CH2Cl2 (6 mL) was stirred for 30 min at room temperature, and
cooled down to ꢀ708C followed by the addition of AgOTf (68 mg,
264 mmol) in anhydrous acetonitrile (0.1 mL) directly to the solution
without touching the wall of reaction flask. After 5 min, p-TolSCl
(12.8 mL, 88 mmol) was added via a microsyringe. The reaction mixture
was stirred for 1.5 h until the temperature reached ꢀ208C and triethyla-
mine (30 mL) was added. The mixture was diluted with CH2Cl2 (50 mL)
filtered through Celite. The filtrate was concentrated and purified by
flash column chromatography (hexanes/EtOAc 3:2) to give 15 (116 mg,
86%). [a]2D0 =ꢀ28.0 (c=1.0 in CHCl3); 1H NMR (600 Hz, CDCl3): d=
7.85–7.81 (m, 1H), 7.80–7.76 (m, 1H), 7.72–7.60 (m, 4H), 7.59–7.56 (m,
1H), 7.52–7.40 (m, 6H), 7.36–7.20 (m, 18H), 7.17–7.12 (m, 3H), 7.05–
7.02 (m, 3H), 6.98–6.94 (m, 4H), 6.92–6.88 (m, 2H), 6.80–6.71 (m, 8H),
5.52 (d, 1H, J=8.4 Hz), 4.92–4.70 (m, 10H), 4.68 (s, 1H), 4.66–4.47 (m,
phy (hexanes/EtOAc 3:2) to give compound 16 (32 mg, 77%). [a]D20
=
+4.5 (c=1.0 in CHCl3); 1H NMR (600 Hz, CDCl3): d=7.76–7.71 (m,
2H), 7.69–7.58 (m, 4H), 7.50–7.45 (m, 4H), 7.41–7.16 (m, 42H), 7.14–
7.01 (m, 11H), 6.99–6.93 (m, 4H), 6.93–6.88 (m, 2H), 6.78–6.68 (m, 6H),
6.62–6.58 (m, 2H), 5.49–5.46 (m, 2H), 5.30–5.28 (m, 1H), 5.14 (s, 1H),
4.93 (d, 3J
10.8 Hz, 1H), 4.71 (d, J
1H), 4.64–4.52 (m, 9H), 4.50–4.43 (m, 4H), 4.42–4.33 (m, 5H), 4.29 (d,
3J
(H,H)=12.6 Hz, 1H), 4.28–4.12 (m, 6H), 4.10–4.04 (m, 2H), 3.99–3.87
(m, 6H), 3.85–3.70 (m, 7H), 3.67–3.56 (m, 7H), 3.5–3.48 (m, 2H), 3.32–
3.28 (dd, 3J(H,H)=3.0, 10.2 Hz, 1H), 3.21 (d, 3J
(H,H)=9.6 Hz, 1H),
3.17–3.13 (m, 1H), 2.10 (s, 3H), 1.80 (s, 3H), 0.96 ppm (d, 3J
(H,H)=
(H,H)=8.4 Hz, 1H), 4.92–4.80 (m, 10H), 4.77 (d, 3J
ACHTREUNG
3
3
ACHTREUNG
(H,H)=11.4 Hz, 1H), 4.67 (d, J
8H), 4.39 (d, 3J
G
G
ACHTREUNG
4.30–4.10 (m, 8H), 4.09–4.04 (m, 1H), 3.95–3.92 (m, 3H), 3.84–3.76 (m,
4H), 3.68 (d, 3J
G
A
ACHTREUNG
3
2H), 3.22 (d, J
A
ACHTREUNG
(s, 3H), 0.95 ppm (d, 3J
6.6 Hz, 3H); 13C NMR (150 Hz, CDCl3): d=170.4, 169.9, 168.8, 167.78,
139.2, 139.0, 138.9, 138.85, 138.6, 138.3, 138.2, 138.15, 137.8, 137.7, 137.3,
133.9, 133.7, 132.1, 131.9, 131.7, 128.9, 128.7, 128.61, 128.5, 128.4, 128.3,
128.1, 128.0, 127.8, 127.7, 127.6, 127.4, 127.3, 127.1, 127.0, 123.7, 123.4,
102.0, 99.7, 99.6, 98.5, 97.1, 96.8, 81.5, 79.8, 79.5, 78.9, 78.4, 77.98, 77.9,
76.7, 76.4, 75.7, 75.3, 75.2, 75.19, 75.0, 74.9, 74.85, 74.8, 74.7, 74.69, 74.67,
74.6, 74.5, 74.4, 74.2, 74.0, 73.7, 73.6, 73.5, 73.46, 72.6, 72.5, 72.0, 71.6,
71.4, 70.1, 70.0, 69.0, 68.9, 68.34, 68.129, 66.7, 66.2, 65.3, 64.0, 56.8, 56.0,
24.9, 21.3, 21.2, 16.7 ppm; MALDI-MS: m/z: calcd for C161H162N2O34Na:
2690.10 [M+Na]+, found: 2690.49.
d=206.9, 206.4, 172.5, 172.3, 168.4, 168.0, 167.94, 167.85, 139.3, 139.2,
139.1, 139.00, 138.86, 138.85, 138.2, 138.1, 137.3, 134.1, 133.9, 132.1, 131.9,
131.8, 128.9, 128.8, 128.68, 128.67, 128.6, 128.40, 128.35, 128.3, 128.13,
128.11, 128.08, 128.04, 128.00, 127.99, 127.9, 127.81, 127.77, 127.74, 127.71,
127.6, 127.50, 127.48, 127.2, 127.1, 123.7, 123.5, 100.8, 97.1, 96.92, 96.85,
79.8, 79.2, 77.9, 77.1, 76.9, 76.5, 76.2, 75.4, 75.3, 75.0, 74.96, 74.7, 74.6,
74.6, 73.9, 73.5, 73.46, 73.3, 73.28, 72.7, 70.1, 68.2, 66.2, 64.0, 63.3, 60.6,
56.8, 56.1, 38.0, 37.99, 30.0, 29.9, 28.2, 28.1, 16.6 ppm; HRMS: m/z: calcd
for C113H114N2O26Na: 1938.7592 [M+Na]+, found: 1938.7534.
Benzyl
2,4-di-O-benzyl-b-d-mannopyranosyl-(1!4)-3,6-di-O-benzyl-2-
Benzyl
3,4,6-tri-O-benzyl-a-d-mannopyranosyl-(1!6)-(3,4,6-tri-O-
deoxy-2-phthalimido-b-d-glucopyranosyl-(1!4)-(2,3,4-tri-O-benzyl-a-l-
fucopyranosyl-(1!6))-3-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyra-
noside (6): Hydrazine acetate (60 mg, 0.65 mmol) was added to a solution
of 15 (300 mg, 0.157 mmol) in CH2Cl2/MeOH 1:1 (8 mL), and stirred at
room temperature for 90 min. The reaction mixture was diluted with
CH2Cl2 (100 mL) and extracted with satd. NH4Cl solution. The organic
layer was dried over anhydrous sodium sulfate and concentrated under
reduced pressure. The compound 6 (217 mg, 81%) was isolated by flash
column chromatography (hexanes/EtOAc 2:3). [a]2D0 =ꢀ16.5 (c=1.0 in
CHCl3); 1H NMR (600 MHz, CDCl3): d=7.92–7.88 (m, 1H), 7.84–7.78
(m, 1H), 7.76–7.62 (m, 5H), 7.58–7.55 (m, 1H), 7.52–7.42 (m, 1H), 7.40–
7.22 (m, 22H), 7.08–7.00 (m, 7H), 6.98–6.90 (m 7H), 6.81–6.76 (m, 3H),
benzyl-a-d-mannopyranosyl-(1!3))-2,4-di-O-benzyl-b-d-mannopyrano-
syl-(1!4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl-
(1!4)-(2,3,4-tri-O-benzyl-a-l-fucopyranosyl-(1!6))-3-O-benzyl-2-deoxy-
2-phthalimido-b-d-glucopyranoside (4): The solution of compound 16
(242 mg, 90.6 mmol) in methanol (5 mL) and CH2Cl2 (2 mL) was stirred
with NaOMe (12 mg, 0.5 mmol) at room temperature for 3 h, which was
then neutralized to pH 5–6 with acetic acid. After evaporating the sol-
vent, the residue was purified by flash column chromatography (hexanes/
EtOAc 1:1) to give 4 (184 mg, 79%). [a]2D0 =+4.0 (c=1.0 in CHCl3);
1H NMR (600 Hz, CDCl3): d=7.83–7.77 (m, 2H), 7.72–7.61 (m, 4H),
7.56–7.47 (m, 4H), 7.45–7.39 (m, 4H), 7.36–7.10 (m, 53H), 7.09–7.03 (m,
2H), 7.02–6.97 (m, 4H), 6.94–6.91 (m, 2H), 6.80–6.73 (m, 6H), 6.70–6.66
(m, 2H), 5.52 (d, 1H, J=8.4 Hz), 5.19 (s, 1H), 5.00–4.82 (m, 11H), 4.79–
4.73 (m, 2H), 4.67–4.45 (m, 15H), 4.45–4.37 (m, 3H), 4.36–4.23 (m, 6H),
4.21–4.10 (m, 4H), 4.04–3.96 (m, 4H), 3.92–3.50 (m, 20H), 3.35 (dd,
5.64 (d, 3J
4.67–4.60 (m, 5H), 4.57–4.51 (m, 2H), 4.46–4.26 (m, 6H), 4.24–4.16 (m,
A
3H), 4.10–4.00 (m, 3H), 3.93 (d, 3J
ACHTREUNG
3J
3J
A
N
3J
3J
3J
A
N
G
E
ACHTREUNG
1H), 3.55–3.49 (m, 1H), 3.45–3.40 (m, 3H), 3.29 (d, 3J
G
ACHTREUNG
7078
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 7072 – 7081

Oligosaccharides
FULL PAPER
139.2, 139.15, 139.1, 138.9, 138.8, 138.75, 138.6, 138.4, 138.3, 138.2, 138.16,
138.1, 137.3, 134.1, 133.8, 132.1, 131.7, 128.9, 128.73, 128.70, 128.69,
128.67, 128.6, 128.5, 128.48, 128.46, 128.4, 128.3, 128.2, 128.16, 128.1,
128.09, 128.07, 128.04, 128.02, 127.98, 127.95, 127.93, 127.89, 127.85, 127.8,
127.76, 127.74, 127.72, 127.67, 127.6, 127.57, 127.3, 127.1, 127.06, 123.7,
123.4, 101.8, 101.5, 100.1, 97.2, 97.1, 96.8, 81.9, 80.3, 80.0, 79.56, 79.3, 78.8,
77.9, 76.5, 75.8, 75.5, 75.3, 75.1, 75.07, 75.0, 74.8, 74.75, 74.7, 74.6, 74.5,
74.4, 74.3, 74.0, 73.7, 73.5, 73.47, 72.7, 72.3, 72.2, 71.8, 71.4, 70.1, 69.0,
68.9, 68.2, 67.9, 66.7, 66.2, 64.0, 56.8, 56.1, 16.7 ppm; HRMS: m/z: calcd
for C157H158N2O32Na: 2607.0730 [M+Na]+, found: 2607.0669.
7.57 (m, 2H), 7.38–7.30 (m, 5H), 7.07–7.04 (m, 2H), 6.52 (d, 3J
9.6 Hz, 1H), 5.43–5.39 (m, 1H), 5.32 (s, 1H), 5.27–5.24 (dd, 1H, J=1.8,
9.6 Hz), 5.11–5.05 (m, 1H), 4.63 (d, 3J
(H,H)=9.6 Hz, 1H), 4.32 (dd,
(H,H)=1.8,
(H,H)=3.0 Hz, 1H), 3.82 (q,
(H,H)=
AHCTREUNG
3J
(H,H)=1.2, 12 Hz, 1H), 4.29–4.24 (m, 2H), 4.22 (dd, 3J
ACHTREUNG
10.8 Hz, 1H), 4.10–4.05 (m, 2H), 3.97 (d, 3J
ACHTREUNG
3J
A
G
3
3.54 (s, 1H), 2.72 (dd, 3J
1.8 Hz, 1H), 2.32 (s, 3H), 2.19 (s, 3H), 2.18 (s, 3H), 2.00 (s, 3H), 1.98 (s,
3H), 1.94 ppm (dd, 3J(H,H)=6.0, 12.6 Hz, 1H); 13C NMR (150 MHz,
CDCl3): d=170.9, 170.7, 170.5, 170.4, 168.4, 162.3, 138.2, 138.15, 134.42,
134.41, 129.81, 129.77, 129.2, 128.3, 127.4, 126.8, 101.1, 97.4, 92.3, 86.8,
76.4, 74.0, 71.9, 69.7, 69.5, 68.1, 67.6, 67.3, 66.0, 62.5, 53.3, 51.8, 38.7, 21.6,
21.5, 21.04, 21.01, 20.97 ppm; HRMS: m/z: calcd for C40H46Cl3NO17SNa:
972.1444 [M+Na]+, found: 972.1448. The sialyl a2!3 galactose linkage
was confirmed by a HMBC correlation between C2 of the sialic acid and
H3 of the galactose unit. Stereochemistry of the linkage was established
using compound 30.
Methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trichloroacetamido-d-glycero-
a-d-galacto-2-nonulopyranosonate-2-(N-phenyl)-trifluoroacetimidate
(17): Methanesulfonic acid (1.95 mL, 30 mmol) was added at room tem-
perature to a solution of methyl (p-tolyl 5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-2-thio-d-glycero-d-galacto-2-nonulopyranosid)onate
(1.8 g, 3 mmol)[67] in methanol (30 mL). After being stirred at 608C for
24 h, the reaction mixture was neutralized with triethylamine and concen-
trated in vacuo. The residue was used for next reaction without further
purification. Methyl trichloroacetate (3.57 mL, 30 mmol) and triethyl-
amine (0.84 mL, 6 mmol) were added to a solution of the residue in
methanol (30 mL) at 08C. The reaction mixture was stirred at room tem-
perature for 6 h and all volatile solvents were removed. The remaining
residue was dissolved in pyridine (10 mL), to which acetic anhydride
(2 mL) and a catalytic amount of DMAP were added at 08C. After being
stirred at room temperature overnight, the reaction mixture was diluted
with ethyl acetate, washed with water, 1m HCl, saturated aq. NaHCO3,
and brine, dried over anhydrous Na2SO4, filtered and concentrated in
vacuo. The residue was purified by column chromatography on silica gel
(EtOAc/hexanes 2:3) to give methyl (p-tolyl 4,7,8,9-tetra-O-acetyl-3,5-di-
deoxy-2-thio-5-trichloroacetamido-d-glycero-d-galacto-2-nonulopyrano-
sid)onate (1.40 g, 2 mmol, 67%).
p-Tolyl (methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trichloroacetamido-
d-glycero-a-d-galacto-2-nonulopyranosonate-(2!3))-2-O-benzoyl-4,6-O-
benzylidene-1-thio-b-d-galactopyranoside (30): The mixture of 24
(475 mg, 0.5 mmol), pyridine (2 mL) and benzoyl chloride (1 mL) was
stirred overnight at room temperature. The mixture was then diluted
with CH2Cl2 (100 mL) and washed with HCl (1m), water and satd.
sodium bicarbonate solution. The organic layer was dried over anhydrous
sodium sulfate and concentrated under reduced pressure. After flash
column chromatography (EtOAc/CH2Cl2/hexanes 3:3:4), compound 30
was obtained (501 mg, 95%). [a]2D0 =ꢀ15.0 (c=1.0 in CHCl3); 1H NMR
(600 MHz, CDCl3): d=8.16–8.12 (m, 2H), 7.60–7.56 (m, 1H), 7.52–7.46
(m, 2H), 7.45–7.42 (m, 2H), 7.39–7.35 (m, 2H), 7.35–7.30 (m, 3H), 7.02–
3
7.00 (m, 2H), 6.58 (d, J
ACHTREUNG
3J
U
ACHTREUNG
To a solution of this thioglycoside (1.4 g, 2 mmol) in acetone (20 mL) and
water (1 mL) was added NBS (930 mg, 5.23 mmol).[72] After being stirred
for 0.5 h at room temperature, the solution was concentrated. The residue
was diluted with CH2Cl2 and washed with saturated aq. NaHCO3. The or-
ganic layer was dried over Na2SO4 and concentrated leading to methyl
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trichloroacetamido-b-d-glycero-d-gal-
3J
(m, 2H), 4.02–3.96 (m, 2H), 3.72 (q, 3J
1H), 3.54 (s, 3H), 2.58 (dd, J
(s, 3H), 2.00 (s, 3H), 1.89 (s, 3H), 1.76 (s, 3H), 1.64 ppm (t, 3J
ACHTREUNG
13.2 Hz, 1H); 13C NMR (150 MHz, CDCl3): d=170.9, 170.6, 170.1, 168.8,
165.1, 162.3, 138.4, 138.0, 134.6, 133.3, 130.8, 130.7, 130.2, 129.7, 129.3,
129.2, 128.7, 128.3, 127.7, 126.8, 101.3, 96.9, 92.2, 85.5, 73.73, 73.68, 71.9,
69.7, 69.5, 68.5, 68.0, 67.9, 67.2, 62.7, 53.2, 51.5, 38.5, 21.7, 21.5, 21.01,
20.93, 20.6 ppm; HRMS: m/z: calcd for C47H50Cl3NO18SNa: 1076.1712
[M+Na]+, found: 1076.1676. Three-bond coupling between CO2Me and
H3ax of sialic acid was determined to be 5.8 Hz indicating a sialyl linkage.
acto-2-nonulopyranosonate. To
a solution of methyl 4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-5-trichloroacetamido-d-glycero-d-galacto-2-nonulopyr-
anosonate in acetone was added K2CO3 (3 equiv) and N-phenyl trifluor-
oacetimidoyl chloride[66] (10 equiv). After stirred at room temperature
for 3 h, the mixture was filtered and then concentrated. The residue was
subject to chromatography on silica gel column (hexanes/EtOAc 3:2, con-
taining 0.5% of triethylamine) leading to imidate 17 (a/b 1:2, 79%). a/b
mixture 1H NMR (400 MHz, CDCl3): d=7.32–7.25 (m, 1.5H), 7.15–7.08
(m, 1.5H), 6.83–6.77 (m, 1.5H), 6.76–6.61 (m, 3H), 5.50–5.47 (m, 1H, b),
5.46–5.40 (m, 1H, b), 5.36–5.33 (m, 0.5H, a), 5.33–5.28 (m, 0.5H, a),
5.18–5.13 (m, 1H, b), 4.91–4.84 (m, 0.5H, a), 4.55–4.49 (m, 1H, b), 4.46–
Benzyl (methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trichloroacetamido-
d-glycero-a-d-galacto-2-nonulopyranosonate-(2!3))-2-O-benzoyl-4,6-O-
benzylidene-b-d-galactopyranosyl-(1!4)-3,6-di-O-benzyl-2-deoxy-2-
phthalimido-b-d-glucopyranoside-(1!2)-3,4,6-tri-O-benzyl-a-d-manno-
pyranosyl-(1!6)-((methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trichloro-
acetamido-d-glycero-a-d-galacto-2-nonulopyranosonate-(2!3))-2-O-ben-
zoyl-4,6-O-benzylidene-b-d-galactopyranosyl-(1!4)-3,6-di-O-benzyl-2-
deoxy-2-phthalimido-b-d-glucopyranoside-(1!2)-3,4,6-tri-O-benzyl-a-d-
mannopyranosyl-(1!3))-2,4-di-O-benzyl-b-d-mannopyranosyl-(1!4)-3,6-
di-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl-(1!4)-(2,3,4-tri-
O-benzyl-a-l-fucopyranosyl-(1!6))-3-O-benzyl-2-deoxy-2-phthalimido-
b-d-glucopyranoside (2): A mixture of donor 30 (53 mg, 0.05 mmol) and
freshly activated molecular sieves 4 Š (800 mg) in CH2Cl2 (6 mL) was
stirred at room temperature for 30 minutes and then cooled to ꢀ788C,
which was followed by the addition of AgOTf (38 mg, 0.15 mmol) dis-
solved in acetonitrile (0.1 mL) without touching the wall of the flask.
After 5 minutes, orange colored p-TolSCl (7.6 mL, 0.05 mmol) was added
through a microsyringe. Since the reaction temperature was lower than
the freezing point of p-TolSCl, p-TolSCl was added directly into the reac-
tion mixture to prevent it from freezing on the flask wall. The character-
istic yellow color of p-TolSCl in the reaction solution dissipated rapidly
within a few seconds indicating its depletion. After the donor was com-
pletely consumed according to TLC analysis (about 5 min at ꢀ788C), a
solution of acceptor 31 (21 mg, 0.04 mmol) and TTBP (20 mg,
0.08 mmol) in CH2Cl2 (1 mL) was slowly added dropwise via a syringe.
The reaction mixture was stirred for 1.5 h until the temperature reached
4.41 (m, 1H, b), 4.38–4.32 (m, 0.5H, a), 4.29–4.23 (m, 0.5H, a), 4.18–4.12
3
(m, 2H, b), 4.10–3.97 (m, 1H, a), 2.94–2.87 (dd, J
A
H-3eq, 1H), 2.83–2.77 (dd, 3J
A
3
3
ACHTREUNG
2.30 (dd, J
A
11.6, 13.6 Hz, 3-Hax, b 1H), 2.20 (s, 1.5H, a), 2.19 (s, 3H, b), 2.09, 2.05,
1.84, 1.62 (each 3H, each s, OAc from b isomer), 2.04 (s, 3H, from 2
OAc of a isomer), 1.98 (s, 1.5H, a), 1.96 ppm (1.5H, a); HRMS: m/z:
calcd for C28H30Cl3F3N2O13Na: 787.0663 [M+Na]+, found 787.0626.
p-Tolyl (methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trichloroacetamido-
d-glycero-a-d-galacto-2-nonulopyranosonate-(2!3))-4,6-O-benzylidene-
1-thio-b-d-galactopyranoside (24): A mixture of the donor 17 (514 mg,
0.67 mmol) and acceptor 18 (411 mg, 1.1 mmol) and MS 3 Š in anhy-
drous CH2Cl2/CH3CN 1:1 (30 mL) was stirred at room temperature
under N2 for 30 min and then cooled to ꢀ658C. TMSOTf (0.2 equiv) was
added. After stirred at ꢀ658C for 2 h, the mixture was warmed up to
room temperature and quenched with triethylamine (50 mL). The result-
ing mixture was filtered and concentrated. The residue was chromato-
graphed on a silica gel column to afford the desired coupling product 24
(346 mg, 68% based on acceptor consumed, recover acceptor 210 mg).
[a]2D0 =ꢀ11.2 (c=1.0 in CHCl3); 1H NMR (600 MHz, CDCl3): d=7.61–
Chem. Eur. J. 2008, 14, 7072 – 7081
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7079

X. Huang et al.
08C, then cooled to ꢀ788C again. A solution of diol acceptor 4 (32 mg,
0.0125 mmol) in CH2Cl2 (1 mL) was added, followed by AgOTf (26 mg,
0.1 mmol) in acetonitrile (0.1 mL). After 5 minutes, p-TolSCl (6.1 mL,
0.04 mmol) was added and the reaction mixture was stirred for 1.5 h until
the temperature reached 08C, at which point triethylamine (30 mL) was
added to quench the reaction. The reaction mixture was diluted with
CH2Cl2 (50 mL) and filtered through Celite. The Celite was washed ex-
tensively with CH2Cl2 until TLC showed no products in the filtrate. The
filtrate was combined, concentrated and purified by flash column chro-
matography (hexanes/EtOAc 2:3) to give compound 2 (42 mg, 64.5%).
[a]2D0 =ꢀ2.5 (c=1.0 in CHCl3); 1H NMR (600 Hz, CDCl3): d=8.20–8.12
(m, 4H), 7.82–7.76 (m, 3H), 7.72–6.84 (m, 103H), 6.84–6.79 (m, 4H),
6.78–6.74 (m, 3H), 6.70–6.66 (m, 1H), 6.49–6.45 (m, 1H), 6.44–6.34 (m,
4H), 5.56–5.40 (m, 5H), 5.34 (s, 1H), 5.32 (s, 1H), 5.34–5.31 (m, 1H),
5.24–5.19 (m, 2H), 4.96- 4.85 (m, 6H), 4.83–4.65 (m, 10H), 4.63–4.41 (m,
12H), 4.37–4.29 (m, 5H), 4.28–4.19 (m, 8H), 4.17–3.98 (m, 23H), 3.94–
3.83 (m, 7H), 3.81–3.72 (m, 5H), 3.68 (s, 2H), 3.66–3.57 (m, 7H), 3.56 (s,
3H), 3.54–3.48 (m, 5H), 3.47 (s, 3H), 3.45–3.37 (m, 4H), 3.33–3.15 (m,
syl-(1!4)-2-amino-2-deoxy-b-d-glucopyranosyl-(1!2)-a-d-mannopyra-
nosyl-(1!3))-b-d-mannopyranosyl-(1!4)-2-amino-2-deoxy-b-d-glucopyr-
anosyl-(1!4)-(a-l-fucopyranosyl-(1!6))-2-amino-2-deoxy-d-glucopyra-
noside (1): Lithium iodide (200 mg) was added to a solution of compound
2 (80 mg) in dry pyridine (7 mL). The reaction mixture was heated under
reflux at 1208C for 8 h under nitrogen atmosphere. The dark yellow solu-
tion was then evaporated to dryness and co-evaporated with toluene to
yield a dark yellow amorphous solid which was directly used for next re-
action. A solution of the above solid in ethanol (5 mL) was treated with
NH2-NH2·H2O (1 mL) at 858C for 48 h. The reaction mixture was con-
centrated and co-evaporated with toluene then selective acetylated with
Ac2O (0.3 mL), Et3N (0.3 mL) in methanol (3 mL) at room temperature
overnight. The acetylated mixture was concentrated and passed through
a short column of silica gel and eluted with CH2Cl2/MeOH to give com-
pound 22. A mixture of 22, 10% Pd(OH)2/C (30 mg) in methanol (3 mL)
and water (1 mL) was stirred for 24 h at room temperature under H2 at-
mosphere. The solid was filtered off and the solution was concentrated to
give compound 1 (18 mg, 49%). [a]2D0 =ꢀ4.2 (c=1.0 in H2O); 1H NMR
(600 Hz, CDCl3): a/b mixture at the reducing end fucose d = 4.97 (d,
8H), 3.13–3.05 (m, 2H), 2.90 (d, 3J
ACHTREUNG
3J
(H,H)=2.4 Hz, 0.5H), 4.91 (s, 1H), 4.72 (s, 1H), 4.70–4.68 (m, 1H),
A
3J
3J
C
ACHTREUNG
4.57–4.54 (m, 1H), 4.47–4.44 (m, 1H), 4.38–4.33 (m, 4H), 4.08 (brs, 1H),
4.02 (brs, 1H), 3.94–3.88 (m, 4H), 3.80–3.28 (m, 68H), 2.58 (dd,
1.97 (s, 6H), 1.95 (s, 3H), 1.94 (s, 3H), 1.91 (s, 3H), 1.90 (s, 3H), 1.65–
3J
N
N
1.56 (m, 2H), 0.93 ppm (d, 3J(H,H)=6.6 Hz, 3H); 13C NMR (150 Hz,
AHCTREUNG
12.0 Hz, 2H), 1.06–1.02 ppm (m, 3H); ESI-MS (neg. mode): m/z: calcd
CDCl3): d=170.9, 170.6, 170.5, 170.4, 170.3, 168.68, 168.65, 168.5, 168.11,
168.05, 168.00, 164.98, 164.90, 162.3, 139.4, 139.3, 139.1, 139.04, 138.99,
138.90, 138.82, 138.6, 138.5, 138.3, 138.2, 137.9, 137.8, 137.3, 133.7, 133.6,
132.1, 132.0, 131.1, 130.3, 130.2, 130.0, 129.2, 129.1, 129.0, 128.9, 128.6,
128.5, 128.42, 128.41, 128.36, 128.33, 128.27, 128.24, 128.22, 128.17, 128.14,
128.07, 128.02, 127.90, 127.82, 127.79, 127.76, 127.66, 127.57, 127.54,
127.45, 127.34, 127.23, 127.17, 127.00, 126.97, 126.91, 126.61, 126.59,
126.52, 125.41, 123.31, 101.9, 101.5, 101.5, 101.0, 98.5, 97.7, 97.10, 97.08,
96.98, 96.96, 96.85, 95.7, 92.4, 92.1, 81.6, 81.4, 80.3, 79.3, 78.3, 78.0, 77.0,
76.2, 76.1, 75.5, 75.0, 74.9, 74.7, 74.4, 74.3, 74.1, 74.0, 73.8, 73.40, 73.35,
73.33, 73.24, 73.1, 72.8, 72.7, 72.54, 72.45, 72.1, 72.0, 70.9, 70.6, 70.3, 70.1,
69.7, 69.4, 68.8, 68.4, 68.3, 67.8, 67.6, 67.3, 66.7, 66.6, 66.1, 62.7, 62.6, 56.8,
56.2, 56.0, 53.3, 53.2, 51.6, 38.9, 38.9, 21.6, 20.9, 20.9, 20.82, 20.76,
for C90H148N6O66: 1183.42 [Mꢀ2H]2ꢀ, found: 1183.83.
Acknowledgements
This work was supported by the National Institute of General Medical
Sciences (NIH, R01-GM72667) and a CAREER award from the Nation-
al Science Foundation (CHE 0547504).
16.7 ppm; MALDI-MS: m/z: calcd for C279H280Cl6N6NaO80
: 5232.61
[1] R. A. Dwek, Chem. Rev. 1996, 96, 683–720.
[M+Na]+ (one of the highest isotope peaks), found: 5232.24.
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p-Tolyl (methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trichloroacetamido-
d-glycero-a-d-galacto-2-nonulopyranosonate-(2!3))-2-O-benzoyl-4,6-O-
benzylidene-b-d-galactopyranosonyl-(1!4)-3,6-di-O-benzyl-2-deoxy-2-
phthalimido-1-thio-b-d-glucopyranoside (3): See procedure for com-
pound 2. The one-pot reaction for synthesis of compound 2 was terminat-
ed prior to addition of acceptor 4. Compound 3 was purified from the re-
action mixture by flash column chromatography (hexanes/EtOAc/CH2Cl2
4:3:3) in 50 mg (82% yield). 1H NMR (600 MHz, CDCl3): d=8.15–8.12
(m, 2H), 7.87–7.84 (m, 1H), 7.81–7.77 (m, 1H), 7.70–7.66 (m, 2H), 7.63–
7.59 (m, 1H), 7.51–7.47 (m, 2H), 7.46–7.42 (m, 2H), 7.36–7.28 (m, 6H),
7.28–7.24 (m, 2H), 7.20–7.16 (m, 2H), 6.93–6.90 (m, 2H), 6.58 (d,
[4] T. Shinkawa, K. Nakamura, N. Yamane, E. Shoji-Hosaka, Y. Kanda,
M. Sakurada, K. Uchida, H. Anazawa, M. Satoh, M. Yamasaki, N.
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[5] Compound 1 has been characterized within an N-glycan mixture iso-
lated from carbohydrates attached to human g-interferon, see:
J. H. G. M. Mutsaers, J. P. Kamerling, R. Devos, Y. Guisez, W. Fiers,
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3J
3J
ACHTREUNG
A
ACHTREUNG
1H), 4.53 (dd, 3J
ACHTREUNG
[7] K. Taketa, Electrophoresis 1998, 19, 2595–2602.
3J
ACHTREUNG
[8] M. Tajiri, C. Ohyama, Y. Wada, Glycobiology 2008, 18, 2–8.
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2644.
AHCTREUNG
AHCTREUNG
[10] E. Watson, A. Bhide, H. van Halbeek, Glycobiology 1994, 4, 227–
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Danishefsky, Angew. Chem. 2007, 119, 7527–7531; Angew. Chem.
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12.6 Hz, 1H); 13C NMR (150 MHz, CDCl3): d=171.0, 170.59, 170.55,
170.1, 168.7, 168.2, 168.0, 165.1, 162.3, 138.8, 138.2, 137.7, 134.24, 134.18,
133.6, 133.4, 132.1, 132.0, 130.2, 130.1, 129.7, 129.2, 128.8, 128.5, 128.41,
128.35, 127.68, 127.66, 126.6, 123.9, 123.5, 101.7, 101.0, 96.9, 92.1, 83.6,
82.1, 78.2, 73.1, 72.8, 72.3, 72.0, 71.2, 70.0, 68.7, 68.6, 67.7, 67.5, 67.3, 66.7,
62.9, 60.7, 55.4, 53.2, 51.6, 38.8, 21.7, 21.31, 21.30, 20.9, 20.8 ppm; HRMS:
m/z: calcd for C68H69Cl3N2O24SNa: 1457.2924 [M+Na]+, found:
1457.2908. The Galb1!4GlcN linkage was confirmed by a HMBC corre-
lation between C1 of the galactose and H4 of the glucosamine unit.
5-Acetamido-3,5-dideoxy-d-glycero-a-d–galacto-2-nonulopyranosylo-
nate-(2!3)-b-d-galactopyranosyl-(1!4)-2-amino-2-deoxy-b-d-glucopyra-
nosyl-(1!2)-a-d-mannopyranosyl-(1!6)-(5-acetamido-3,5-dideoxy-d-
glycero-a-d-galacto-2-nonulopyranosylonate-(2!3)-b-d-galactopyrano-
7080
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 7072 – 7081

Oligosaccharides
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Received: April 21, 2008
Published online: July 7, 2008
Chem. Eur. J. 2008, 14, 7072 – 7081
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7081