From polymer to size-defined oligomers: A step economy process for the efficient and stereocontrolled construction of chondroitin oligosaccharides and biotinylated conjugates thereof: Part 1

Article abstract of DOI:10.1002/chem.200900740

Controlled acid hydrolysis of polymeric chondroitin sulfate of bovine origin afforded in good yield a basic disaccharide fragment that was used for the first time as a starting material for the expeditious preparation of a set of building blocks that in t

Full text of DOI:10.1002/chem.200900740

FULL PAPER
DOI: 10.1002/chem.200900740
From Polymer to Size-Defined Oligomers: A Step Economy Process for the
Efficient and Stereocontrolled Construction of Chondroitin Oligosaccharides
and Biotinylated Conjugates Thereof: Part 1
Aude Vibert, Chrystel Lopin-Bon, and Jean-Claude Jacquinet*[a]
Abstract: Controlled acid hydrolysis of
polymeric chondroitin sulfate of bovine
origin afforded in good yield a basic
disaccharide fragment that was used
for the first time as a starting material
for the expeditious preparation of a set
of building blocks that in turn act as
versatile synthons for the efficient and
stereocontrolled construction of a col-
lection of size-defined chondroitin olig-
omers (from di- to octasaccharides).
This step economy process allows their
preparation as reducing species, fitted
with a fluorophore, or as biotinylated
conjugates; all useful tools for the
preparation of microarrays, or as
probes for the study of the biosynthesis
of chondroitin sulfate.
Keywords: biotinylated sugars
carbohydrates chondroitin
glycosylation · oligosaccharides
·
·
·
Introduction
(ChPF).[6] However, the substrate specificity of these en-
zymes—that is, from which structure polymerization
begins—remains a matter of controversy.[7] Therefore, a
facile and efficient method for the synthesis of size-defined
chondroitin oligomers is urgently required. An in vitro enzy-
matic synthesis of chondroitin based on the polymerization
of an oxazoline derived from the basic disaccharide cata-
lyzed by hyaluronidase has been reported,[8] but this tech-
nique did not allow the preparation of size-defined small
fragments.
A defect in the biosynthesis of GAGs has been observed
in several pathological conditions such as cancer, atheroscle-
rosis, fibrosis, and osteoarthritis (OA), thereby leading, in
the latter case, to a progressive loss of cartilage tissue and
joint functions. OA is a very common degenerative joint dis-
ease and a leading cause of disability with an increasing so-
cioeconomic impact. Current pharmaceutical interventions
that address chronic pain are insufficient, and no proven dis-
ease-modifying therapy is available to date. Therefore, there
is an urgent need to develop new therapies by identifying
pharmacological targets to design new drugs. Aggrecan, a
CS polymer, is the major component of cartilage matrix,
and its anabolism is greatly affected by OA. In this context,
the glycosyltransferases involved in the polymerization of
CS chains are crucial enzymes whose activity determines the
biosynthesis rate.
Chondroitin sulfates (CSs) are naturally occurring heteropo-
lysaccharides that belong to a family of complex, polyanion-
ic, linear polymers called glycosaminoglycans (GAGs). They
exist predominantly as polysaccharide side chains of proteo-
glycans (PGs) in extracellular matrixes. They are built from
a repeating dimeric unit composed of d-glucuronic acid
(GlcA) and 2-acetamido-2-deoxy-d-galactose (GalNAc) ar-
ranged in the sequence [!4)-b-d-GlcpA-(1!3)-b-d-Galp-
NAc-(1!]n, and bearing sulfate groups at various posi-
tions.[1] They are widely distributed among various tissues
and exhibit a large variety of biological functions.[2] Biosyn-
thesis of chondroitin is catalyzed by specific glycosyltransfer-
ases[3] using UDP-GlcA and UDP-GalNAc as glycosyl
donors in the Golgi apparatus.[4] Recently, a human chon-
droitin synthase (ChSy) having both glucuronyltransferase
and N-acetylgalactosaminyltransferase activity was ex-
pressed and cloned,[5] but chondroitin polymerization was
not demonstrated. In fact, polymerizing activity requires
concomitant expression of a chondroitin polymerizing factor
[a] A. Vibert, Dr. C. Lopin-Bon, Dr. J.-C. Jacquinet
Institut de Chimie Organique et Analytique
UMR 6005 Universitꢀ d’Orlꢀans et CNRS
Universitꢀ d’Orlꢀans, BP 6759
45067 Orlꢀans Cedex (France)
Within a program devoted to the study of the biosynthetic
pathways of CS chains in relation with OA, we recently dis-
closed the possible chemical synthesis of size-defined chon-
droitin oligomers by semisynthesis starting from a CS poly-
Fax : (+33)2-3841-7281
E-mail: Jean-Claude.Jacquinet@univ-orleans.fr
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200900740.
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mer.[9] Herein we describe the development of this step
economy process for the straightforward construction of
nonsulfated chondroitin oligomers and their biotinylated
conjugates as probes for the study of the biosynthesis of CS.
pling reactions were planned to be performed using
Schmidtꢂs trichloroacetimidate glycosylation procedure,[14]
acetal 3 was transformed in a first route into the crystalline
a-imidate 5 by a classical sequence. This easily available gly-
cosyl donor was used systematically to introduce the last di-
saccharide unit at the nonreducing end in the synthesis of
oligomers. Reaction of imidate 5 with a set of alcohols
under the catalysis of trimethylsilyl triflate (TMSOTf) af-
forded exclusively the 1,2-trans-linked glycosides 6–8 in ex-
cellent yields. The 2-naphthylmethyl group[15] in 6 allowed
for flexibility because it should either be retained at the end
of the syntheses and serve as a fluorophore in biological
assays or easily removed to afford reducing species, and the
2-benzyloxycarbonylaminoethyl group in 8 was installed to
gain access to biotinylated conjugates, all these species
being useful tools for the preparation of microarrays. In an
alternative route, and within the scope to reduce the
number of steps, acetal 3 was transformed into crystalline a-
Results and Discussion
To prepare chondroitin oligomers, synthetic routes should
ideally provide ready access to starting materials and in-
volve a limited number of steps. The basic disaccharide re-
peating unit of CS is composed of d-galactosamine, a rare
and expensive sugar, and of d-glucuronic acid, a not easily
available species. A practical approach was found in the use
of natural CS polymer of bovine origin, now commercially
available at reasonable cost. In their seminal studies on the
structure of CS, Levene[10] and Meyer and Davidson[11] dem-
onstrated that acid hydrolysis of the native polymer afford
the basic disaccharide unit, a
process that also results in com-
plete desulfation and N-deace-
tylation. Thus, starting from a
heterogeneously sulfated poly-
mer of variable length, a single
synthon that still has the correct
backbone of the basic disac-
charide can be obtained in
good yield in a simple proce-
dure, and is easily transformed
into the crystalline methyl ester
1 (Scheme 1). This protocol was
reinvestigated and improved,
and can be now easily per-
formed on
a 50–100 g scale
without chromatographic sepa-
ration. Also relevant in 1 is the
possibility to substitute the free
amine group with a powerful
stereocontrolling auxiliary such
as the trichloroacetyl group,[12]
a process successfully applied
to the highly stereoselective
synthesis of 1,2-trans-2-amino-
2-deoxy-d-glycosides.[13] Conse-
quently, amine
1 was trans-
formed into trichloroacetamide
2, this latter compound being
obtained routinely in a 50–55%
overall yield from the polymer.
The necessity to lock the d-gal-
actosamine unit in the pyranose
Scheme 1. Semisynthesis of building blocks 6–8, 10, and 11. a) IR-120 [H+] resin, H2O; then 0.5m H2SO4,
1008C, 6 h; then 0.02m HCl in MeOH, 08C, 4 d; b) Cl3CCOCl, pyridine, 08C, 1 h; then CH2Cl2/MeOH/pyri-
dine, 4 h, 52% from the polymer; c) PhCHO, TFA, 24 h; then Ac2O/pyridine, 16 h, 68%; d) 75% TFA,
CH2Cl2, 08C, 4 h; then Ac2O/pyridine, 16 h, 80%; e) hydrazine acetate, DMF, 30 min; then Cl3CCN, DBU,
CH2Cl2, 30 min, 70% for 5, 60% for 9; f) alcohol, TMSOTf, CH2Cl2, 30 min, 87% for 6, 80% for 7 and 8;
g) alcohol, BF3·OEt2, CH2Cl2, ꢀ608!ꢀ208C, 3 h, 79% for 10, 72% for 11. TCA=trichloroacetyl, TFA=tri-
conformation through
a 4,6-
benzylidene acetal before ace-
tylation, thus avoiding the ex-
tensive formation of furanose
derivatives, is still being dis-
fluoroacetic acid, DBU=1,8-diazabicycloACTHNUGTRNEUNG[5.4.0]undec-7-ene, TMSOTf=trimethylsilyl trifluoromethanesulfo-
cussed.[9] Because crucial cou- nate, NAP=2-naphthylmethyl, Z=benzyloxycarbonyl.
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Stereocontrolled Construction of Size-Defined Oligomers
FULL PAPER
imidate 9. However, conformationally constrained activated
4,6-benzylidene derivatives in the d-galacto series are
known to lead to mixtures of glycosides despite the presence
of a participating group at C-2.[16] Indeed, attempted glyco-
sylation of 9 with several alcohols under the catalysis of
TMSOTf, in various solvents and different temperatures af-
forded, as expected, a,b mixtures of glycosides. But chang-
ing the catalyst for a softer Lewis acid (boron trifluoride
ethyl etherate) and lowering the temperature (ꢀ608C) al-
lowed imidate 9 to react smoothly through an SN2-like
mechanism and to afford the 1,2-trans-linked glycosides 10
and 11 in 79 and 72% yields, respectively.
With building blocks 6 and 10 in hand, we first focused on
their transformation into glycosyl donors and acceptors suit-
able for an expeditious construction of target oligosacchar-
ides (Scheme 2). For comparative reasons, both acetyl- and
benzoyl-protected derivatives were prepared. Zemplꢃn
transesterification of 6 and 10 followed by isopropylidena-
tion under kinetic control afforded the acetals 12 and 24, re-
spectively, as major products. The selectively removable lev-
ulinoyl (Lev) group was then installed at the C-4 position of
the d-GlcA moiety to allow further elongation at the nonre-
ducing end. Acid hydrolysis of 13 and 25 followed either by
acetylation or benzoylation gave the esters 14 and 15, re-
spectively, which were now ready to be transformed into a
set of glycosyl donors and acceptors. Interestingly, the 2,3-
isopropylidene acetal in 25 could be selectively removed to
give the crystalline diol 26 in 79% yield. This tailor-made
derivative, easily prepared in only 11 steps from the poly-
mer, will be used as a single starting material for the highly
divergent chemical synthesis of all known variants (A, C, D,
E, K, L, and M) of CS oligomers (see Part 2).[17] Both deriv-
atives 14 and 15 were either transformed into acceptors 16
and 17 through selective cleavage of the levulinoyl group
with hydrazine acetate,[18] or
into donors 18 and 19 through
oxidative removal[19] of the
NAP glycoside with 2,3-di-
chloro-5,6-dicyanobenzoqui-
none (DDQ) followed by imi-
doylation. Noticeable was the
possibility to install the 2-ben-
zyloxycarbonylaminoethyl
group at this point, thus avoid-
ing having to repeat the same
sequence of reactions starting
from glycoside 11. Delevulinoy-
lation of 20 and 21 afforded the
acceptors 22 and 23, respective-
ly, in good yields.
The construction of size-de-
fined reducing chondroitin olig-
omers was then achieved as fol-
lows (Scheme 3). A coupling re-
action of acceptor 16 with a
moderate excess (1.5 equiv) of
imidate 5 under the catalysis of
TMSOTf gave the crystalline
tetrasaccharide derivative 27,
whereas its reaction with the
levulinoylated imidate 18 under
similar conditions afforded the
corresponding derivative 28,
both in 57% yield. It is to be
noted that no 1,2-cis-linked spe-
cies could be isolated in these
and further described coupling
reactions, and that characteris-
Scheme 2. Transformation of building blocks into glycosyl donors and acceptors. a) NaOMe, MeOH; then 2-
methoxypropene, CSA, DMF, 2 h, 65% for 12, 70% for 24; b) LevOH, DCC, DMAP, CH2Cl2, 1 h, 90% for
13, 92% for 25; c) 60% AcOH, 1008C, 1 h; then Ac2O/pyridine, 16 h, 71% for 14; or PhCOCl/pyridine, 08C,
2 h, 56% for 15; d) 80% AcOH, CH2Cl2, 24 h, 79%; e) hydrazine acetate, pyridine, 8 min, 89% for 16 and 17,
80% for 22, 75% for 23; f) DDQ, CH2Cl2/MeOH, 24 h; then Cl3CCN, DBU, CH2Cl2, 30 min, 66% for 18,
74% for 19; g) 2-benzyloxycarbonylaminoethanol, TMSOTf, CH2Cl2, 30 min, 54% for 20, 89% for 21.
LevOH=4-oxopentanoic acid, DCC=N,N-dicyclohexylcarbodiimide, DMAP=4-dimethylaminopyridine,
DDQ=2,3-dichloro-5,6-dicyanobenzoquinone.
tic coupling constants (J1,2
ꢁ7.5–8.0 Hz) were observed in
1
the H NMR spectra of all olig-
omers for the newly established
1,2-trans-linkages. Delevulinoy-
lation of 28 gave the crystalline
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J.-C. Jacquinet et al.
suitable in terms of reactivity,
stability, and ease of handling
than their acetylated congeners.
For these reasons, the crystal-
line hexasaccharide acceptor 34
was readily prepared through a
similar sequence starting from
the benzoylated acceptor 23
and using imidate 19 in an iter-
ative manner in more than
60% yield for each coupling re-
action. Condensation of 34 with
imidate 5 afforded the crystal-
line octasaccharide derivative
35 in a moderate 40% yield
(not optimized).
Final deprotection of oligo-
mers 6, 27, 30, and 35
(Scheme 4) was achieved first
through transformation of their
N-trichloroacetyl groups into
their N-acetyl congeners by
radical reduction with tri-n-
butyl stannane[12] to give the
crystalline acetamides 36–39,
followed by a two-step saponifi-
cation process avoiding an b-
elimination reaction at the d-
GlcA units with lithium hydro-
peroxide[20] and sodium hydrox-
ide to afford the target glyco-
sides 40–43. The 1H and
13C NMR spectra for 40–43
Scheme 3. Construction of the size-defined oligomers. a) TMSOTf, CH2Cl2, 30 min, 57% for 27 and 28, 62%
for 31; b) hydrazine acetate, pyridine, 8 min, 77% for 29, 69% for 32, 72% for 34; c) 5, TMSOTf, CH2Cl2,
30 min, 65% for 30, 40% for 35; d) 19, TMSOTf, CH2Cl2, 30 min, 65%.
alcohol 29, which upon further coupling with imidate 5 gave
the crystalline hexasaccharide derivative 30 in 65% yield.
However, attempted coupling of 29 with the levulinoylated
imidate 18 gave low yield of the expected corresponding
hexasaccharide derivative. At this point, it became clear
that benzoylated species (donors and acceptors) were more
showed high purity and were in full agreement with the ex-
pected structures. Catalytic hydrogenation of 40–43 with
10% palladium on carbon in water smoothly afforded the
reducing oligomers 44–47 in excellent yields. It was the first
time that these oligomers were obtained as pure species and
fully characterized.
Scheme 4. Access to target chondroitin oligomers. a) Bu3SnH, AIBN, benzene/DMAC, 808C, 2 h, 74% for 36, 81% for 37, 64% for 38, 62% for 39;
b) LiOH/H2O2, THF, ꢀ108C!RT, 16 h; then 4m NaOH, MeOH, 4 h, 69% for 40, 71% for 41, 51% for 42, 60% for 43; c) H2, Pd/C, H2O, 20 h, 86% for
44, 87% for 45, 74% for 46, 67% for 47. AIBN=2,2’-azobis-2-methylpropionitrile, DMAC=N,N-dimethylacetamide.
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Stereocontrolled Construction of Size-Defined Oligomers
FULL PAPER
In light of the above results, the preparation of size-de-
fined biotinylated chondroitin oligomers was next undertak-
en (Scheme 5). According to our synthetic plan, a similar
strategy should be used for the construction of even-num-
bered oligomers (from di- to octasaccharides), and several
common intermediates should be employed for the prepara-
tion of odd-numbered oligomers (tri-, penta-, and heptasac-
charides), in which the introduction of a d-GalNAc residue
at the nonreducing end should be achieved by the use of
known a-imidate 49.[16] A coupling reaction of imidate 49
with alcohol 23 under the catalysis of TMSOTf afforded the
trisaccharide derivative 50 as a single isomer, albeit in a
moderate 55% yield due to difficulties in purification of the
reaction mixture. The acetylated tetrasaccharide derivative
52 was similarly obtained in 67% yield from imidate 5 and
alcohol 22. For the construction of larger molecules, benzoy-
lated species were previously found to be more efficient.
Thus, a coupling reaction between imidate 19 and alcohol
23, both benzoylated, provided the crystalline tetrasacchar-
ide derivative 54 in 87% yield, which upon delevulinoyla-
tion gave the pivotal acceptor 55. The latter was condensed
with imidates 49 and 5, respectively, to afford the crystalline
pentasaccharide 56 and hexasaccharide derivative 58 in 85
and 55% yields, respectively. Further coupling of 55 with
Scheme 5. Preparation of precursors of odd- and even-numbered biotinylated chondroitin oligomers. a) Bu3SnH, AIBN, benzene/DMAC, 808C, 4 h, 53 %
for 48, 55% for 51, 65% for 53, 59% for 57, 78% for 59, 77% for 63, 53% for 65; b) TMSOTf, CH2Cl2, 30 min, 55% for 50, 67% for 52, 87% for 54;
c) hydrazine acetate, pyridine, 8 min, 75% for 55, 83% for 61; d) 49, TMSOTf, CH2Cl2, 30 min, 85% for 56, 67% for 62; e) 5, TMSOTf, CH2Cl2, 30 min,
55% for 58, 65% for 64; f) 19, TMSOTf, CH2Cl2, 30 min, 66%.
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J.-C. Jacquinet et al.
imidate 19 gave the crystalline hexasaccharide 60 in 66%
yield. Delevulinoylation of 60 afforded the crystalline alco-
hol 61, which was similarly condensed with imidates 49 and
5, respectively, to provide the crystalline heptasaccharide 62
and octasaccharide derivative 64 in 67 and 65% yields, re-
spectively. Radical reduction of the N-trichloroacetyl groups
in oligomers 8, 50, 52, 56, 58, 62, and 64 gave the corre-
sponding crystalline acetamides 48, 51, 53, 57, 59, 63, and 65
in 53–78% yields, depending on the ease of purification of
these complex molecules. Saponification of the latter group
(Scheme 6), as described above, provided the sodium salts
Conclusion
To conclude, we designed a step economy process for the
expeditious preparation for the first time of size-defined
chondroitin oligomers and their biotinylated conjugates.
This semisynthetic route starting from an abundant natural
polymer should also be applied to other biologically impor-
tant members of the GAG family such as dermatan sulfate
and hyaluronic acid. The glycosylating efficiency and stereo-
selectivity exhibited by 2-deoxy-2-trichloroacetamido-d-gal-
acto trichloroacetimidate derivatives allowed the straightfor-
ward construction in good
yields of oligomers up to octa-
saccharides, and the crystallini-
ty of most intermediates ren-
dered this route very attractive.
For the first time, all target
compounds, reducing species,
and glycosides as well as biotin-
ylated conjugates, were ob-
tained in high purity and fully
characterized through 1H and
13C NMR spectrometry. We do
hope that with such a collection
of potential probes we shall be
able to determine the starting
point of the polymerization re-
action in the biosynthesis of
chondroitin and identify new
pharmacological targets. All
these structures are currently
being investigated in biological
assays, the results of which will
be reported elsewhere in due
course.
Scheme 6. Access to target biotinylated chondroitin oligomers. a) LiOH/H2O2, THF, ꢀ108C!RT, 16 h; then
4m NaOH, MeOH, 4 h, 67% for 66, 68% for 76, 62% for 68, 58% for 69, 71% for 70, 95% for 71, 84% for
72; b) H2, Pd/C, H2O, 20 h; then 6-biotinylamidohexanoic acid N-hydroxysuccinimidyl ester, NEt3, DMF/H2O,
1 h, 67% for 73, 71% for 74, 72% for 75 and 77, 75% for 76, 85% for 78, 89% for 79.
Experimental Section
General methods: To prepare chon-
droitin sulfate,
a sodium salt from
bovine trachea and 6-biotinylamido-
hexanoic acid N-hydroxysuccinimidyl
ester were purchased from Sigma
66–72 in good yields. Finally, catalytic hydrogenation of 66–
SAFC (St-Quentin-Fallavier, France). Solvents were dried by standard
methods, and molecular sieves were activated prior to use by heating for
4 h at 5008C. Melting points were determined in capillary tubes with a
Bꢄchi apparatus and are uncorrected. Optical rotations were measured at
72 led to the corresponding free amine intermediates, which
were readily treated with 6-biotinylamidohexanoic acid N-
hydroxysuccinimidoyl ester and triethylamine in N,N-dime-
thylformamide/water to afford, after purification using a Se-
phadex LH-20 in water, the target molecules 73–79 in 67–
89% yields, for which the 1H and 13C NMR spectroscopic
data showed high purity and were in full agreement with the
expected structures.
1
20–258C using a Perkin–Elmer 341 polarimeter. H and 13C NMR spectra
were recorded at 258C with Bruker DPX 250 and Advance II 400 instru-
ments, with Me4Si as internal standard, unless otherwise stated. Assign-
ments were based on homo- and heteronuclear correlations using the
supplierꢂs software. In the NMR data for oligomers, GlcAI and GalNI
(with roman numeral superscripts) refer to d-glucuronic acid and d-gal-
actosamine residues, respectively, in the reducing disaccharide unit. Low-
resolution mass spectra were obtained on a Perkin–Elmer SCIEX API
300 spectrometer operating in the ion-spray mode or on a Micromass
Quattro Ultima spectrometer equipped with a Z-spray ionization source
operating in the positive or negative mode. HRMS data were obtained
from the Centre Regional de Mesure Physique (Universitꢀ Blaise Pascal,
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Stereocontrolled Construction of Size-Defined Oligomers
FULL PAPER
Clermont-Ferrand, France). Flash-silica chromatography was performed
on silica gel 60 (0.040–0.063 mm, Merck, Darmstadt). The reactions were
ture was extracted with CH2Cl2 (2ꢅ300 mL), and the organic layer was
washed with water, saturated aqueous NaHCO3, and water, and dried
(MgSO4) and concentrated. Flash silica chromatography (CH2Cl2!
CH2Cl2/acetone 15:1) gave first the a-acetate 3a (24.4 g, 60%): m.p. 126–
1278C (from EtOAc/petroleum ether); [a]2D2 =+49 (c=1 in chloroform);
monitored by TLC on coated aluminum sheets (silica gel 60 GF254
,
Merck), and spots were detected under UV light and by charring with a
95:5 mixture of ethanol and sulfuric acid. Elemental analyses were car-
ried out at the Service Central de Microanalyse du CNRS (Vernaison,
France).
1H NMR (250 MHz, CDCl3): d=7.50 (m, 5H; arom. H), 6.78 (d, J2,NH
=
7.5 Hz, 1H; GalN NH), 6.48 (d, J1,2 =3.6 Hz,1H; GalN H-1), 5.54 (s, 1H;
CHPh), 5.24 (m, 2H; GlcA H-3, H-4), 5.08 (dd, J1,2 =8.0 Hz, J2,3 =9.0 Hz,
1H; GlcA H-2), 4.95 (d, 1H; GlcA H-1), 4.64 (dd, J2,3 =11.0 Hz; GalN
H-2), 4.51 (dd, J3,4 =3.5 Hz, J4,5 <1.0 Hz; GalN H-4), 4.45 (dd, 1H; GalN
H-3), 4.20 (m, 3H; 2GalN H-6, GlcA H-5), 3.85 (m, 1H; GalN H-5),
3.74 (s, 3H; COOCH3), 2.18, 2.05 ppm (2s, 12H; COCH3); MS: m/z: 762
[M+Na]+ (for 35Cl); elemental analysis calcd (%) for C30H34Cl3NO16: C
46.74, H 4.44, N 1.81; found: C 46.50, H 4.33, N 1.78. Next eluted
(CH2Cl2/acetone 12:1) was the b-acetate 3b (3.46 g, 8%): m.p. 237–2398C
(from EtOH); [a]2D2 =+25 (c=1 in chloroform); 1H NMR (250 MHz,
CDCl3): d=7.50 (m, 5H; arom. H), 6.90 (d, J2,NH =8.0 Hz, 1H; GalN
NH), 6.05 (d, J1,2 =8.5 Hz; GalN H-1), 5.58 (s, 1H; PhCH), 5.20 (m, 2H;
GlcA H-3,H-4), 5.05 (dd, J1,2 =7.5 Hz, J2,3 =9.0 Hz, 1H; GlcA H-2), 4.93
(d, 1H; GlcA H-1), 4.55 (dd, J2,3 =11.0 Hz, J3,4 =3.5 Hz, 1H; GalN H-3),
O-(Methyl b-d-glucopyranosyluronate)-(1!3)-2-amino-2-deoxy-d-galac-
topyranose hydrochloride (1): A solution of chondroitin sulfate A (50 g)
in water (500 mL) was brought to pH 1.6 with Amberlite IR-120 [H+]
resin (pH meter monitoring), and was then filtered. The resin was
washed with water (4ꢅ100 mL), and the volume of the filtrate was ad-
justed to 970 mL. Concentrated H2SO4 (18m, 27.8 mL, 0.5m final concen-
tration) was added, and the mixture was stirred for 6 h at 1008C, then
was cooled. Solid Ba(OH)2·8H2O was added portionwise under vigorous
stirring to pH 3.5 (pH meter monitoring), and the slurry was allowed to
settle overnight. The solids were filtered off through a Celite pad,
washed with water, and the yellow filtrate was concentrated to approxi-
mately 500 mL and slowly applied to a column of Amberlite IR-120 [H+]
resin (500 mL, settled volume). The column was washed with water (1 L),
AcOH/water (3:1, 1 L), then with aqueous HCl (1m, 3 L). The fractions
containing ninhydrin-positive material were pooled, concentrated, evapo-
rated with water (2ꢅ500 mL), and dried under vacuum over P2O5. The
residue was treated with methanolic HCl (0.02m, 500 mL) for 4 d at 08C,
then was concentrated. Repeated additions of absolute EtOH and con-
centrations gave crude 1 (~38 g). A portion was recrystallized from
EtOH to give pure 1. Rf =0.3 (EtOAc/MeOH/water 1:1:1); m.p. 160–
1638C (ref. [11]: 159–1618C); [a]2D2 =+40 (c=1, equil., in water)
(ref. [11]: +42 (c=2 in water); 1H NMR (400 MHz, D2O, internal H2O,
dH =4.79): d=5.48 (d, J1,2 =3.5 Hz; GalN H-1a), 4.72 (d, J1,2 =8.0 Hz;
GalN H-1b), 4.73, 4.72 (2d, J1,2 =7.5 Hz, 1H; GlcA H-1a,b), 4.30–4.20
(m; GalN H-3a, H-4, GlcA H-5), 4.15–4.06 (m; GalN H-3b, GlcA H-4),
4.47 (dd, J4,5 <1.0 Hz, 1H; GalN H-4), 4.32 (dd, J5,6a =2.5 Hz, J6a,6b
=
12.0 Hz, 1H; GalN H-6a), 4.10 (m, 3H; GalN H-2, H-6b, GlcA H-5),
3.73 (s, 3H; COOCH3), 3.67 (m, 1H; GalN H-5), 2.16, 2.01 ppm (2s,
12H; COCH3); MS: m/z: 762 [M+Na]+ (for 35Cl); elemental analysis
calcd (%) for C30H34Cl3NO16: C 46.74, H 4.44, N 1.81; found: C 46.60, H
4.51, N 1.81.
O-(Methyl 2,3,4-tri-O-acetyl-b-d-glucopyranosyluronate)-(1!3)-1,4,6-tri-
O-acetyl-2-deoxy-2-trichloroacetamido-d-galactopyranose (4): A mixture
of 3a,b (19.25 g, 25 mmol), CH2Cl2 (400 mL), and TFA/water (3:1,
15 mL) was stirred for 4 h at 08C, then was washed with 10% aqueous
NaOAc, saturated aqueous NaHCO3 and water, dried (MgSO4), and con-
centrated. The solid residue was stirred with pyridine (150 mL) and Ac2O
(100 mL) for 16 h at RT, then was concentrated and evaporated with tol-
uene. Flash-silica chromatography (EtOAc/petroleum ether 3:2) gave the
peracetate 4 (15.5 g, 80%) as a white solid. a-anomer 4a: Rf =0.4
(EtOAc/petroleum ether 2:1); m.p. 181–1828C (from EtOAc/petroleum
ether); [a]2D2 =+41 (c=1 in chloroform); 1H NMR (250 MHz, CDCl3):
d=6.97 (d, J2,NH =7.5 Hz, 1H; GalN NH), 6.37 (d, J1,2 =3.5 Hz, 1H;
GalN H-1), 5.35 (dd, J3,4 =3.5 Hz, J4,5 <1.0 Hz, GalN H-4), 5.15 (m, 3H;
3.82 (s, 3H; COOCH3), 3.80–3.70 (m; GalN H-5, 2H-6), 3.65 (dd, J2,3
=
11.0 Hz; GalN H-2a), 3.56 (m; GlcA H-3a,b), 3.45 (m; GlcA H-2a,b),
3.34 ppm (dd, J2,3 =11.0 Hz; GalN H-2b); 13C NMR (100 MHz, D2O, in-
ternal acetone, dC =30.83): d=171.60, 171.54 (GlcA C-6), 104.16, 103.91
(GlcA C-1), 93.46 (GalN C-1b), 89.93 (GalN C-1a), 80.11, 78.01 (GalN
C-3), 75.43, 75.36 (GalN C-5), 75.02, 74.96 (GlcA C-5), 73.20, 73.18
(GlcA C-3), 71.67, 71.61 (GlcA C-2), 70.75, 70.71 (GlcA C-4), 68.04, 68.0
(GalN C-4), 61.55, 61.31 (GalN C-6), 53.73 (COOCH3), 53.50, 50.40 ppm
(GalN C-2).
GlcA H-2, H-3, H-4), 4.84 (d, J1,2 =8.0 Hz; GlcA H-1), 4.51 (m, J2,3
=
11.0 Hz, 1H; GalN H-2), 4.41 (dd, 1H; GalN H-3), 4.27 (m, 1H; GalN
H-5), 4.08 (m, 3H; GalN H-6a,H-6b, GlcA H-5), 3.76 (s, 3H; COOCH3),
2.15, 2.12, 2.02 ppm (3s, 18H; COCH3); MS: m/z: 788 [M+Na]+ (for
35Cl); elemental analysis calcd (%) for C27H34Cl3NO18: C 42.28, H, 4.47,
N 1.83; found: C 42.12, H 4.60, N 1.71. b-anomer 4b: Rf =0.35 (EtOAc/
petroleum ether 2:1); m.p. 228–2298C (from EtOAc-petroleum ether);
[a]2D2 =+10 (c=1 in chloroform); 1H NMR (250 MHz, CDCl3): d=6.83
(d, J2,NH =8.5 Hz, 1H; GalN NH), 5.89 (d, J1,2 =8.5 Hz, 1H; GalN H-1),
5.44 (dd, J3,4 =3.5 Hz, J4,5 <1.0 Hz, 1H; GalN H-4), 5.20–5.16 (m, 2H;
GlcA H-3, H-4), 4.97 (dd, J1,2 =7.5 Hz, J2,3 =9.0 Hz, 1H; GlcA H-2), 4.76
(d, 1H; GlcA H-1), 4.36 (dd, J2,3 =11.0 Hz, 1H; GalN H-3), 4.25–4.05 (m,
5H; GalN H-2,H-5, H-6a,H-6b, GlcA H-5), 3.76 (s, 3H; COOCH3), 2.12,
2.08, 1.96 ppm (3 s, 18H; COCH3); MS: m/z: 788 [M+Na]+ (for 35Cl); el-
emental analysis calcd (%) for C27H34Cl3NO18: C 42.28, H 4.47, N 1.83;
found: C 42.30, H 4.56, N 1.77.
O-(Methyl b-d-glucopyranosyluronate)-(1!3)-2-deoxy-2-trichloroacet-
amido-d-galactopyranose (2): Trichloroacetyl chloride (112 mL, 1 mol)
was added slowly at 08C to a solution of crude 1 (36.2 g, 89 mmol) in pyr-
idine (400 mL), and the mixture was stirred for 1 h at 08C. Water
(30 mL) was carefully added dropwise, and the mixture was diluted with
CH2Cl2 (800 mL), rapidly washed with water, saturated aqueous NaCl,
and water, and dried (MgSO4) and concentrated. A solution of the resi-
due in CH2Cl2/MeOH/pyridine (1:1:1, 300 mL) was stirred for 4 h at RT,
then was concentrated. Flash silica chromatography (CH2Cl2/MeOH 4:1)
gave the trichloroacetamide 2 (26.85 g, 52% from the polymer) as a pale
yellow foam. [a]2D2 =+35 (c=1 in methanol); 1H NMR (400 MHz, D2O,
internal H2O, dH =4.79): d=5.30 (d, J1,2 3.5 Hz; GalN H-1a), 4.72 (d, J1,2
8.0 Hz; GalN H-1b), 4.68, 4.59 (2d, J1,2 =8.0 Hz, 1H; GlcA H-1), 4.34
(dd,
J2,3 =11.0 Hz; GalN H-2a), 4.24 (dd, J2,3 =11.0 Hz, J3,4 <1.0 Hz;
O-(Methyl 2,3,4-tri-O-acetyl-b-d-glucopyranosyluronate)-(1!3)-4,6-di-O-
acetyl-2-deoxy-2-trichloroacetamido-1-O-trichloroacetimidoyl-a-d-galac-
topyranose (5): A mixture of 4a,b (1.92 g, 2.5 mmol) and hydrazine ace-
tate (0.35 g, 3.75 mmol) in DMF (15 mL) was stirred for 30 min at RT,
then was diluted with EtOAc (80 mL), washed with water, saturated
aqueous NaCl and water, dried (MgSO4), and concentrated. A mixture of
the residue, CCl3CN (2 mL, 20 mmol), and DBU (57 mL, 0.38 mmol) in
CH2Cl2 (15 mL) was stirred for 30 min at RT, then was concentrated.
Flash-silica chromatography (EtOAc/petroleum ether 1:1, containing
0.1% of Et3N) and crystallization from EtOAc/petroleum ether gave the
imidate 5 (1.52 g, 70%). M.p. 117–1188C; [a]2D2 =+62 (c=1 in chloro-
form); 1H NMR (250 MHz, CDCl3): d=8.78 (s, 1H; C=NH); 6.88 (d,
GalN H-3a), 4.12 (m, 1H; GalN H-4), 4.08–4.0 (m; GalN H-2b, H-3b, H-
5, GlcA H-5), 3.82, 3.81 (2s, 3H; COOCH3), 3.80–3.68 (m, 2H; 2 GalN
H-6), 3.54 (dd, J3,4 =J4,5 =9.5 Hz, 1H; GlcAH-4), 3.48, 3.77 (2dd, J2,3
=
9.5 Hz,1H; GlcA H-3), 3.37, 3.35 ppm (2dd, 1H; GlcA H-2); MS: m/z:
535 [M+Na]+ (for 35Cl); elemental analysis calcd (%) for C15H21Cl3NO12
:
C 35.07, H 4.12, N 2.73; found: C 34.80, H 4.31, N 2.54.
O-(Methyl
2,3,4-tri-O-acetyl-b-d-glucopyranosyluronate)-(1!3)-1-O-
acetyl-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-d-galactopyra-
nose (3): A mixture of 2 (26.8 g, 52 mmol), benzaldehyde (200 mL), and
TFA (10 mL) was stirred for 24 h at RT under an atmosphere of dry
argon. Anhydrous NaOAc (16.4 g, 0.2 mol), pyridine (200 mL), and
Ac2O (120 mL) were added sequentially, and the mixture was stirred
overnight, then poured into ice-cold water and stirred for 2 h. The mix-
J
2,NH =8.5 Hz, 1H; GalN NH), 6.54 (d, J1,2 =3.5 Hz, 1H; GalN H-1), 5.46
(dd, J3,4 =3.5 Hz, J4,5 <1.0 Hz, 1H; GalN H-4), 5.18 (m, 2H; GlcA H-3,
Chem. Eur. J. 2009, 15, 9561 – 9578
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9567

J.-C. Jacquinet et al.
H-4), 5.05 (d, J1,2 =8.0 Hz, J2,3 =9.0 Hz, 1H; GlcA H-2), 4.81 (d, 1H;
GlcA H-1), 4.64 (m, 1H; GalN H-2), 4.30 (m, 2H; GalN H-3, H-5), 4.10
(m, 3H; GalN H-6a, H-6b, GlcA H-5), 3.74 (s, 3H; COOCH3), 2.15, 2.08,
2.01 ppm (3s, 15H; COCH3); MS: m/z: 885 [M+NH4]+ (for 35Cl); ele-
mental analysis calcd (%) for C27H32Cl6N2O17: C 37.31, H 3.71, N 3.12;
found: C 37.19, H 3.82, N 3.01.
ethyl ether afforded the imidate 9 (10.48 g, 60%). M.p. 131–1328C;
[a]2D0 =+67 (c=1 in chloroform); 1H NMR (250 MHz, CDCl3): d=8.75
(s, 1H; C=NH), 7.50–7.20 (m, 5H; arom. H), 6.79 (d, J2,NH =8.0 Hz, 1H;
GalN NH), 6.62 (d, J1,2 =3.5 Hz, 1H; GalN H-1), 5.57 (s, 1H; CHPh),
5.26 (dd, J2,3 =J3,4 =9.5 Hz, 1H; GlcA H-3), 5.20 (dd, J4,5 =9.5 Hz, 1H;
GlcA H-4), 5.09 (d, J1,2 =7.5 Hz, 1H; GlcA H-2), 4.95 (d, 1H; GlcA H-
1), 4.77 (m, J2,3 =11.0 Hz, 1H; GalN H-2), 4.56 (dd, J3,4 =3.0 Hz, J4,5
<
2-Naphthylmethyl O-(methyl 2,3,4-tri-O-acetyl-b-d-glucopyranosyluro-
nate)-(1!3)-4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido-b-d-galacto-
pyranoside (6): A mixture of imidate 5 (4.0 g, 4.60 mmol), 2-naphthalene-
methanol (1.46 g, 9.2 mmol), and 4 ꢆ powdered molecular sieves (1.0 g)
in dry CH2Cl2 (40 mL) was stirred for 1 h at RT under dry argon. A solu-
tion of TMSOTf in toluene (1m, 0.69 mL) was added, and the mixture
was stirred for 30 min at RT, then was quenched with NEt3 (0.3 mL), fil-
tered and concentrated. Flash-silica chromatography (EtOAc/petroleum
ether 1:1, containing 0.1% of NEt3) gave the disaccharide 6 (3.46 g,
87%) as a white powder. M.p. 120–1228C (from 2-propanol); [a]2D2 =ꢀ21
1.0 Hz, 1H; GalN H-4), 4.40–4.30 (m, 2H; GalN H-3, H-6), 4.12–4.08 (m,
2H; GalN H-6, GlcA H-5), 3.92 (m, 1H; GalN H-5), 3.72 (s, 3H;
COOCH3), 2.02 ppm (s, 9H; COCH3); MS: m/z: 893 [M+H]+ (for 35Cl);
elemental analysis calcd (%) for C30H32Cl6N2O15: C 41.26, H 3.69, N 3.21;
found: C 41.11, H 3.58, N 3.09.
2-Naphthylmethyl O-(methyl 2,3,4-tri-O-acetyl-b-d-glucopyranosyluro-
nate)-(1!3)-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-b-d-galac-
topyranoside (10): A mixture of imidate 9 (16.60 g, 19 mmol), 2-naphtha-
lenemethanol (4.75 g, 30 mmol), and 4 ꢆ powdered molecular sieves
(2.0 g) in dry CH2Cl2 (150 mL) was stirred for 1 h at RT under dry argon,
then was cooled to ꢀ608C. A solution of BF3·OEt2 in toluene (1m,
1.9 mL) was added, and the mixture was stirred from ꢀ60 to ꢀ208C
within 3 h, then was quenched with NEt3 (1 mL), filtered, and concentrat-
ed. Flash-silica chromatography (CH2Cl2/EtOAc 10:1, containing 0.1%
of NEt3) gave the glycoside 10 (13.05 g, 79%) as a white solid. Rf =0.35
(EtOAc/petroleum ether 3:2); m.p. 235–2368C (from EtOAc/petroleum
ether); [a]2D2 =ꢀ12 (c=1 in chloroform); 1H NMR (250 MHz, CDCl3):
d=7.70–7.30 (m, 12H; arom. H), 7.07 (d, J2,NH =7.5 Hz, 1H; GalN NH),
5.61 (s, 1H; CHPh), 5.22 (dd, J2,3 =J3,4 =9.5 Hz, 1H; GlcA H-3), 5.16 (d,
1
(c=1 in chloroform); H NMR (250 MHz, CDCl3): d=7.78–7.40 (m, 7H;
arom. H), 6.91 (d, J2,NH =7.5 Hz, 1H; GalN NH), 5.45 (dd, J3,4 =3.0 Hz,
J
4,5 <1.0 Hz, 1H; GalN H-4), 5.20–5.10 (m, 2H; GlcA H-3, H-4), 4.95
(dd, 1H; GlcA H-2), 4.92 (d, J1,2 =8.5 Hz, 1H; GalN H-1), 4.91 (ABq,
2H; CH2Ar), 4.69 (d, J1,2 =7.5 Hz, 1H; GlcA H-1), 4.51 (dd, J2,3
11.0 Hz, 1H; GalN H-3), 4.20–4.08 (m, 2H; GalN H-6a, H-6b), 3.97 (d,
4,5 =9.5 Hz, 1H; GlcA H-5), 3.89–3.83 (m, 2H; GalN H-2, H-5), 3.73 (s,
=
J
3H; COOCH3), 2.13, 2.10, 2.02, 1.99, 1.98 ppm (5s, 15H; COCH3); MS:
m/z: 886 [M+Na]+ (for 35Cl); elemental analysis calcd (%) for
C36H40Cl3NO17: C 49.98, H 4.66, N 1.62; found: C 49.84, H 4.82, N 1.55.
J
1,2 =8.0 Hz, 1H; GalN H-1), 5.15 (dd, J4,5 =9.5 Hz, 1H; GlcA H-4), 5.04
4-Pentenyl O-(methyl 2,3,4-tri-O-acetyl-b-d-glucopyranosyluronate)-(1!
3)-4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido-b-d-galactopyranoside
(d, J1,2 =7.5 Hz, 1H; GlcA H-2), 4.95 (ABq, 2H; CH2Ar), 4.90 (d, 1H;
GlcA H-1), 4.67 (dd, J2,3 =11.0 Hz, J3,4 =3.5 Hz, 1H; GalN H-3), 4.46–
4.28 (m, 2H; GalN H-4, H-6a), 4.03 (dd, J5,6a =2.5 Hz, J6a,6b =12.0 Hz,
1H; GalN H-6b), 4.0 (d, 1H; GlcA H-58, 3.90 (m, 1H; GalN H-2), 3.71
(s, 3H; COOCH3), 3.54 (m, 1H; GalN H-5), 2.05, 2.03, 2.01 ppm (3s,
9H; COCH3); MS: m/z: 891 [M+Na]+ (for 35Cl); elemental analysis
calcd (%) for C39H40Cl3NO15: C 53.90, H 4.64, N 1.61; found: C 53.79, H
4.68, N 1.49.
(7): From imidate
5 (1.2 g, 1.38 mmol) and 4-penten-1-ol (290 mL,
2.76 mmol) as described for the preparation of disaccharide 6. Flash-
silica chromatography (EtOAc/petroleum ether 1:1, containing 0.1% of
NEt3) afforded the disaccharide 7 (874 mg, 80%) as a white powder.
[a]2D0 =+1.5 (c=1 in chloroform); 1H NMR (250 MHz, CDCl3): d=7.01
(d, J2,NH =7.5 Hz, 1H; GalN NH), 5.77 (m, 1H; Pentenyl H-d), 5.47 (dd,
J
3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalN H-4), 5.23–5.11 (m, 2H; GlcA H-4,
Pentenyl H-e), 5.03–4.90 (m, 3H; GlcA H-2, H-3, Pentenyl H-e), 4.86 (d,
1,2 =8.0 Hz, 1H; GalN H-1), 4.74 (d, J1,2 =7.5 Hz, 1H; GlcA H-1), 4.60
2-Benzyloxycarbonylaminoethyl O-(methyl 2,3,4-tri-O-acetyl-b-d-gluco-
pyranosyluronate)-(1!3)-4,6-O-benzylidene-2-deoxy-2-trichloroacetami-
do-b-d-galactopyranoside (11): From imidate 9 (419 mg, 0.48 mmol) as
described for the preparation of 10. Flash silica chromatography
(CH2Cl2/2-propanol 19:1, containing 0.1% of NEt3) afforded the glyco-
side 11 (312 mg, 72%) as a white powder. M.p. 190–1928C (from EtOAc/
petroleum ether); [a]2D0 =+14 (c=1 in chloroform); 1H NMR (400 MHz,
CDCl3): d=7.54–7.22 (m, 10H; arom. H), 7.06 (d, J2,NH =6.5 Hz, 1H;
J
(dd, J2,3 =11.0 Hz, 1H; GalN H-3), 4.16–3.98 (m, 2H; GalN H-6a, H-6b),
4.01 (d, J4,5 =9.5 Hz, 1H; GlcA H-5), 3.94–3.84 (m, 2H; GalN H-5, Pen-
tenyl H-a), 3.79–3.65 (m, 4H; GalN H-2, COOCH3), 3.50 (m, 1H; Pen-
tenyl H-a), 2.12, 2.07, 2.04, 2.03, 1.99 (5 s, 15H; COCH3), 2.10–2.00 (m,
2H; Pentenyl H-c), 1.73–1.66 ppm (m, 2H; Pentenyl H-b); MS: m/z: 814
[M+Na]+ (for 35Cl); elemental analysis calcd (%) for C30H40Cl3NO17: C
45.44, H 5.08, N 1.77; found: C 45.21, H 5.14, N 1.74.
GalN NH), 5.58 (s, 1H; CH-Ph), 5.27 (m, 1H; NHCOO), 5.25 (dd, J3,4
4,5 =9.5 Hz, 1H; GlcA H-4), 5.17 (dd, J2,3 =8.5 Hz, 1H; GlcA H-3),
5.11–5.01 (m, 3H; GlcA H-2, CH2-Ph), 5.03 (d, J1,2 =8.0 Hz, 1H; GalN
H-1), 4.94 (d, J1,2 =7.5 Hz, 1H; GlcA H-1), 4.58 (dd, J2,3 =11.0 Hz, J3,4
3.5 Hz, 1H; GalN H-3), 4.44 (dd, J4,5 <1 Hz, 1H; GalN H-4), 4.31 (d,
5,6a <1 Hz, J6a,6b =12.0 Hz, 1H; GalN H-6a), 4.07 (dd, J5,6b <1.0 Hz, 1H;
=
J
2-Benzyloxycarbonylaminoethyl O-(methyl 2,3,4-tri-O-acetyl-b-d-gluco-
pyranosyluronate)-(1!3)-4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido-
b-d-galactopyranoside (8): From imidate 5 (100 mg, 0.11 mmol) and 2-
benzyloxycarbonylaminoethanol (40 mg, 0.21 mmol) as described for the
preparation of disaccharide 6. Flash-silica chromatography (CH2Cl2/2-
propanol 19:1, containing 0.1% of NEt3) afforded the disaccharide 8
(83 mg, 80%) as a white powder. Rf =0.52 (EtOAc/toluene 3:1); m.p.
192–1938C (from EtOAc/petroleum ether); [a]2D0 =+6.0 (c=1 in chloro-
form); 1H NMR (250 MHz, CDCl3): d=7.35 (s, 5H; arom. H), 6.85 (d,
=
J
GalN H-6b), 4.02 (d, 1H; GlcA H-5), 3.93 (m, 1H; CH2-O), 3.82 (m,
1H; GalN H-2), 3.71 (s, 3H; COOCH3), 3.69 (m, 1H; CH2-O), 3.49 (m,
1H; GalN H-5), 3.45–3.33 (m, 2H; CH2-N), 2.01, 2.00, 1.99 ppm (3s, 9H;
COCH3); MS: m/z: 922 [M+NH4]+ (for 35Cl); elemental analysis calcd
(%) for C36H43Cl3N2O17: C 50.37, H 4.78, N 3.09; found: C 50.45, H 4.96,
N 2.95.
J
2,NH =7.5 Hz, 1H; GalN NH), 5.44 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H;
GalN H-4), 5.21–5.15 (m, 3H; GlcA H-3, H-4, NHCOO), 5.08 (s, 2H;
CH2Ph), 4.95 (m, 1H; GlcA H-2), 4.84 (d, J1,2 =8.5 Hz, 1H; GalN H-1),
4.72 (d, J1,2 =7.5 Hz, 1H; GlcA H-1), 4.50 (dd, J2,3 =11.0 Hz, 1H; GalN
H-3), 4.16 (dd, J6a,6b =11.0 Hz, J5,6a =5.0 Hz, 1H; GalN H-6a), 4.09–3.97
(m, 2H; GalN H-6b, GlcA H-5), 3.91–3.82 (m, 2H; GalN H-5, CH2-O),
3.76 (s, 3H; COOCH3), 3.74–3.65 (m, 2H; GalN H-2, CH2-O), 3.45–3.35
(m, 2H; CH2-N), 2.12, 2.05, 2.03, 2.01, 1.99 ppm (5s, 15H; COCH3); MS:
m/z: 899 [MꢀH]ꢀ (for 35Cl); elemental analysis calcd (%) for
C35H43Cl3N2O19: C 46.60, H 4.80, N 3.11; found: C 46.44, H 4.92, N 3.15.
2-Naphthylmethyl O-(methyl 2,3-O-isopropylidene-b-d-glucopyranosylur-
onate)-(1!3)-2-deoxy-4,6-O-isopropylidene-2-trichloroacetamido-b-d-
galactopyranoside (12): Methanolic sodium methoxide (1m, 20 mL) was
added to a solution of 6 (8.75 g, 10 mmol) in dry methanol (85 mL), and
the mixture was stirred for 3 h at room temperature, then was neutralized
with Amberlite IR-120 [H+] resin, filtered, concentrated, and dried in
vacuo. Aliquots of 2-methoxypropene (1.25 mL, 13 mmol, 6 additions)
were added every 20 min to a solution of the residue (6.5 g, 9.92 mmol)
and CSA (700 mg) in DMF (70 mL). Triethylamine (3.5 mL) was added
and the mixture was concentrated. A solution of the residue in EtOAc
(100 mL) was washed with saturated aqueous NaHCO3, then several
times with saturated aqueous NaCl, dried (MgSO4), and concentrated.
Flash-silica chromatography (EtOAc/toluene 3:2, containing 0.2% of
O-(Methyl 2,3,4-tri-O-acetyl-b-d-glucopyranosyluronate)-(1!3)-4,6-O-
benzylidene-2-deoxy-2-trichloroacetamido-1-O-trichloroacetimidoyl-a-d-
galactopyranose (9): From ester 3 (14.82 g, 20 mmol) as described for the
preparation of imidate 5. Flash-silica chromatography (EtOAc/petroleum
ether 4:3, containing 0.1% of NEt3) followed by crystallization from di-
9568
www.chemeurj.org
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 9561 – 9578

Stereocontrolled Construction of Size-Defined Oligomers
FULL PAPER
NEt3) afforded the bis-acetal 12 (4.81 g, 65%) as a white powder. M.p.
218–2198C (from 2-propanol); [a]2D0 =ꢀ28 (c=1 in chloroform);
1H NMR (250 MHz, CDCl3): d=7.86–7.46 (m, 7H; arom. H), 7.08 (d,
4), 5.30 (dd, J1,2 =7.0 Hz, 1H; GlcA H-2), 5.12 (d, J1,2 =8.0 Hz, 1H; GalN
H-1), 4.94 (d, 1H; GlcA H-1), 4.89 (ABq, 2H; CH2Ar), 4.88 (dd, J2,3
=
11.0 Hz, 1H; GalN H-3), 4.58–4.50 (m, 2H; GalN H-6a, H-6b), 4.16 (d,
1H; GlcA H-5), 4.13 (m, 1H; GalN H-5), 3.81 (m, 1H; GalN H-2), 3.71
(s, 3H; COOCH3), 2.60–2.35 (m, 4H; CH2CO), 2.10 ppm (s, 3H;
COCH3); MS: m/z: 1190 [M+Na]+ (for 35Cl); elemental analysis calcd
(%) for C59H52Cl3NO18: C 60.60, H 4.48, N 1.20; found: C 60.35, H 4.56,
N 1.27.
J
2,NH =7.0 Hz, 1H; GalN NH), 5.24 (d, J1,2 =8.5 Hz, 1H; GalN H-1), 4.96
(ABq, 2H; CH2Ar), 4.85 (d, J1,2 =7.5 Hz, 1H; GlcA H-1), 4.63 (dd, J2,3
=
11.0 Hz, J3,4 =3.5 Hz, 1H; GalN H-3), 4.52 (dd, J4,5 <1.0 Hz, 1H; GalN
H-4), 4.20–4.02 (m, 3H; GalN H-6a, H-6b, GlcA H-4), 3.90–3.79 (m, 4H;
GalN H-2, COOCH3), 3.85 (d, J4,5 =9.0 Hz, 1H; GlcA H-5), 3.59–3.43
(m, 2H; GlcA H-2, H-3), 3.46 (m, 1H; GalN H-5), 3.23 (d, J4,OH =2.5 Hz,
2-Naphthylmethyl O-(methyl 2,3-di-O-acetyl-b-d-glucopyranosyluro-
nate)-(1!3)-4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido-b-d-galacto-
pyranoside (16): A freshly prepared mixture of pyridine/acetic acid/hy-
drazine hydrate (6:4:0.5, 17 mL) was added to a solution of disaccharide
14 (748 mg, 0.81 mmol) in pyridine (7.5 mL), and the mixture was stirred
for 8 min at RT, then was diluted with CH2Cl2 (50 mL), washed with
water and saturated aqueous NaHCO3, dried (MgSO4), concentrated,
and evaporated with toluene. Flash-silica chromatography (EtOAc/petro-
leum ether 3:2) afforded the disaccharide 16 (592 mg, 89%) as a white
powder. M.p. 1838C (from 2-propanol); [a]2D0 =ꢀ30 (c=1 in chloroform);
1H NMR (250 MHz, CDCl3): d=7.85–7.35 (m, 7H; arom. H), 6.81 (d,
1H; OH), 1.51, 1.50, 1.43, 1.40 ppm (4s, 12H; CACHTNUGTRNEG(UN CH3)2); MS: m/z: 732
[MꢀH]ꢀ (for 35Cl); elemental analysis calcd (%) for C32H38Cl3NO12: C
52.29, H 5.21, N 1.91; found: C 52.47, H 5.32, N 1.78.
2-Naphthylmethyl O-(methyl 2,3-O-isopropylidene-4-O-levulinoyl-b-d-
glucopyranosyluronate)-(1!3)-2-deoxy-4,6-O-isopropylidene-2-trichloro-
acetamido-b-d-galactopyranoside (13):
A
mixture of 12 (2.78 g,
3.8 mmol), levulinic acid (0.56 g, 4.75 mmol), and DMAP (122 mg,
1 mmol) in anhydrous CH2Cl2 (40 mL) was treated portionwise with
DCC (0.98 g, 4.75 mmol), and the mixture was stirred for 1 h at RT. The
solids were filtered off, washed with CH2Cl2, and the filtrate was washed
with saturated aqueous NaHCO3 and water, dried (MgSO4), and concen-
trated. Flash-silica chromatography (EtOAc/toluene 3:2, containing 0.2%
of NEt3) afforded the ester 13 (2.84 g, 90%) as a white powder. M.p.
188–1908C (from EtOAc/petroleum ether); [a]2D0 =ꢀ33 (c=1 in chloro-
J
2,NH =7.5 Hz, 1H; GalN NH), 5.49 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H;
GalN H-4), 4.90 (ABq, 2H; CH2Ar), 5.01–4.80 (m, 2H; GlcA H-2, H-3),
4.95 (d, J1,2 =8.5 Hz, 1H; GalN H-1), 4.66 (d, J1,2 =7.5 Hz, 1H; GlcA H-
1), 4.52 (dd, J2,3 =11.0 Hz, 1H; GalN H-3), 4.25–4.10 (m, 2H; GalN H-
6a, H-6b), 3.92–3.80 (m, 3H; GalN H-5, GlcA H-4, H-5), 3.83 (s, 3H;
COOCH3), 3.80 (m, 1H; GalN H-2), 3.11 (d, J4,OH =3.0 Hz, 1H; GlcA
HO-4), 2.13, 2.10, 2.05, 2.02ppm (4s, 12H; OCOCH3); MS: m/z: 844
[M+NH4]+, 822 [M+H]+ (for 35Cl); elemental analysis calcd (%) for
C34H38Cl3NO16: C 49.62, H 4.65, N 1.70; found: C 49.40, H 4.52, N 1.58.
1
form); H NMR (250 MHz, CDCl3): d=7.80–7.43 (m, 7H; arom. H), 7.22
(d, J2,NH =7.0 Hz, 1H; GalN NH), 5.27 (m, 1H; GlcA H-4), 5.17 (d, J1,2
=
8.0 Hz, 1H; GalN H-1), 4.92 (ABq, 2H; CH2Ar), 4.83 (d, J1,2 =7.5 Hz,
1H; GlcA H-1), 4.57 (dd, J2,3 =11.0 Hz, J3,4 =3.5 Hz, 1H; GalN H-3),
4.41 (dd, J4,5 <1.0 Hz, 1H; GalN H-4), 4.15–4.01 (m, 2H; GalN H-6a, H-
6b), 3.90 (d, J4,5 =9.0 Hz, 1H; GlcA H-5), 3.83 (m, 1H; GalN H-2), 3.72
(s, 3H; COOCH3), 3.61–3.54 (m, 2H; GlcA H-2, H-3), 3.40 (m, 1H;
GalN H-5), 2.75–2.60 (m, 4H; CH2CO), 2.18 (s, 3H; COCH3), 1.50, 1.47,
1.39, 1.36 ppm (4s, 12H; CACHTGNUTRENNUNG
mental analysis calcd (%) for C37H44Cl3NO14: C 53.34, H 5.32, N 1.68;
found: C 53.55, H 5.45, N 1.72.
2-Naphthylmethyl O-(methyl 2,3-di-O-benzoyl-b-d-glucopyranosyluro-
nate)-(1!3)-4,6-di-O-benzoyl-2-deoxy-2-trichloroacetamido-b-d-galacto-
pyranoside (17): From disaccharide 15 (880 mg, 0.75 mmol) as described
for the preparation of disaccharide 16. Flash-silica chromatography
(EtOAc/petroleum ether 1:1) afforded the disaccharide 17 (720 mg,
89%) as
a white powder. M.p. 179–1818C (from EtOAc/petroleum
2-Naphthylmethyl O-(methyl 2,3-di-O-acetyl-4-O-levulinoyl-b-d-gluco-
pyranosyluronate)-(1!3)-4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido-
ether); [a]2D0 =+19 (c=1 in chloroform); 1H NMR (250 MHz, CDCl3):
d=8.09–7.26 (m, 27H; arom. H), 6.86 (d, J2,NH =7.0 Hz, 1H; GalN NH),
b-d-galactopyranoside (14):
A
solution of disaccharide 16 (3.16 g,
5.88 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz 1H; GalN H-4), 5.43 (dd, J2,3 =J3,4
9.0 Hz, 1H; GlcA H-3), 5.25 (dd, J1,2 =7.0 Hz, 1H; GlcA H-2), 5.15 (d,
1,2 =8.5 Hz, 1H; GalN H-1), 4.91 (d, 1H; GlcA H-1), 4.89 (ABq, 2H;
CH2Ar), 4.88 (dd, J2,3 =11.0 Hz, 1H; GalN H-3), 4.54–4.50 (m, 2H; GalN
H-6a, H-6b), 4.19–4.08 (m, 2H; GalN H-5, GlcA H-4), 4.04 (d, J4,5
=
3.8 mmol) in 60% aqueous AcOH (30 mL) was stirred for 1 h at 1008C,
then was cooled, concentrated, evaporated with water (3ꢅ30 mL), and
dried under vacuum. A solution of the residue and in pyridine (30 mL)
and Ac2O (15 mL) was stirred for 16 h at RT, then was concentrated and
evaporated with toluene (3ꢅ20 mL). Flash-silica chromatography
(EtOAc/petroleum ether 3:2) gave the disaccharide 14 (2.48 g, 71%) as a
white powder. M.p. 168–1698C (from 2-propanol); [a]2D0 =ꢀ19 (c=1 in
chloroform); 1H NMR (250 MHz, CDCl3): d=7.80–7.43 (m, 7H; arom.
J
=
10.0 Hz, 1H; GlcA H-5), 3.80 (m, 1H; GalN H-2), 3.75 (s, 3H;
COOCH3), 3.34 ppm (d, J4,OH =3.0 Hz, 1H; GlcA HO-4); MS: m/z: 1092
[M+Na]+ (for 35Cl); elemental analysis calcd (%) for C54H46Cl3NO16: C
60.54, H 4.33, N 1.31; found: C 60.70, H 4.21, N 1.24.
H), 6.85 (d, J2,NH =8.0 Hz, 1H; GalN NH), 5.47 (d, J3,4 =3.5 Hz, J4,5
1.0 Hz, 1H; GalN H-4), 5.24–5.12 (m, 3H; GlcA H-2, H-3, H-4), 4.93 (d,
1,2 =8.0 Hz, 1H; GalN H-1), 4.91 (ABq, 2H; CH2Ar), 4.71 (d, J1,2
7.5 Hz, 1H; GlcA H-1), 4.52 (dd, J2,3 =11.0 Hz, J3,4 =3.5 Hz, 1H; GalN
H-3), 4.17 (m, 2H; GalN H-6a, H-6b), 3.97 (d, J4,5 =9.0 Hz, 1H; GlcA H-
5), 3.85 (m, 2H; GalN H-2, H-5), 3.74 (s, 3H; COOCH3), 2.80–2.40 (m,
4H; CH2CO), 2.14 (s, 3H; COCH3), 2.13, 2.11, 2.02 ppm (3s, 12H;
COCH3); MS: m/z: 937 [M+NH4]+ (for 35Cl); elemental analysis calcd
(%) for C39H44Cl3NO18: C 50.85, H 4.81, N 1.52; found: C 50.82, H 4.92,
N 1.43.
<
O-(Methyl
2,3-di-O-acetyl-4-O-levulinoyl-b-d-glucopyranosyluronate)-
(1!3)-4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido-1-O-trichloroacet-
imidoyl-a-d-galactopyranose (18): DDQ (480 mg, 2.12 mmol) was added
to a solution of disaccharide 14 (651 mg, 0.71 mmol) in CH2Cl2/MeOH
(9:1, 7 mL), and the mixture was stirred for 24 h at RT under dry argon,
then was diluted with CH2Cl2 (40 mL), washed with saturated aqueous
NaHCO3 and water, dried (MgSO4), and concentrated. Flash-silica chro-
matography (CH2Cl2/acetone 9:1) gave the corresponding free hemiacetal
(471 mg, 85%) as a pale yellow powder. A mixture of the above isolated
hemiacetal (471 mg, 0.60 mmol), CCl3CN (0.61 mL, 6 mmol), and DBU
(18 mL, 0.12 mmol) in dry CH2Cl2 (5 mL) was stirred for 30 min at RT,
then was concentrated. Flash-silica chromatography (EtOAc/petroleum
ether 1:1, containing 0.1% of NEt3) and crystallization from EtOAc/pe-
troleum ether gave the imidate 18 (368 mg, 66%). M.p. 100–1018C;
[a]2D2 =+55 (c=1 in chloroform); 1H NMR (250 MHz, CDCl3): d=8.75
(s, 1H; C=NH), 6.87 (d, J2,NH =8.2 Hz, 1H; NH), 6.52 (d, J1,2 =3.5 Hz,
1H; GalN H-1), 5.42 (dd, J3,4 =3.5 Hz, J4,5 =0.8 Hz, 1H; GalN H-4), 5.17
(m, 2H; GlcA H-3, H-4), 5.05 (m, 1H; GlcA H-2), 4.78 (d, J1,2 =8.0 Hz,
1H; GalN H-1), 4.60 (m, J2,3 =11.0 Hz, 1H; GalN H-2), 4.31 (m, 1H;
J
=
2-Naphthylmethyl O-(methyl 2,3-di-O-benzoyl-4-O-levulinoyl-b-d-gluco-
pyranosyluronate)-(1!3)-4,6-di-O-benzoyl-2-deoxy-2-trichloroacetam-
ido-b-d-galactopyranoside (15): From acetal 13 (1.20 g, 1.44 mmol) as de-
scribed for the preparation of 14. The solid residue and benzoyl chloride
(1.23 mL, 10.6 mmol) in pyridine (12 mL) were stirred for 2 h at 08C.
Methanol (1.2 mL) was added, and the mixture was diluted with CH2Cl2
(100 mL), washed with water, saturated aqueous NaHCO3 and water,
dried (MgSO4), and concentrated. Flash-silica chromatography (EtOAc/
petroleum ether 1:1) afforded the disaccharide 15 (955 mg, 56%) as a
white powder. [a]2D0 =+24 (c=1 in chloroform); 1H NMR (250 MHz,
CDCl3): d=8.09–7.26 (m, 27H; arom. H), 6.85 (d, J2,NH =7.0 Hz, 1H;
GalN NH), 5.87 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalN H-4), 5.56 (dd,
GalN H-5), 4.30 (dd, 1H; GalN H-3), 4.16 (dd, J5,6a =6.0 Hz, J6a,6b
11.5 Hz, 1H; GalN H-6a), 4.00 (d, J4,5 =9.5 Hz, 1H; GlcA H-5), 3.97 (dd,
5,6b =7.0 Hz, 1H; GalN H-6b), 3.72 (s, 3H; COOCH3), 2.68, 2.45 (2 m,
4H; CH2CO), 2.14, 2.13, 2.05, 2.03, 2.01 ppm (5s, 15H; COCH3,
=
J
J
2,3 =J3,4 =9.5 Hz, 1H; GlcA H-3), 5.42 (dd, J4,5 =10.0 Hz, 1H; GlcA H-
Chem. Eur. J. 2009, 15, 9561 – 9578
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9569

J.-C. Jacquinet et al.
CH3CO); ISMS: m/z: 940 [M+NH4]+ (for 35Cl); elemental analysis calcd
(%) for C30H36Cl6N2O18: C 38.94, H 3.92, N 3.03; found: C 38.76, H 3.78,
N, 2.88.
1H; GalN H-3), 4.20–3.99 (m, 3H; GalN H-6a, H-6b, GlcA H-5), 3.92–
3.82 (m, 3H; GalN H-5, GlcA H-4, CH2O), 3.79 (s, 3H; COOCH3), 3.78–
3.67 (m, 2H; GalN H-2, CH2O), 3.45–3.31 (m, 2H; CH2N), 3.13 (d,
J
4,OH =3.0 Hz, 1H; GlcA HO-4), 2.11, 2.05, 2.04, 2.02 ppm (4s, 12H;
O-(Methyl 2,3-di-O-benzoyl-4-O-levulinoyl-b-d-glucopyranosyluronate)-
(1!3)-4,6-di-O-benzoyl-2-deoxy-2-trichloroacetamido-1-O-trichloroacet-
imidoyl-a-d-galactopyranose (19): From disaccharide 17 (3.91 g,
3.34 mmol) as described for the preparation of 18. Flash-silica chroma-
tography (petroleum ether/EtOAc 3:2, containing 0.1% of NEt3) afford-
ed the imidate 19 (2.15 g, 74%) as a white powder. M.p. 112–1158C
(from petroleum ether/diethyl ether); [a]2D0 =+89 (c=1 in chloroform);
1H NMR (250 MHz, CDCl3): d=8.72 (s, 1H; C=NH), 8.01–7.26 (m, 20H;
arom. H), 6.95 (d, J2,NH =7.5 Hz, 1H; GalN NH), 6.69 (d, J1,2 =3.5 Hz,
1H; GalN H-1), 5.99 (dd, J3,4 =2.5 Hz, J4,5 <1.0 Hz, 1H; GalN H-4), 5.64
(dd, J2,3 =J3,4 =9.5 Hz, 1H; GlcA H-3), 5.45 (dd, J1,2 =8.0 Hz, 1H; GlcA
H-2), 5.39 (dd, J4,5 =9.5 Hz, 1H; GlcA H-4), 5.16 (d, 1H; GlcA H-1),
4.81 (dd, J2,3 =11.0 Hz, 1H; GalN H-2), 4.62–4.58 (m, 2H; GalN H-3, H-
6a), 4.44–4.38 (m, 2H; GalN H-5, H-6b), 4.25 (d, 1H; GlcA H-5), 3.67 (s,
3H; COOCH3), 2.62–234 (m, 4H; CH2 CO), 2.03 ppm (s, 3H; COCH3);
OCOCH3); MS: m/z: 857 [MꢀH]ꢀ (for 35Cl); elemental analysis calcd
(%) for C33H41Cl3N2O18: C 46.09, H 4.81, N 3.26; found: C 46.39, H 4.81,
N 3.19.
2-Benzyloxycarbonylaminoethyl O-(methyl 2,3-di-O-benzoyl-b-d-gluco-
pyranosyluronate)-(1!3)-4,6-di-O-benzoyl-2-deoxy-2-trichloroacetami-
do-b-d-galactopyranoside (23): From disaccharide 21 (882 mg,
0.73 mmol) as described for the preparation of 8. Flash-silica chromatog-
raphy (petroleum ether/EtOAc 1:1) afforded the alcohol 23 (621 mg,
75%) as a white powder. [a]2D0 =+39 (c=1 in chloroform); 1H NMR
(250 MHz, CDCl3): d=8.09–7.71 (m, 25H; arom. H), 6.88 (d, J2,NH
=
7.0 Hz, 1H; GalN NH), 5.89 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalN H-
4), 5.43 (dd, J2,3 =J3,4 =9.5 Hz, 1H; GlcA H-3), 5.26 (m, 1H; GlcA H-2),
5.12–5.09 (m, 1H; NHCOO), 5.09 (d, J1,2 =8.0 Hz, 1H; GalN H-1), 5.01
(s, 2H; CH2Ph), 4.96 (d, J1,2 =7.0 Hz, 1H; GlcA H-1), 4.84 (dd, J2,3
=
MS: m/z: 1026 [MꢀC(NH)CCl3]
H
11.0 Hz, 1H; GalN H-3), 4.49–4.38 (m, 2H; GalN H-6a, H-6b), 4.57–4.51
(m, 2H; GalN H-5, GlcA H-4), 4.08 (d, J4,5 =9.5 Hz, 1H; GlcA H-5),
3.91–3.80 (m, 1H; CH2O), 3.77 (s, 3H; COOCH3), 3.72–3.61 (m, 2H;
GalN H-2, CH2O), 3.42–3.34 (m, 2H; CH2N), 3.33 ppm (d, J4,OH =3.0 Hz,
1H; GlcA HO-4); MS: m/z: 1129 [M+Na]+ (for 35Cl); elemental analysis
calcd (%) for C53H49Cl3N2O18: C 57.44, H 4.46, N 2.53; found: C 57.79, H
4.62, N 2.45.
2-Benzyloxycarbonylaminoethyl O-(methyl 2,3-di-O-acetyl-4-O-levulino-
yl-b-d-glucopyranosyluronate)-(1!3)-4,6-di-O-acetyl-2-deoxy-2-trichloro-
acetamido-b-d-galactopyranoside (20): From imidate 18 (262 mg,
0.28 mmol) as described for the preparation of 8. Flash-silica chromatog-
raphy (EtOAc/acetone 12:1, containing 0.1% of NEt3) afforded the dis-
accharide 20 (147 mg, 54%) as a white powder. Rf =0.22 (CH2Cl2/acetone
12:1); [a]2D0 =+8 (c=1 in chloroform); 1H NMR (250 MHz, CDCl3): d=
7.33–7.21 (m, 5H; arom. H), 7.09 (d, J2,NH =7.5 Hz, 1H; GalN NH), 5.33
(dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalN H-4), 5.10–4.97 (m, 5H; GlcA
2-Naphthylmethyl O-(methyl 2,3-O-isopropylidene-b-d-glucopyranosylur-
onate)-(1!3)-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-b-d-galac-
topyranoside (24): From disaccharide 10 (8.69 g, 10 mmol) as described
for the preparation of 12. Flash-silica chromatography (CH2Cl2/acetone
9:1, containing 0.2% of Et3N) gave the alcohol 24 (5.49 g, 70%) as a
white powder. Rf =0.27 (toluene/EtOAc 3:2 containing 0.2% of NEt3);
m.p. 188–1908C (from petroleum ether/EtOAc); [a]2D0 =ꢀ8.5 (c=1 in
chloroform); 1H NMR (250 MHz, CDCl3): d=7.83–7.27 (m, 12H; arom.
H), 7.24 (d, J2,NH =6.5 Hz, 1H; GalN NH), 5.61 (s, 1H; CHPh), 5.34 (d,
H-3, H-4, CH2Ph, NHCOO), 4.85 (m, 1H; GlcA H-2), 4.71 (d, J1,2
8.0 Hz, 1H; GalN H-1), 4.64 (d, J1,2 =8.0 Hz, 1H; GlcA H-1), 4.39 (dd,
2,3 =11.0 Hz, 1H; GalN H-3), 4.08 (dd, J6a,6b =11.5 Hz, J5,6a =6.0 Hz, 1H;
=
J
GalN H-6a), 3.99–3.95 (m, 2H; GalN H-6b, GlcA H-5), 3.81–3.71 (m,
2H; GalN H-5, CH2O), 3.63 (s, 3H; COOCH3), 3.62–3.57 (m, 2H; GalN
H-2, CH2O), 3.31–3.21 (m, 2H; CH2N), 2.63–2.59 (m, 2H; CH2CO),
2.42–2.33 (m, 2H; CH2CO), 2.07, 2.03, 1.96, 1.95 ppm (4s, 15H;
OCOCH3); MS: m/z: 955 [MꢀH]ꢀ (for 35Cl); elemental analysis calcd
(%) for C38H47Cl3N2O20: C 47.64, H 4.94, N 2.92; found: C 47.56, H 5.02,
N, 2.85.
J
1,2 =8.5 Hz, 1H; GalN H-1), 4.96 (ABq, 2H; CH2Ar), 4.93 (d, J1,2 =
7.5 Hz, 1H; GlcA H-1), 4.75 (dd, J2,3 =11.0 Hz, J3,4 =3.0 Hz, 1H; GalN
H-3), 4.48 (dd, J4,5 <1.0 Hz, 1H; GalN H-4), 4.42 (d, J6a,6b =12.5 Hz, 1H;
GalN H-6a), 4.15–4.05 (m, 2H; GalN H-6b, GlcA H-4), 3.96–3.83 (m,
5H; GalN H-2, GlcA H-5, COOCH3), 3.57 (m, 1H; GalN H-5), 3.58–
3.39 (m, 2H; GlcA H-2, H-3), 3.24 (d, J4,OH =2.5 Hz, 1H; GlcA HO-4),
1.42, 1.39 ppm (2s, 6H; CACHTNUTGRNEUNG
(CH3)2); MS: m/z: 799 [M+NH4]+ (for 35Cl); el-
2-Benzyloxycarbonylaminoethyl O-(methyl 2,3-di-O-benzoyl-4-O-levuli-
noyl-b-d-glucopyranosyluronate)-(1!3)-4,6-di-O-benzoyl-2-deoxy-2-tri-
chloroacetamido-b-d-galactopyranoside (21): From imidate 19 (1.0 g,
0.85 mmol) as described for the preparation of 8. Flash-silica chromatog-
raphy (toluene/EtOAc 1:1, containing 0.1% of NEt3) afforded the disac-
charide 21 (918 mg, 89%) as a white powder. [a]2D0 =+44 (c=1 in chloro-
form); 1H NMR (250 MHz, CDCl 3): d=8.09–7.31 (m, 25H; arom. H),
6.88 (d, J2,NH =6.5 Hz, 1H; GalN NH), 5.88 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz,
1H; GalN H-4), 5.58 (dd, J2,3 =10.0 Hz, J3,4 =9.5 Hz, 1H; GlcA H-3),
5.42 (dd, J4,5 =10.0 Hz, 1H; GlcA H-4), 5.21 (dd, J1,2 =7.5 Hz, 1H; GlcA
H-2), 5.14 (m, 1H; NHCOO), 5.08 (d, J1,2 =8.0 Hz, 1H; GalN H-1), 5.04
(s, 2H; CH2Ph), 4.98 (d, 1H; GlcA H-1), 4.82 (dd, J2,3 =11.0 Hz, 1H;
GalN H-3), 4.45–4.40 (m, 2H; GalN H-6a, H-6b), 4.21 (d, 1H; GlcA H-
5), 4.11 (m, 1H; GalN H-5), 3.89–3.71 (m, 2H; GalN H-2, CH2O), 3.72
(s, 3H; COOCH3), 3.67 (m, 1H; CH2O), 3.41–3.29 (m, 2H; CH2N), 2.62–
2.37 (m, 4H; CH2CO), 2.02 ppm (s, 3H; COCH3); MS: m/z: 1227
[M+Na]+ (for 35Cl); elemental analysis calcd (%) for C58H55Cl3N2O20: C
57.74, H 4.60, N 2.32; found: C 57.87, H 4.09, N 2.55.
emental analysis calcd (%) for C36H38Cl3NO12: C 55.22, H 4.89, N 1.79;
found: C 54.97, H 4.96, N 1.70.
2-Naphthylmethyl O-(methyl 2,3-O-isopropylidene-4-O-levulinoyl-b-d-
glucopyranosyluronate)-(1!3)-4,6-O-benzylidene-2-deoxy-2-trichloroace-
tamido-b-d-galactopyranoside (25): From disaccharide 24 (5.02 g,
6.4 mmol) as described for the preparation of 13. Flash-silica chromatog-
raphy (CH2Cl2/acetone 12:1, containing 0.2% of NEt3) gave the disac-
charide 25 (5.19 g, 92%) as a white powder. Rf =0.25 (toluene/EtOAc 3:2
containing 0.2% of NEt3); m.p. 200–2038C (from EtOAc); [a]2D0 =ꢀ10
(c=1 in chloroform); 1H NMR (250 MHz, CDCl3): d=7.82–7.35 (m,
12H; arom. H), 7.22 (d, J2,NH =7.0 Hz, 1H; GalN NH), 5.63 (s, 1H;
CHPh), 5.30 (d, J1,2 =8.0 Hz, 1H; GalN H-1), 5.28 (m, 1H; GlcA H-4),
4.95 (ABq, 2H; CH2Ar), 4.91 (d, J1,2 =7.5 Hz, 1H; GlcA H-1), 4.72 (dd,
J
2,3 =11.0 Hz, J3,4 =3.5 Hz, 1H; GalN H-3), 4.44 (dd, J4,5 <1.0 Hz, 1H;
GalN H-4), 4.40 (m, 1H; GalN H-6a), 4.13 (m, 1H; GalN H-6b), 3.93 (d,
4,5 =8.0 Hz, 1H; GlcA H-5), 3.91 (m, 1H; GalN H-2), 3.71 (s, 3H;
COOCH3), 3.62–3.56 (m, 3H; GalN H-5, GlcA H-2, H-3), 2.85–2.55 (m,
J
4H; CH2CO), 2.18 (s, 3H; COCH3), 1.40, 1.37 ppm (2s, 6H; CACHTUNGTRENNUNG(CH3)2);
2-Benzyloxycarbonylaminoethyl O-(methyl 2,3-di-O-acetyl-b-d-glucopyr-
anosyluronate)-(1!3)-4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido-b-d-
galactopyranoside (22): From disaccharide 20 (365 mg, 0.38 mmol) as de-
scribed for the preparation of 8. Flash-silica chromatography (EtOAc/tol-
uene 2:1) afforded the alcohol 22 (263 mg, 80%) as a white powder.
[a]2D0 =+1 (c=1 in chloroform); 1H NMR (250 MHz, CDCl3): d=7.39–
7.32 (m, 5H; arom. H), 6.72 (d, J2,NH =7.5 Hz, 1H; GalN NH), 5.49 (dd,
MS: m/z: 897 [M+NH4]+ (for 35Cl); elemental analysis calcd (%) for
C41H44Cl3NO14: C 55.89, H 5.03, N 1.59; found: C 55.63, H 5.07, N 1.52.
2-Naphthylmethyl O-(methyl 4-O-levulinoyl-b-d-glucopyranosyluronate)-
(1!3)-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-b-d-galactopyra-
noside (26): A solution of 25 (4.05 g, 4.6 mmol) in CH2Cl2/AcOH/water
(5:4:1, 80 mL) was stirred for 24 h at RT, then was concentrated, evapo-
rated with water (3ꢅ20 mL), and dried in vacuo. Flash-silica chromatog-
raphy (CH2Cl2/MeOH 19:1!12:1) gave the diol 26 (3.06 g, 79%) as a
white solid. Rf =0.40 (CH2Cl2/MeOH 12:1); m.p. 194–1958C (from
J
3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalN H-4), 5.19 (m, 1H; NHCOO), 5.18
(s, 2H; CH2Ph), 5.00 (m, 1H; GlcA H-3), 4.91–4.83 (m, 2H; GalN H-1,
GlcA H-2), 4.69 (d, J1,2 =7.5 Hz, 1H; GlcA H-1), 4.39 (dd, J2,3 =11.0 Hz,
9570
www.chemeurj.org
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 9561 – 9578

Stereocontrolled Construction of Size-Defined Oligomers
FULL PAPER
1
EtOAc); [a]2D0 =ꢀ8.5 (c=1 in chloroform); H NMR (250 MHz, CD3OD/
1H NMR (250 MHz, CDCl3): d=7.85–7.35 (m, 7H; arom. H), 6.98, 6.91,
CDCl3 3:1): d=7.83–7.30 (m, 12H; arom. H), 5.63 (s, 1H; CHPh), 4.95
(ABq, 2H; CH2Ar), 4.93 (d, J1,2 =7.5 Hz, 1H; GalN H-1), 4.85 (dd, J3,4
6.90 (3d, J2,NH =7.0 Hz, 3H; 3GalN NH), 5.45, 5.41, 5.38 (3dd, J3,4
3.0 Hz, J4,5 <1.0 Hz, 3H; 3GalN H-4), 5.23–5.12 (m, 3H; 3GlcA H-3),
5.03–4.85 (m, 4H; GlcAIII H-4, 3GlcA H-2), 4.94, 4.92, 4.91 (3d, J1,2
8.0 Hz, 3H; 3GalN H-1), 4.91 (ABq, 2H; CH2Ar), 4.68, 4.66, 4.65 (3d,
1,2 =7.5 Hz, 3H; 3GlcA H-1), 4.48, 4.27, 4.23 (3dd, J2,3 =11.0 Hz, 3H;
=
=
9.5 Hz, J4,5 =10.0 Hz, 1H; GlcA H-4), 4.45 (m, 2H; GlcA H-1, GalN H-
4), 4.24 (dd, J2,3 =11.0 Hz, J3,4 =3.5 Hz; GalN H-3), 4.20–4.10 (m, 3H;
GalN H-2, H-6a, H-6b), 3.94 (d, 1H; GlcA H-5), 3.69 (s, 3H; COOCH3),
3.55 (m, 2H; GalN H-5, GlcA H-3), 3.35 (dd, J1,2 =7.5 Hz, J2,3 =9.5 Hz;
GlcA H-2), 2.87–2.55 (m, 4H; CH2CO), 2.16 ppm (s, 3H; COCH3); MS:
m/z: 862 [M+Na]+ (for 35Cl); elemental analysis calcd (%) for
C38H40Cl3NO14: C 54.26, H 4.79, N 1.66; found: C 54.19, H 4.86, N 1.52.
=
J
3GalN H-3), 4.15–3.80 (m, 14H; 3GalN H-5, H-6a, H-6b, GlcAI H-4,
GlcAII H-4, 3GlcA H-5), 3.85, 3.83, 3.76 (3s, 9H; COOCH3), 3.78–3.68
(m, 3H; 3GalN H-2), 2.35, 2.11, 2.09, 2.04, 2.01, 2.00, 1.99 ppm (7s, 39H;
OCOCH3); MS: m/z: 2188 [MꢀH]ꢀ (for 35Cl); elemental analysis calcd
(%) for C82H95Cl9N3O47: C 44.88, H 4.41, N 1.91; found: C 44.61, H 4.47,
N 1.91.
Tetrasaccharide 27: From imidate 5 (480 mg, 0.55 mmol) and alcohol 16
(300 mg, 0.36 mmol) as described for the preparation of 6. Flash-silica
chromatography (EtOAc/petroleum ether 2:1, containing 0.1% of NEt3)
afforded the tetrasaccharide 27 (316 mg, 57%) as a white powder. M.p.
1408C (from 2-propanol); [a]2D0 =ꢀ9.5 (c=1 in chloroform); 1H NMR
Tetrasaccharide 31: From imidate 19 (1.01 g, 0.86 mmol) and alcohol 23
(710 mg, 0.66 mmol) as described for the preparation of disaccharide 6.
Flash-silica chromatography (toluene/EtOAc 3:1, containing 0.1% of
NEt3) afforded the tetrasaccharide 31 (850 mg, 62%) as a white powder.
M.p. 163–1658C (from 2-propanol); [a]2D0 =+12 (c=1 in chloroform);
1H NMR (250 MHz, CDCl3): d=8.12–7.10 (m, 47H; arom. H), 6.87, 6.80
(2d, J2,NH =8.0 Hz, 2H; 2GalN NH), 5.82 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz,
1H; GalNI H-4), 5.61 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNII H-4),
5.53 (dd, J2,3 =J3,4 =9.0 Hz, 1H; GlcAI H-3), 5.46 (dd, J2,3 =J3,4 =9.5 Hz,
1H; GlcAII H-3), 5.38 (dd, J4,5 =10.0 Hz, 1H; GlcAII H-4), 5.27–5.20 (m,
2H; 2GlcA H-2), 5.15, 5.13 (2d, J1,2 =8.0 Hz, 2H; 2GalN H-1), 4.89 (d,
(250 MHz, CDCl3): d=7.70–7.21 (m, 7H; arom. H), 6.98, 6.91 (2d, J2,NH
=
7.0 Hz, 2H; 2GalN NH), 5.44, 5.39 (2dd, J3,4 =3.5 Hz, J4,5 <1.0 Hz, 2H;
2GalN H-4), 5.14 (dd, J3,4 =8.0 Hz, J4,5 =8.5 Hz, 1H; GlcAII H-4), 5.05–
4.96 (m, 2H; 2GlcA H-3), 4.91, 4.90 (2d, J1,2 =8.5 Hz, 2H; 2GalN H-1),
4.89 (ABq, 2H; CH2Ar), 4.88–4.83 (m, 2H; 2GlcA H-2), 4.66, 4.63 (2d,
J
1,2 =7.5 Hz, 2H; 2GlcA H-1), 4.46, 4.28 (2dd, J2,3 =11.0 Hz, 2H; 2GalN
H-3), 4.17–4.07 (m, 5H; 2GalN H-6a, H-6b, GlcAI H-4), 3.96 (d, 1H;
GlcAII H-5), 3.90 (d, J4,5 =9.5 Hz, 1H; GlcAI H-5), 3.87–3.80 (m, 2H;
2GalN H-5), 3.82, 3.75 (2s, 6H; COOCH3), 3.80–3.65 (m, 2H; 2GalN H-
2), 2.09, 2.02, 2.00, 1.99, 1.98, 1.97 ppm (6s, 27H; OCOCH3); MS: m/z:
1525 [MꢀH]ꢀ (for 35Cl); elemental analysis calcd (%) for
C59H68Cl6N2O32: C 46.32, H 4.48, N 1.83; found: C 46.19, H 4.44, N 1.82.
J
1,2 =7.0 Hz, 1H; GlcA H-1), 4.87 (ABq, 2H; CH2Ar), 4.86 (d, J1,2 =
7.0 Hz, 1H; GlcA H-1), 4.84 (dd, J2,3 =11.0 Hz, 1H; GalNI H-3), 4.68
(dd, J2,3 =11.0 Hz, 1H; GalNII H-3), 4.54–4.49 (m, 2H; GalNI H-6a, H-
6b), 4.31 (dd, J4,5 =9.5 Hz, 1H; GlcAI H-4), 4.15–4.08 (m, 2H; GalNI H-
5, GlcAII H-5), 4.02 (d, 1H; GlcAI H-5), 3.88 (m, 1H; GalNII H-5), 3.78
(m, 1H; GalNI H-2), 3.72, 3.65 (2 s, 6H; COOCH3), 3.70–350 (m, 2H;
GalNII H-2, H-6a), 3.32 (dd, J6a,6b =11.0 Hz, J5,6b =7.0 Hz, 1H; GalNII H-
6b), 2.42 (m, 4H; CH2CO), 2.00 ppm (s, 3H; COCH3); MS: m/z: 2077
[MꢀH]ꢀ (for 35Cl); elemental analysis calcd (%) for C102H88Cl6N2O33: C
58.83, H 4.26, N 1.35; found: C 58.56, H 4.43, N 1.34.
Tetrasaccharide 28: From imidate 18 (168 mg, 0.18 mmol) and alcohol 16
(100 mg, 0.12 mmol) as described for the preparation of disaccharide 6.
Flash-silica chromatography (EtOAc/petroleum ether 2:1, containing
0.1% of NEt3) afforded the tetrasaccharide 28 (110 mg, 57%) as a white
powder. M.p. 143–1458C (from 2-propanol); [a]2D0 =ꢀ6 (c=1 in chloro-
form); 1H NMR (250 MHz, CDCl3): d=7.80–7.31 (m, 7H; arom. H),
6.97, 6.92 (2d, J2,NH =7.5 Hz, 2H; 2GalN NH), 5.44, 5.39 (2dd, J3,4
=
Tetrasaccharide acceptor 32: From tetrasaccharide 31 (634 mg,
0.31 mmol) as described for the preparation of disaccharide 16. Flash-
silica chromatography (petroleum ether/EtOAc 1:1) afforded the alcohol
38 (415 mg, 69%) as a white powder. M.p. 173–1758C (from 2-propanol);
[a]2D0 =+2 (c=1 in chloroform); 1H NMR (250 MHz, CDCl3): d=8.20–
7.25 (m, 47H; arom. H), 6.86 (d, J2,NH =8.0 Hz, 1H; GalNI NH), 6.74 (d,
3.5 Hz, J4,5 <1.0 Hz, 2H; 2GalN H-4), 5.19 (dd, J3,4 =8.0 Hz, J4,5 =9.5 Hz,
1H; GlcAII H-4), 5.20–5.10 (m, 2H; 2GlcA H-3), 4.91 (d, J1,2 =8.0 Hz,
1H; GalN H-1), 4.90 (d, J1,2 =8.5 Hz, 1H; GalN H-1), 4.89 (ABq, 2H;
CH2Ar), 4.86–4.82 (m, 2H; 2GlcA H-2), 4.67 (d, J1,2 =7.0 Hz, 1H; GlcA
H-1), 4.64 (d, J1,2 =7.5 Hz, 1H; GlcA H-1), 4.47, 4.28 (2dd, J2,3 =11.0 Hz,
2H; 2GalN H-3), 4.17–4.00 (m, 5H; 2GalN H-6a, H-6b, GlcAI H-4),
3.96, 3.91 (2d, J4,5 =9.5 Hz, 2H; 2GlcA H-5), 3.89–3.81 (m, 2H; 2GalN
H-5), 3.83, 3.75 (2s, 6H; COOCH3), 3.80–3.68 (m, 2H; 2GalN H-2),
2.75–2.68, 2.50–2.40 (2 m, 4H; CH2CO), 2.11 (s, 3H; COCH3), 2.09, 2.07,
2.05, 2.04, 2.03, 2.02, 2.00 ppm (8s, 24H; OCOCH3); MS: m/z: 1581
[MꢀH]ꢀ (for 35Cl); elemental analysis calcd (%) for C62H72Cl6N2O33: C
46.95, H 4.58, N 1.77; found: C 46.75, H 4.72, N 1.70.
J
2,NH =7.5 Hz, 1H; GalNII NH), 5.82 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H;
GalNI H-4), 5.63 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNII H-4), 5.47
(dd, J2,3 =J3,4 =9.0 Hz, 1H; GlcAI H-3), 5.38 (dd, J2,3 =J3,4 =9.0 Hz, 1H;
GlcAII H-3), 5.20 (m, 2H; 2GlcA H-2), 5.16 (d, J1,2 =8.0 Hz, 1H; GalN
H-1), 5.14 (d, J1,2 =8.5 Hz, 1H; GalN H-1), 4.90 (d, J1,2 =7.0 Hz, 1H;
GlcA H-1), 4.88 (ABq, 2H; CH2Ar), 4.87 (m, 1H; GalNI H-3), 4.84 (d,
J
1,2 =7.0 Hz, 1H; GlcA H-1), 4.69 (dd, J2,3 =11.0 Hz, 1H; GalNII H-3),
4.55–4.45 (m, 2H; GalNI H-6a, H-6b), 4.32 (dd, J4,5 =9.5 Hz, 1H; GlcAI
Tetrasaccharide acceptor 29: From tetrasaccharide 28 (152 mg, 90 mmol)
as described for the preparation of disaccharide 16. Flash-silica chroma-
tography (EtOAc/petroleum ether 3:1) afforded the alcohol 29 (110 mg,
77%) as a white powder. M.p. 1428C (from 2-propanol); [a]2D0 =ꢀ13 (c=
1 in chloroform); 1H NMR (250 MHz, CDCl3): d=7.80–7.35 (m, 7H;
arom. H), 6.80, 6.78 (2d, J2,NH =7.5 Hz, 2H; 2GalN NH), 5.45, 5.44 (2dd,
H-4), 4.15–4.05 (m, 2H; GalNI H-5, GlcAII H-4), 4.02, 3.99 (2d, J4,5
=
9.5 Hz, 2H; 2GlcA H-5), 3.90 (m, 1H; GalNII H-5), 3.74 (m, 1H; GalNI
H-2), 3.72, 3.69 (2s, 6H; COOCH3), 3.68–3.52 (m, 2H; GalNII H-2, H-
6a), 3.34 (d, J4,OH =3.0 Hz, 1H; GlcAII HO-4), 3.30 ppm (dd, J6a,6b
=
11.0 Hz, J5,6b =7.0 Hz, 1H; GalNII H-6b); MS: m/z: 1979 [MꢀH]ꢀ (for
35Cl); elemental analysis calcd (%) for C97H82Cl6N2O31: C 58.71, H 4.17,
N 1.41; found: C 58.72, H 4.37, N 1.36.
J
3,4 =3.0 Hz, J4,5 <1.0 Hz, 2H; 2GalN H-4), 5.15–5.05 (m, 2H; 2GlcA H-
3), 4.93, 4.91 (2d, 1,2 =8.5 Hz, 2H; 2GalN H-1), 4.89 (ABq, 2H;
J
CH2Ar), 4.86–4.82 (m, 2H; 2GlcA H-2), 4.64, 4.60 (2d, J1,2 =7.5 Hz, 2H;
2GlcA H-1), 4.51, 4.26 (2dd, J2,3 =11.0 Hz, 2H; 2GalN H-3), 4.19–4.00
(m, 5H; 2GalN H-6a, 2H-6b, GlcAI H-4), 3.95–3.70 (m, 5H; 2GalN H-5,
GlcAII H-4, 2GlcA H-5), 3.86, 3.84 (2s, 6H; COOCH3), 3.75–3.65 (m,
2H; 2GalN H-2), 3.14 (d, J4,OH =3.0 Hz, 1H; GlcAII HO-4), 2.35, 2.12,
2.10, 2.05, 2.04, 2.00, 1.98 ppm (7s, 24H; OCOCH3); MS: m/z: 1485
[M+H]+, 1502 [M+NH4]+, 1507 [M+Na]+ (for 35Cl); elemental analysis
calcd (%) for C57H66Cl6N2O31: C 46.01, H 4.47, N 1.88; found: C 46.17, H
4.38, N 1.78.
Hexasaccharide 33: From imidate 19 (270 mg, 0.23 mmol) and alcohol 32
(283 mg, 0.14 mmol) as described for the preparation of disaccharide 6.
Flash-silica chromatography (toluene/EtOAc 1:1, containing 0.1% of
NEt3) afforded the hexasaccharide 33 (277 mg, 65%) as a white powder.
M.p. 184–1868C (from CH2Cl2/diethyl ether/petroleum ether); [a]2D0 =ꢀ1
(c=1 in chloroform); 1H NMR (400 MHz, CDCl3): d=8.10–7.05 (m,
67H; arom. H), 6.84, 6.81, 6.72 (3d, J2,NH =7.5 Hz, 3H; 3GalN NH), 5.81
(dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNI H-4), 5.60, 5.55 (2dd, J3,4
3.0 Hz, J4,5 <1.0 Hz, 2H; GalNII H-4, GalNIII H-4), 5.52 (dd, J2,3 =J3,4
=
=
Hexasaccharide 30: From imidate 5 (116 mg, 0.13 mmol) and alcohol 29
(100 mg, 70 mmol) as described for the preparation of disaccharide 6.
Flash-silica chromatography (EtOAc/toluene 3:1, containing 0.1% of
NEt3) afforded the hexasaccharide 30 (100 mg, 65%) as a white powder.
M.p. 157–1588C (from 2-propanol); [a]2D0 =ꢀ7 (c=1 in chloroform);
9.5 Hz, 1H; GlcAI H-3), 5.45, 5.42 (2dd, J2,3 =J3,4 =9.5 Hz, 2H; GlcAII H-
3, GlcAIII H-3), 5.37 (dd, J4,5 =10.0 Hz, 1H; GlcAIII H-4), 5.22 (dd, J1,2
=
7.5 Hz, 1H; GlcAI H-2), 5.21 (dd, J1,2 =7.5 Hz, 1H; GlcA H-2), 5.15 (m,
1H; GlcA H-2), 5.14, 5.13, 5.10 (3d, J1,2 =8.0 Hz, 3H; 3GalN H-1), 4.88
(d, J1,2 =7.5 Hz, 1H; GlcA H-1), 4.87 (ABq, 2H; CH2Ar), 4.86 (m, 1H;
Chem. Eur. J. 2009, 15, 9561 – 9578
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9571

J.-C. Jacquinet et al.
1
GalNI H-3), 4.85, 4.81 (2d, J1,2 =7.5 Hz, 2H; 2GlcA H-1), 4.68, 4.65 (2dd,
2,3 =11.0 Hz, 2H; GalNII H-3, GalNIII H-3), 4.51–4.49 (m, 2H; GalNI H-
(c=1 in chloroform); H NMR (250 MHz, CDCl3): d=7.85–7.42 (m, 7H;
arom. H), 5.72 (d, J2,NH =7.0 Hz, 1H; GalN NH), 5.41 (dd, J3,4 =3.0 Hz,
J
6a, H-6b), 4.31, 4.26 (2dd, J4,5 =9.5 Hz, 2H; GlcAI H-4, GlcAII H-4), 4.12
(m, 1H; GalNI H-5), 4.10 (d, 1H; GlcAIII H-5), 4.00, 3.96 (2d, 2H;
GlcAI H-5, GlcAII H-5), 3.86–3.83 (m, 2H; GalNII H-5, GalNIII H-5),
3.75 (m, 1H; GalNI H-2), 3.71, 3.64, 3.63 (3s, 9H; COOCH3), 3.66–3.50
(m, 4H; GalNII H-2, H-6a, GalNIII H-2, H-6a), 3.30–3.22 (m, 2H; GalNII
H-6b, GalNIII H-6b), 2.57–2.33 (m, 4H; CH2CO), 2.00 ppm (s, 3H;
COCH3); MS: m/z: 2988 [MꢀH]ꢀ (for 35Cl); elemental analysis calcd
(%) for C145H124Cl9N3O48: C 58.14, H 4.17, N 1.40; found: C 57.99, H
4.37, N 1.33.
J
J
4,5 <1.0 Hz, 1H; GalN H-4), 5.21–5.17 (m, 2H; GlcA H-3, H-4), 5.07 (d,
1,2 =8.0 Hz, 1H; GalN H-1), 4.97 (m, 1H; GlcA H-2), 4.91 (ABq, 2H;
CH2Ar), 4.72 (d, J1,2 =7.5 Hz, 1H; GlcA H-1), 4.65 (dd, J2,3 =11.0 Hz,
1H; GalN H-3), 4.19 (dd, J6a,6b =11.5 Hz, J5,6a =6.0 Hz, 1H; GalN H-6a),
4.11 (dd, J5,6b =7.0 Hz, 1H; GalN H-6b), 3.99 (d, J4,5 =9.5 Hz, 1H; GlcA
H-5), 3.89 (m, 1H; GalN H-5), 3.75 (s, 3H; COOCH3), 3.52 (m, 1H;
GalN H-2), 2.10, 2.09, 2.04, 2.02, 2.00 (5s, 15H; OCOCH3), 1.89 ppm (s,
3H; NHCOCH3); MS: m/z: 784 [M+Na]+; elemental analysis calcd (%)
for C36H43NO17: C 56.76, H 5.69, N 1.84; found: C 56.88, H 5.67, N 1.77.
Hexasaccharide acceptor 34: From hexasaccharide 33 (285 mg, 95 mmol)
as described for the preparation of disaccharide 16. Flash-silica chroma-
tography (petroleum ether/EtOAc 1:1) afforded the hexasaccharide 34
(198 mg, 72%) as a white powder. M.p. 163–1658C (from 2-propanol);
[a]2D0 =ꢀ5 (c=1 in chloroform); 1H NMR (400 MHz, CDCl3): d=8.10–
7.10 (m, 67H; arom. H), 6.82, 6.73, 6.72 (3d, J2,NH =7.5 Hz, 3H; 3GalN
NH), 5.81 (dd, J3,4 =3.5 Hz, J4,5 <1.0 Hz, 1H; GalNI H-4), 5.61, 5.55 (2dd,
Tetrasaccharide acetamide 37: From tetrasaccharide 27 (495 mg,
0.32 mmol) in N,N-dimethylacetamide (10 mL) as described for the prep-
aration of the disaccharide 36. Flash-silica chromatography (CH2Cl2/
MeOH 13:1) afforded the acetamide 37 (345 mg, 81%) as a white
powder. M.p. 145–1468C (from 2-propanol); [a]2D0 =ꢀ8.5 (c=1 in chloro-
form); 1H NMR (250 MHz, CDCl3): d=7.75–7.20 (m, 7H; arom. H),
5.92, 5.86 (2d, J2,NH =7.0 Hz, 2H; 2GalN NH), 5.40, 5.34 (2dd, J3,4
=
J
3,4 =3.0 Hz, J4,5 <1.0 Hz, 2H; GalNII H-4, GalNIII H-4), 5.45, 5.43, 5.37
3.0 Hz, J4,5 <1.0 Hz, 2H; 2GalN H-4), 5.24–5.17 (m, 2H; 2GlcA H-3),
5.06 (d, J1,2 =8.0 Hz, 1H; GalNI H-1), 5.05 (m, 1H; GlcAII H-4), 4.97–
4.84 (m, 2H; 2GlcA H-2), 4.89 (ABq, 2H; CH2Ar), 4.76 (d, J1,2 =8.0 Hz,
1H; GalNII H-1), 4.76, 4.68 (2d, J1,2 =7.5 Hz, 2H; 2GlcA H-1), 4.61, 4.56
(2dd, J2,3 =11.0 Hz, 2H; 2GalN H-3), 4.20–3.95 (m, 9H; 2GalN H-5, H-
6a, H-6b, GlcAI H-4, 2GlcA H-5), 3.85, 3.75 (2s, 6H; COOCH3), 3.56–
3.30 (m, 2H; 2GalN H-2), 2.10, 2.07, 2.05, 2.03, 2.02, 2.01, 1.94, 1.89 ppm
(8s, 33H; OCOCH3, NHCOCH3); MS: m/z: 1323 [M+H]+; elemental
analysis calcd (%) for C59H74N2O32: C 53.55, H 5.64, N 2.12; found: C
53.28, H 5.60, N 2.05.
(3dd, J2,3 =J3,4 =9.5 Hz, 3H; 3GlcA H-3), 5.24–5.19 (m, 3H; 3GlcA H-2),
5.16, 5.14, 5.12 (3d, J1,2 =8.0 Hz, 3H; 3GalN H-1), 4.88 (d, J1,2 =7.5 Hz,
1H; GlcA H-1), 4.87 (ABq, 2H; CH2Ar), 4.85 (m, 1H; GalNI H-3), 4.82,
4.81 (2d, J1,2 =7.5 Hz, 2H; 2GlcA H-1), 4.68, 4.65 (2dd, J2,3 =11.0 Hz,
2H; GalNII H-3, GalNIII H-3), 4.51–4.49 (m, 2H; GalNI H-6a, H-6b),
4.30, 4.26 (2dd, J4,5 =9.5 Hz, 2H; GlcAI H-4, GlcAII H-4), 4.14–4.08 (m,
2H; GalNI H-5, GlcAIII H-4), 4.0, 3.99, 3.96 (3d, J4,5 =9.5 Hz, 3H; 3GlcA
H-5), 3.89–3.83 (m, 2H; GalNII H-5, GalNIII H-5), 3.76 (m, 1H; GalNI H-
2), 3.71, 3.69, 3.67 (3s, 9H; COOCH3), 3.60–3.52 (m, 4H; GalNII H-2, H-
6a, GalNIII H-2, H-6a), 3.29 (d, J4,OH =3.0 Hz, 1H; GlcAIII HO-4), 3.28–
3.23 ppm (m, 2H; GalNII H-6b, GalNIII H-6b); MS: m/z: 2890 [MꢀH]ꢀ
(for 35Cl); elemental analysis calcd (%) for C140H118Cl9N3O46: C 58.03, H
4.10, N 1.45; found: C 58.03, H 4.10, N 1.39.
Hexasaccharide acetamide 38: From hexasaccharide 30 (200 mg,
90 mmol) in N,N-dimethylacetamide (5 mL) as described for the prepara-
tion of the disaccharide 36. Flash-silica chromatography (CH2Cl2/MeOH
13:1) afforded the acetamide 38 (110 mg, 64%) as a white powder. M.p.
159–1608C (from 2-propanol); [a]2D0 =ꢀ1.5 (c=1 in chloroform);
1H NMR (250 MHz, CDCl3): d=7.80–6.90 (m, 7H; arom. H), 5.86, 5.84,
5.78 (3d, J2,NH =7.0 Hz, 3H; 3GalN NH), 5.43, 5.40 (2dd, J3,4 =3.0 Hz,
Octasaccharide 35: From imidate 5 (160 mg, 180 mmol) and acceptor 34
(260 mg, 90 mmol) as described for the preparation of disaccharide 6.
Flash-silica chromatography (toluene/EtOAc 1:1, containing 0.1% of
NEt3) afforded the octasaccharide 35 (130 mg, 40%) as a white powder.
M.p. 170–1728C (from 2-propanol); [a]2D0 =ꢀ7 (c=1 in chloroform);
1H NMR (400 MHz, CDCl3): d=8.10–7.05 (m, 67H; arom. H), 6.83, 6.77,
6.72, 6.70 (4d, J2,NH =7.5 Hz, 4H; 4GalN NH), 5.81 (dd, J3,4 =3.5 Hz,
J
J
4,5 <1.0 Hz, 3H; 3GalN H-4), 5.10–5.04 (m, 3H; 3GlcA H-3), 5.07 (d,
1,2 =8.0 Hz, 1H; GalNI H-1), 5.04 (m, 1H; GlcAIII H-4), 4.89 (ABq, 2H;
CH2Ar), 4.97–4.80 (m, 3H; 3GlcA H-2), 4.75, 4.74 (2d, J1,2 =7.5 Hz, 2H;
GalNII H-1, GalNIII H-1), 4.68, 4.65, 4.63 (3d, J1,2 =7.5 Hz, 3H; 3GlcA H-
1), 4.63–4.50 (m, 3H; 3GalN H-3), 4.20–3.75 (m, 14H; 3GalN H-5, H-6a,
H-6b, GlcAI H-4, GlcAII H-4, 3GlcA H-5), 3.86, 3.84, 3.74 (3s, 9H;
COOCH3), 3.45–3.25 (m, 3H; 3GalN H-2), 2.10, 2.09, 2.07, 2.05, 2.03,
2.02, 2.00, 1.97 ppm (8s, 48H; OCOCH3, NHCOCH3); MS: m/z: 1882
[MꢀH]ꢀ; elemental analysis calcd (%) for C82H105N3O47: C 52.26, H 5.62,
N 2.23; found: C 52.33, H 5.80, N 2.09.
J
4,5 <1.0 Hz, 1H; GalNI H-4), 5.57, 5.54 (2dd, J3,4 =3.0, J4,5 <1.0 Hz, 2H;
GalNII H-4, GalNIII H-4), 5.46, 5.45, 5.43 (3dd, J2,3 =J3,4 =9.5 Hz, 3H;
GlcAI H-3, GlcAII H-3, GlcAIII H-3), 5.22 (dd, J1,2 =7.5 Hz, 1H; GlcAI
H-2), 5.18 (dd, J3,4 =3.5 Hz, J4,5 <1.0 Hz, 1H; GalNIV H-4), 5.17–5.06 (m,
7H; GalNI H-1, GalNII H-1, GalNIII H-1, GlcAII H-2, GlcAIII H-2,
GlcAIV H-3, H-4), 4.89 (d, J1,2 =8.5 Hz, 1H; GalNIV H-1), 4.88 (d, 1H;
GlcAI H-1), 4.87 (ABq, 2H; CH2Ar), 4.87–4.83 (m, 2H; GalNI H-3,
GlcAIV H-2), 4.83, 4.82 (2d, J1,2 =7.5 Hz, 2H; GlcAII H-1, GlcAIII H-1),
4.65, 4.64 (2dd, J2,3 =11.0 Hz, 2H; GalNII H-3, GalNIII H-3), 4.58 (d,
Octasaccharide acetamide 39: From octasaccharide 35 (124 mg, 34 mmol),
in benzene (3.5 mL) and N,N-dimethylacetamide (1.5 mL) as described
for the preparation of 36, except that more tributylstannane (110 mL,
0.41 mmol) and AIBN (50 mg) were added after 2 h and 4 h. Flash-silica
chromatography (CH2Cl2/acetone 3:1, then CH2Cl2/MeOH 12:1) afforded
the acetamide 39 (85 mg, 62%) as a white powder. Rf =0.25 (dichlorome-
thane/acetone 3:1); m.p. 176–1788C (from 2-propanol); [a]2D0 =ꢀ9 (c=1
in chloroform); 1H NMR (400 MHz, CDCl3): d=8.10–6.90 (m, 67H;
arom. H), 5.72 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNI H-4), 5.68 (d,
J
1,2 =8.0 Hz, 1H; GlcAIV H-1), 4.51–4.49 (m, 2H; GalNI H-6a, H-6b),
4.31, 4.26, 4.22 (3dd, J4,5 =9.5 Hz, 3H; GlcAI H-4, GlcAII H-4, GlcAIII H-
4), 4.20 (m, 1H; GalNIV H-3), 4.12 (m, 1H; GalNI H-5), 4.01, 3.99, 3.96
(3d, 3H; GlcAI H-5, GlcAII H-5, GlcAIII H-5), 3.92, (d, J4,5 =9.5 Hz, 1H;
GlcAIV H-5), 3.87–3.83 (m, 2H; GalNII H-5, GalNIII H-5), 3.75 (m, 1H;
GalNI H-2), 3.71, 3.64, 3.63 (3s, 12H; COOCH3), 3.64–3.52 (m, 6H;
GalNII H-2, H-6a, GalNIII H-2, H-6a, GalNIV H-2, H-5), 3.37 (dd, J6a,6b
=
J
2,NH =7.5 Hz, 1H; GalNIV NH), 5.52, 5.45 (2dd, J3,4 =3.5 Hz, J4,5 <1.0 Hz,
11.5 Hz, J5,6a =6.5 Hz, 1H; GalNIV H-6a), 3.28–3.19 (m, 2H; GalNII H-6b,
GalNIII H-6b), 3.09 (dd, J5,6b =6.5 Hz, 1H; GalNIV H-6b), 2.02, 1.99, 1.97,
1.96, 1.82 ppm (5s, 15H; OCOCH3); MS: m/z: 3595 [MꢀH]ꢀ (for 35Cl);
elemental analysis calcd (%) for C165H148Cl12N4O62: C 54.98, H 4.14, N
1.55; found: C 54.78, H 4.23, N 1.49.
2H; GalNII H-4, GalNIII H-4), 5.49–5.41 (m, 3H; GlcAI H-3, GlcAII H-3,
GlcAIII H-3), 5.40, 5.37, 5.35 (3d, J2,NH =7.0 Hz, 3H; GalNI NH, GalNII
NH, GalNIII NH), 5.30–5.13 (m, 6H; GalNI H-1, GlcAI H-2, GlcAII H-2,
GlcAIII H-2, GlcAIV H-3, H-4), 5.10 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H;
GalNIV H-4), 4.92–4.75(m, 9H; GalNI H-3, GalNII H-1, H-3, GalNIII H-1,
H-3, GlcAI H-1, GlcAII H-1, GlcAIII H-1, GlcAIV H-2), 4.82 (ABq, 2H;
CH2Ar), 4.71 (d, J1,2 =8.5 Hz, 1H; GalNIV H-1), 4.58 (d, J1,2 =7.5 Hz, 1H;
GlcAIV H-1), 4.52–4.40 (m, 3H; GalNI H-6a, H-6b, GalNIV H-3), 4.22,
4.16, 4.13 (3dd, J3,4 =J4, =9.5 Hz, 3H; GlcAI H-4, GlcAII H-4, GlcAIII H-
4), 4.12 (m, 1H; GalNI H-5), 4.01, 4.00, 3.94 (3d, 3H; GlcAI H-5, GlcAII
H-5, GlcAIII H-5), 3.92, (d, J4,5 =9.5 Hz, 1H; GlcAIV H-5), 3.68, 3.67,
3.66, 3.61 (4s, 12H; COOCH3), 3.69–3.51 (m, 5H; GalNII H-5, H-6a, Gal-
NIII H-5, H-6a, GalNIV H-5), 3.40 (m, 1H; GalNI H-2), 3.20–3.00 (m, 7H;
Disaccharide acetamide 36:
A mixture of disaccharide 6 (210 mg,
0.24 mmol), tributylstannane (400 mL, 1.46 mmol), and AIBN (22 mg) in
benzene (4.5 mL) and N,N-dimethylacetamide (1 mL) was stirred for 1 h
at RT under a flow of dry argon, then was heated for 2 h at 808C, cooled
and concentrated. The residue was stirred at 08C with petroleum ether
(20 mL) and the resulting solids were filtered off. Flash-silica chromatog-
raphy (EtOAc/petroleum ether 2:1) afforded the acetamide 36 (137 mg,
74%) as a white powder. M.p. 206–2078C (from MeOH); [a]2D0 =ꢀ22
9572
www.chemeurj.org
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 9561 – 9578

Stereocontrolled Construction of Size-Defined Oligomers
FULL PAPER
GalNII H-2, H-6b, GalNIII H-2, H-6b, GalNIV H-2, H-6a, H-6b), 2.01,
1.98, 1.97, 1.95, 1.92, 1.89, 1.79 ppm (6s, 27H; OCOCH3, NHCOCH3); el-
emental analysis calcd (%) for C165H160N4O62: C 62.11, H 5.05, N 1.76;
found: C 61.90, H 4.95, N 1.57.
(25 mg) for 20 h at RT. The catalyst was removed by filtration through a
Celite pad and the filtrate was concentrated. The residue was eluted
from a column (3ꢅ80 cm) of Sephadex LH-20 with water and lyophilized
to afford the target molecule 44 (19 mg, 86%) as a white powder. Rf =
0.17 (EtOAc/MeOH/water 6:2:1); [a]2D0 =ꢀ7 (c=1, equil., in water);
Disaccharide glycoside 40: A solution of ester 36 (245 mg, 0.32 mmol) in
THF (6 mL) was treated at ꢀ108C with a freshly prepared solution of hy-
drogen peroxide (30 wt% solution in water, 0.8 mL) and lithium hydrox-
ide (1m, 1.6 mL), and the mixture was stirred for 1 h at ꢀ108C and 16 h
at RT, then was cooled to 08C. Methanol (4 mL) and sodium hydroxide
(4m, 2 mL) were added, and the mixture was stirred for 4 h at RT, then
was concentrated and diluted with water (10 mL). The mixture was ad-
justed to pH 3 (pH meter monitoring) with Amberlite IR-120 [H+] resin,
filtered, concentrated, and dried to afford the acid (136 mg, 79%) as a
white powder. The solid was dissolved in water (5 mL), the solution was
adjusted to pH 7 (pH meter monitoring) with diluted aqueous NaOH,
and was then eluted from a column (3ꢅ80 cm) of Sephadex LH-20 with
water and lyophilized to afford the sodium salt 40 (121 mg, 69% from
36) as a white powder. Rf =0.37 (EtOAc/MeOH/water 6:2:1); m.p. 261–
2638C (from MeOH); [a]2D0 =ꢀ38 (c=1 in water); 1H NMR (250 MHz,
D2O, internal HOD, dH =4.79): d=7.79–7.28 (m, 7H; arom. H), 4.76
1H NMR (400 MHz, D2O, internal HOD, dH =4.79): d=5.22 (d, J1,2
3.5 Hz; GalN H-1a), 4.68 (d, J1,2 =8.5 Hz; GalN H-1b), 4.56 (d, J1,2
8.0 Hz; GlcA H-1a), 4.50 (d, J1,2 =8.0 Hz; GlcA H-1b), 4.29 (dd, J2,3
=
=
=
11.0 Hz; GalN H-2a), 4.25 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz; GalN H-4a),
4.18 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz; GalN H-4b), 4.12 (m; GalN H-5a),
4.01 (dd; GalN H-3a), 3.99 (dd, J2,3 =11.0 Hz; GalN H-2b), 3.82 (dd;
GalN H-3b), 3.78–3.67 (m; GalN H-5b, H-6a, H-6b, GlcA H-5), 3.49–3.47
(m, 2H; GlcA H-3, H-4), 3.34 (m, 1H; GlcA H-2), 2.04 ppm (s, 3H;
NHCOCH3); HRMS: m/z: calcd for C14H23NO12Na [M+H]+: 420.1118;
found: 420.1114.
Tetrasaccharide 45: From glycoside 41 (31 mg, 30 mmol) as described for
the preparation of 44 to afford the target molecule 45 (23 mg, 87%) as a
white powder. Rf =0.20 (EtOAc/MeOH/water 4:2:1); [a]2D0 =ꢀ15 (c=1,
1
equil., in water); H NMR (400 MHz, D2O, internal HOD, dH =4.79): d=
5.22 (d, J1,2 =3.5 Hz; GalNI H-1a), 4.69 (d, J1,2 =8.5 Hz; GalNI H-1b),
4.57 (d, J1,2 =8.0 Hz; GlcAI H-1a), 4.52, 4.51, 4.50 (3d, J1,2 =7.5 Hz;
GlcAI H-1b, GlcAII H-1), 4.50 (d, J1,2 =8.5 Hz, 1H; GalNII H-1), 4.29 (dd,
(ABq, 2H; CH2Ar), 4.38 (d, J1,2 =8.5 Hz, 1H; GalN H-1), 4.30 (d, J1,2
=
8.0 Hz, 1H; GlcA H-1), 4.03 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalN H-
4), 3.90 (dd, J2,3 =11.0 Hz, 1H; GalN H-2), 3.77–3.62 (m, 2H; GalN H-
6a, H-6b), 3.55 (d, J4,5 =9.0 Hz, 1H; GlcA H-5), 3.57–3.47 (m, 2H; GalN
H-3, H-5), 3.41–3.29 (m, 2H; GlcA H-3, H-4), 3.20 (m, 1H; GlcA H-2),
1.78 ppm (s, 3H; NHCOCH3); MS: m/z: 536 [MꢀNa]ꢀ; HRMS: m/z:
calcd for C25H31NO12Na [M+H]+: 560.1744; found: 560.1747.
J
2,3 =11.0 Hz; GalNI H-2a), 4.20 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz; GalNI H-
4a), 4.19, 4.13 (2dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz; GalNI H-4b, GalNII H-4),
4.11 (m; GalNI H-5a), 4.04–3.98 (m, 2H; GalNI H-2b, H-3a, GalNII H-2),
3.84–3.70 (m; GalNI H-3b, H-5b, H-6a, H-6b, GalNII H-3, H-5, H-6a, H-
6b, GlcAI H-4, 2GlcA H-5), 3.60, 3.59 (2dd, J2,3 =J3,4 =9.0 Hz, 1H; GlcAI
H-3a, GlcAI H-3b), 3.52–3.46 (m, 2H; GlcAII H-3, H-4), 3.38–3.31 (m,
2H; 2GlcA H-2), 2.04, 2.02 ppm (2s, 6H; NHCOCH3); HRMS: m/z:
calcd for C28H44N2O23Na [MꢀNa+2H]+: 799.2233; found: 799.2221.
Tetrasaccharide glycoside 41: From ester 37 (310 mg, 0.23 mmol) as de-
scribed for the preparation of 40 to afford the sodium salt 41 (160 mg,
71%) as a white powder. Rf =0.26 (EtOAc/MeOH/water 4:2:1); [a]D20
=
1
ꢀ32 (c=1 in water); H NMR (250 MHz, D2O, internal HOD, dH =4.79):
Hexasaccharide 46: From glycoside 42 (30 mg, 20 mmol) as described for
the preparation of 44 to afford the target molecule 46 (20 mg, 74%) as a
white powder. Rf =0.23 (EtOAc/MeOH/water 3:2:1); [a]2D0 =ꢀ10 (c=1,
d=7.84–7.32 (m, 7H; arom. H), 4.79 (ABq, 2H; CH2Ar), 4.52, 4.50 (2d,
J
1,2 =8.5 Hz, 2H; 2GalN H-1), 4.48, 4.42 (2d, J1,2 =7.5 Hz, 2H; 2GlcA H-
1
1), 4.16, 4.10 (2dd, J3, =3.0 Hz, J4,5 <1.0 Hz, 2H; 2GalN H-4), 4.04–3.96
(m, 2H; 2GalN H-2), 3.83–3.57 (m, 11H; 2GalN H-3, H-5, H-6a, H-6b
GlcAI H-4, 2GlcA H-5), 3.56 (m, 1H; GlcAI H-3), 3.54–3.45 (m, 2H;
GlcAII H-3, H-4), 3.34, 3.32 (2dd, J2,3 =9.0 Hz, 2H; 2GlcA H-2), 2.02,
1.81 ppm (2s, 6H; NHCOCH3); MS: m/z: 937 [MꢀNa]ꢀ, 915
[MꢀNa+H]ꢀ, 457 [Mꢀ2Na]2ꢀ; HRMS: m/z: calcd for C39H52N2O23Na
[MꢀNa+2H]+: 939.2859; found: 939.2873.
equil., in water); H NMR (400 MHz, D2O, internal HOD, dH =4.79): d=
5.22 (d, J1,2 =3.5 Hz; GalNI H-1a), 4.69 (d, J1,2 =8.5 Hz; GalNI H-1b),
4.57 (d, J1,2 =8.0 Hz; GlcAI H-1a), 4.55–4.48 (m; GalNII H-1, GalNIII H-1,
GlcAI H-1b, GlcAII H-1, GlcAIII H-1), 4.29 (dd, J2,3 =11.0 Hz; GalNI H-
2a), 4.21 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz; GalNI H-4a), 4.18, 4.13, 4.12
(3dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz; GalNI H-4b, GalNII H-4, GalNIII H-4),
4.12 (m; GalNI H-5a), 4.03–3.97 (m, 3H; GalNI H-2b, H-3a, GalNII H-2,
GalNIII H-2), 3.84–3.68 (m; GalNI H-3b, H-5b, GalNII H-3, H-5, GalNIII
H-3, H-5, 3GalN H-6a, H-6b, GlcAI H-4, GlcAII H-4, 3GlcA H-5), 3.62–
3.57 (m, 2H; GlcAI H-3, GlcAII H-3), 3.51–3.48 (m, 2H; GlcAIII H-3, H-
4), 3.40–3.31 (m, 3H; 3GlcA H-2), 2.04, 2.03 ppm (2s, 9H; NHCOCH3);
HRMS: m/z: calcd for C42H65N3O34Na [Mꢀ2Na+3H]+: 1178.3347;
Hexasaccharide glycoside 42: From ester 38 (82 mg, 40 mmol) as de-
scribed for the preparation of 40 to afford the sodium salt 42 (30 mg,
51%) as a white powder. Rf =0.29 (EtOAc/MeOH/water 3:2:1); [a]D20
=
ꢀ22.5 (c=1 in water); 1H NMR (250 MHz, D2O, internal HOD, dH =
4.79): d=7.93–7.47 (m, 7H; arom. H), 4.94 (ABq, 2H; CH2Ar), 4.54,
found: 1178.3365; calcd for C42H65N3O34 [Mꢀ3Na+3H]2ꢀ
:
576.6646;
4.51, 4.50 (3d, J1,2 =8.5 Hz, 3H; 3GalN H-1), 4.49, 4.48, 4.43 (3d, J1,2
=
7.5 Hz, 3H; 3GlcA H-1), 4.16, 4.10 (2dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 3H;
3GalN H-4), 4.05–3.95 (m, 3H; 3GalN H-2), 3.87–3.63 (m, 17H; 3GalN
H-3, H-5, H-6a, H-6b, GlcAI H-4, GlcAII H-4, 3GlcA H-5), 3.59–3.49 (m,
2H; GlcAI H-3, GlcAII H-3), 3.52–3.46 (m, 2H; GlcAIII H-3, H-4), 3.36,
3.34, 3.33 (3dd, J2,3 =9.0 Hz, 3H; 3GlcA H-2), 2.03, 2.00, 1.81 ppm (3s,
found: 576.6630.
Octasaccharide 47: From glycoside 43 (15 mg, 8 mmol) as described for
the preparation of 44 to afford the target molecule 47 (9.5 mg, 67%) as a
white powder. Rf =0.25 (EtOAc/MeOH/water 2:2:1); [a]2D0 =ꢀ16 (c=1,
1
equil., in water); H NMR (400 MHz, D2O, internal HOD, dH =4.79): d=
9H; NHCOCH3); MS: m/z: 1294 [Mꢀ3Na+2H]ꢀ, 647 [Mꢀ3Na+H]2ꢀ
,
5.22 (d, J1,2 =3.5 Hz; GalNI H-1a), 4.68 (d, J1,2 =8.5 Hz; GalNI H-1b),
4.56 (d, J1,2 =8.0 Hz; GlcAI H-1a), 4.52–4.47 (m; GalNII H-1, GalNIII H-1,
GalNIV H-1, GlcAI H-1b, GlcAII H-1, GlcAIII H-1, GlcAIV H-1), 4.29 (dd,
430.5
[Mꢀ3Na]3ꢀ
;
HRMS:
m/z:
calcd
for
C53H73N3O34Na
[Mꢀ2Na+3H]+: 1318.3973; found: 1318.3994.
J
2,3 =11.0 Hz; GalNI H-2a), 4.21 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz; GalNI H-
Octasaccharide glycoside 43: From ester 39 (78 mg, 25 mmol) as described
for the preparation of 26 to afford the sodium salt 43 (26 mg, 60%) as a
white powder. [a]2D0 =ꢀ23.5 (c=1 in water); 1H NMR (400 MHz, D2O, in-
ternal HOD, dH =4.79): d=8.05–7.21 (m, 7H; arom. H), 4.95 (ABq, 2H;
CH2Ar), 4.56–4.50 (m, 4H; 4GalN H-1), 4.50–4.42 (m, 4H; 4GlcA H-1),
4.17, 4.11 (2dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 4H; 4GalN H-4), 4.03–3.97 (m,
4H; 4GalN H-2), 3.86–3.67 (m, 23H; 4GalN H-3, H-5, H-6a, H-6b,
GlcAI H-4, GlcAII H-4, GlcAIII H-4, 4GlcA H-5), 3.62–3.55 (m, 3H;
GlcAI H-3, GlcAII H-3, GlcAIII H-3), 3.50–3.48 (m, 2H; GlcAIV H-3, H-
4), 3.30–3.40 (m, 4H; 4GlcA H-2), 2.03, 2.00, 1.81 ppm (3s, 12H;
4a), 4.18–4.17, 4.14–4.13 (2m, 4H; GalNI H-4b, H-5a, GalNII H-4, Gal-
NIII H-4, GalNIV H-4), 4.04–3.98 (m, 4H; GalNI H-2b, H-3a, GalNII H-2,
GalNIII H-2, GalNIV H-2), 3.85–3.65 (m; GalNI H-3b, H-5b, GalNII H-3,
H-5, GalNIII H-3, H-5, GalNIV H-3, H-5, 4GalN H-6a, H-6b, GlcAI H-4,
GlcAII H-4, GlcAIII H-4, 4GlcA H-5), 3.62–3.53 (m, 3H; GlcAI H-3,
GlcAII H-3, GlcAIII H-3), 3.51–3.48 (m, 2H; GlcAIV H-3, H-4), 3.39–3.31
(m, 4H; 4GlcA H-2), 2.04, 2.03 ppm (2s, 12H; NHCOCH3); 13C NMR
(100 MHz, D2O, internal acetone, dC =30.83): d=175.61, 175.19, 175.17
(4GlcA C-6, NHCOCH3), 104.97, 104.79, 101.51, 101.47 (4GalN C-1,
4GlcA C-1), 81.02, 80.98, 80.36, 80.33, 80.31 (4GalN C-3, GlcAI C-4,
GlcAII C-4, GlcAIII C-4), 77.06, 77.04, 77.02, 76.76, 75.95, 75.62, 74.34,
73.38, 73.13, 73.09, 72.46, 68.39, 68.35 (4GalN C-4, C-5, GlcAIV C-4,
4GlcA C-2, C-3, C-5), 61.74 (4GalN C-6), 51.68, 51.64 (4GalN C-2),
NHCOCH3); HRMS: m/z: calcd for C67H94N4O45 [Mꢀ4Na+4H]2ꢀ
:
836.2517; found: 836.2535.
Disaccharide 44: A solution of the glycoside 40 (30 mg, 50 mmol) in
water (5 mL) was hydrogenated in the presence of 10% Pd/C catalyst
Chem. Eur. J. 2009, 15, 9561 – 9578
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9573

J.-C. Jacquinet et al.
23.17, 23.15 ppm (NHCOCH3); MS: m/z: 788 [Mꢀ2Na]2ꢀ
,
777
H), 6.87, 6.78 (2d, J2,NH =8.0 Hz, 2H; 2GalN NH), 5.46 (dd, J3,4 =3.0 Hz,
[Mꢀ3Na+H]2ꢀ, 767 [Mꢀ4Na+2H]2ꢀ
.
J
4,5 <1.0 Hz, 1H; GalNI H-4), 5.41 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H;
GalNII H-4), 5.23 (m, 1H; NHCOO), 5.19–5.01 (m, 5H; 2GlcA H-3,
GlcAII H-4, CH2Ph), 4.92 (d, J1,2 =8.0 Hz, 1H; GalNII H-1), 4.90–4.82 (m,
3H; GalNI H-1, 2GlcA H-2), 4.69–4.61 (m, 2H; 2GlcA H-1), 4.49, 4.28
(2dd, J2,3 =11.0 Hz, 2H; 2GalN H-3), 4.20–3.70 (m, 7H; 2GalN H-6a,
2H-6b, GlcAI H-4, 2GlcA H-5), 3.82–3.79 (m, 4H; GalN H-2, 2GalN H-
5, CH2O), 3.82, 3.78 (2s, 6H; COOCH3), 3.77–3.65 (m, 2H; GalN H-2,
CH2O), 3.47–3.36 (m, 2H; CH2N), 2.11, 2.04, 2.01, 2.00, 1.99, 1.98 ppm
(6s, 27H; OCOCH3); MS: m/z: 1562 [MꢀH]ꢀ (for 35Cl); elemental analy-
sis calcd (%) for C58H71Cl6N3O34: C 44.46, H 4.57, N 2.68; found: C 44.22,
H 4.47, N 2.48.
Disaccharide acetamide 48: From disaccharide 8 (186 mg, 0.21 mmol) in
N,N-dimethylacetamide (2 mL) as described for the preparation of 39.
Flash-silica chromatography (CH2Cl2/MeOH 19:1) afforded the acet-
amide 48 (88 mg, 53%) as a white powder. M.p. 1248C (diethyl ether/
EtOH); [a]2D0 =+5 (c=1 in chloroform); 1H NMR (250 MHz, CDCl3):
d=7.35 (s, 5H; arom. H), 6.07 (d, J2,NH =7.0 Hz, 1H; GalN NH), 5.35
(m, 1H; NHCOO), 5.33 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalN H-4),
5.22–5.12 (m, 2H; GlcA H-3, H-4), 5.09 (s, 2H; CH2Ph), 4.97 (m, 1H;
GlcA H-2), 4.85 (d, J1,2 =8.5 Hz, 1H; GalN H-1), 4.73 (d, J1,2 =8.0 Hz,
1H; GlcA H-1), 4.47 (dd, J2,3 =11.0 Hz, 1H; GalN H-3), 4.12 (dd, J6a,6b
=
11.5 Hz, J5,6a =6.0 Hz, 1H; GalN H-6a), 4.03–3.99 (m, 2H; GalN H-6b,
GlcA H-5), 3.40–3.30 (m, 2H; GalN H-5, CH2O), 3.74 (s, 3H;
COOCH3), 3.69–3.61 (m, 2H; GalN H-2, CH2O), 3.38–3.32 (m, 2H;
CH2N), 2.07, 2.04, 2.02, 2.01, 1.99 ppm (5s, 18H; OCOCH3, NHCOCH3);
MS: m/z: 821 [M+Na]+; elemental analysis (%) calcd for C35H46N2O19: C
52.63, H 5.80, N 3.51; found: C 52.65, H 5.61, N 3.89.
Tetrasaccharide acetamide 53: From tetrasaccharide 52 (313 mg,
0.2 mmol) as described for the preparation of 39. Flash-silica chromatog-
raphy (CH2Cl2/MeOH 15:1) afforded the acetamide 53 (175 mg, 65%) as
a white powder. M.p. 159–1608C (EtOAc/petroleum ether); [a]2D0 =+8.5
1
(c=1 in chloroform); H NMR (250 MHz, CDCl3): d=7.35 (s, 5H; arom.
H), 5.91, 5.81 (2d, J2,NH =7.0 Hz, 2H; 2GalN NH), 5.39–5.26 (m, 3H;
2GalN H-4, NHCOO), 5.25–5.11 (m, 4H; GlcAII H-3, H-4, CH2Ph), 5.03
(dd, J2,3 =J3,4 =9.0 Hz, 1H; GlcAI H-3), 4.93–4.80 (m, 3H; GalN H-1,
2GlcA H-2), 4.75 (d, J1,2 =8.5 Hz, 1H; GalN H-1), 4.68–4.62 (m, 2H;
2GlcA H-1), 4.57–4.42 (m, 2H; 2GalN H-3), 4.09–3.64 (m, 11H; 2GalN
H-5, 2H-6a, 2H-6b, GlcAI H-4, 2GlcA H-5, CH2O), 3.73, 3.66 (2s, 6H;
COOCH3), 3.54–3.41 (m, 4H; 2GalN H-2, CH2N), 2.11, 2.04, 2.01, 2.00,
1.99, 1.98 ppm (6s, 33H; OCOCH3, NHCOCH3); MS: m/z: 1359
[M+2H]+; elemental analysis calcd (%) for C58H77N3O34: C 51.21, H
5.71, N 3.09; found: C 51.45, H 5.74, N 2.99.
Trisaccharide 50: From alcohol 23 (200 mg, 0.18 mmol) and 3,4,6-tri-O-
acetyl-2-deoxy-2-trichloroacetamido-1-O-trichloroacetimidoyl-a-d-galac-
topyranose[16] (193 mg, 0.33 mmol) as described for the preparation of 6.
Flash-silica chromatography (EtOAc/petroleum ether 3:2, containing
0.1% of NEt3) afforded the trisaccharide 50 (64 mg, 55%) as a white
powder. [a]2D0 =+10 (c=1 in chloroform); 1H NMR (400 MHz, CDCl3):
d=8.12–7.25 (m, 25H; arom. H), 6.98 (d, J2,NH =7.5 Hz, 1H; GalNI NH),
6.83 (d, J2,NH =7.5 Hz, 1H; GalNII NH), 5.88 (dd, J3,4 =3.0 Hz, J4,5
1.0 Hz, 1H; GalNI H-4), 5.54 (dd, J2,3 =J3,4 =9.0 Hz, 1H; GlcA H-3), 5.54
<
(dd, J1,2 =7.0 Hz, 1H; GlcA H-2), 5.17 (m, 1H; NHCOO), 5.12 (dd, J3,4
=
Tetrasaccharide 54: From alcohol 23 (650 mg, 0.59 mmol) and imidate 19
(895 mg, 0.76 mmol) as described for the preparation of 6. Flash-silica
chromatography (CH2Cl2/acetone 12:1, containing 0.1% of NEt3) afford-
ed the tetrasaccharide 54 (1.07 g, 87%) as a white powder. M.p. 149–
1518C (from 2-propanol/petroleum ether); [a]2D0 =+18.5 (c=1 in chloro-
form); 1H NMR (250 MHz, CDCl 3): d=8.12–7.10 (m, 45H; arom. H),
6.80, 6.78 (2d, J2,NH =8.0 Hz, 2H; 2GalN NH), 5.83 (dd, J3,4 =3.0 Hz,
3.0 Hz, J4,5 <1.0 Hz, 1H; GalNII H-4), 5.07 (dd, J2,3 =11.0 Hz, 1H; GalNII
H-3), 5.07 (d, J1,2 =8.0 Hz, 1H; GalNI H-1), 5.02 (s, 2H; CH2Ph), 4.97 (d,
J
1,2 =7.0 Hz, 1H; GlcA H-1), 4.89 (d, J1,2 =8.0 Hz, 1H; GalNII H-1), 4.82
(dd, J2,3 =11.0 Hz, 1H; GalNI H-3), 4.48 (dd, J6a,6b =12.0 Hz, J5,6a =5.0 Hz,
1H; GalNI H-6a), 4.40 (dd, J5,6b =7.0 Hz, 1H; GalNI H-6b), 4.29 (dd,
J
4,5 =10.0 Hz, 1H; GlcA H-4), 4.16–4.09 (m, 2H; GalNI H-5, GlcA H-5),
4.01 (m, 1H; GalNII H-2), 3.88 (m, 1H; CH2O), 3.79–3.62 (m, 3H; GalNI
H-2, GalNII H-5, CH2O), 3.72 (s, 3H; COOCH3), 3.44–3.31 (m, 2H;
CH2N), 3.27 (dd, J6a,6b =12.0 Hz, J5,6a =8.0 Hz, 1H; GalNII H-6a), 3.21
J
4,5 <1.0 Hz, 1H; GalNI H-4), 5.67 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H;
GalNII H-4), 5.53–5.42 (m, 2H; 2GlcA H-3), 5.32 (dd, J3,4 =J4,5 =10.0 Hz,
1H; GlcAII H-4), 5.38–5.24 (m, 2H; 2GlcA H-2), 5.20 (m, 1H;
NHCOO), 5.14 (d, J1,2 =8.0 Hz, 1H; GalNI H-1), 5.06 (d, J1,2 =8.0 Hz,
1H; GalNII H-1), 5.02 (s, 2H; CH2Ph), 4.92, 4.87 (2d, J1,2 =7.0 Hz, 2H;
GlcAI H-1, GlcAII H-1), 4.80, 4.69 (2dd, J2,3 =11.0 Hz, 2H; 2GalN H-3),
(dd,
J
5,6b =6.0 Hz, 1H; GalNII H-6b), 1.95, 1.94, 1.93 ppm (3s, 9H;
OCOCH3); MS: m/z: 1536 [MꢀH]ꢀ (for 35Cl); elemental analysis (%)
calcd for C67H65Cl6N3O26: C 52.22, H 4.25, N 2.73; found: C 51.95, H 4.38,
N 2.63.
4.47 (dd, J6a,6b =12.0 Hz, J5,6a =6.0 Hz, 1H; GalNI H-6a), 4.43 (dd, J5,6b
=
7.0 Hz, 1H; GalNI H-6b), 4.32 (dd, J3,4 =9.5 HzJ4,5 =10.0 Hz, 1H; GlcAI
H-4), 4.16–4.09 (m, 2H; GalNI H-5, GlcAII H-5), 4.05 (d, 1H; GlcAI H-
5), 3.91–3.82 (m, 2H; GalNII H-6a, CH2O), 3.73, 3.66 (2s, 6H;
COOCH3), 3.69–3.52 (m, 4H; GalNII H-6b, 2GalN H-2, CH2O), 3.42–
3.29 (m, 3H; GalNII H-5, CH2N), 3.42–3.29 (m, 4H; CH2CO), 2.00 ppm
(s, 3H; COCH3); MS: m/z: 2144 [MꢀH]ꢀ (for 35Cl); elemental analysis
calcd (%) for C101H91Cl6N3O35: C 57.23, H 4.33, N 1.98; found: C 57.04,
H 4.13, N 1.82.
Trisaccharide acetamide 51: From trisaccharide 50 (135 mg, 90 mmol) in
benzene (3 mL) and N,N-dimethylacetamide (1 mL) as described for the
preparation of 39. Flash-silica chromatography (CH2Cl2/acetone 2:1) af-
forded the acetamide 51 (64 mg, 55%) as a white powder. M.p. 130–
1318C (diethyl ether/petroleum ether); [a]2D0 =+24 (c=1 in chloroform);
1H NMR (400 MHz, CDCl3): d=8.12–7.17 (m, 25H; arom. H), 6.03 (dd,
J
3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNI H-4), 6.98 (d, J2,NH =9.0 Hz, 1H;
GalNII NH), 5.63 (d, J2,NH =7.0 Hz, 1H; GalNI NH), 5.52 (dd, J3,4
9.0 Hz, J2,3 =7.0 Hz, 1H; GlcA H-3), 5.25 (m, 1H; NHCOO), 5.23 (dd,
=
Tetrasaccharide acceptor 55: From tetrasaccharide 54 (1.06 g, 0.50 mmol)
as described for the preparation of 16. Flash-silica chromatography
(CH2Cl2/acetone 12:1) afforded the alcohol 55 (759 mg, 75%) as a white
powder. [a]2D0 =+9.5 (c=1 in chloroform); 1H NMR (250 MHz, CDCl 3):
d=8.12–7.09 (m, 43H; arom. H), 6.88–6.71 (m, 4H; 2arom. H, 2GalN
NH), 5.69, 5.59 (2dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 2H; 2GalN H-4), 5.46,
5.34 (2dd, J2,3 =J3,4 =9.5 Hz, 2H; 2GlcA H-3), 5.27–5.09 (m, 4H; GalNII
H-1, 2GlcA H-2, NHCOO), 5.03 (d, J1,2 =8.0 Hz, 1H; GalNI H-1), 4.98
(s, 2H; CH2Ph), 4.89, 4.82 (2d, J1,2 =7.0 Hz, 2H; 2GlcA H-1), 4.76, 4.67
(2dd, J2,3 =11.0 Hz, 2H; 2GalN H-3), 4.47–4.31 (m, 2H; GalNI H-6a, H-
6b), 4.26 (dd, J4,5 =9.5 Hz, 1H; GlcAI H-4), 4.09–3.93 (m, 2H; GalNI H-
5, GlcAII H-4), 4.02, 3.97 (2d, 2H; 2GlcA H-5), 3.89–3.78 (m, 2H; GalNII
H-6a, CH2O), 3.68, 3.61 (2s, 6H; COOCH3), 3.60–3.50 (m, 4H; GalNII
H-6b, 2GalN H-2, CH2O), 3.34–3.23 ppm (m, 4H; GalNII H-5, GlcAII
HO-4, CH2N); MS: m/z: 2018 [M+H]+ (for 35Cl); elemental analysis
calcd (%) for C96H85Cl6N3O33: C 57.04, H 4.24, N 2.08; found: C 57.02, H
4.11, N 2.05.
J
1,2 =6.0 Hz, 1H; GlcA H-2), 5.18 (d, J1,2 =8.0 Hz, 1H; GalNI H-1), 5.07
(dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNII H-4), 5.03 (s, 2H; CH2Ph), 4.99
(dd, J2,3 =11.0 Hz, 1H; GalNII H-3), 4.97 (d, 1H; GlcA H-1), 4.86 (dd,
J
2,3 =11.0 Hz, 1H; GalNI H-3), 4.60 (d, J1,2 =8.5 Hz, 1H; GalNII H-1),
4.51 (dd, J4,5 =10.0 Hz, 1H; GlcA H-4), 4.46 (dd, J6a,6b =12.0 Hz, J5,6a
=
5.0 Hz, 1H; GalNI H-6a), 4.35 (dd, J5,6b =5.0 Hz, 1H; GalNI H-6b), 4.24
(d, 1H; GlcA H-5), 4.13 (dd, 1H; GalNI H-5), 3.94 (m, 1H; GalNII H-2),
3.83 (m, 1H; CH2O), 3.78–3.66 (m, 2H; GalNII H-5, CH2O), 3.77 (s, 3H;
COOCH3), 3.48–3.31 (m, 4H; GalNI H-2, GalNII H-6a, CH2N), 3.21 (dd,
J
6a,6b =12.0 Hz, J
5,6b =6.0 Hz, 1H; GalNII H-6b), 1.97, 1.95 (2s, 9H;
OCOCH3), 1.90, 1.89 ppm (2s, 6H; NHCOCH3); MS: m/z: 1334 [M+H]+;
elemental analysis (%) calcd for C67H71N3O26: C 60.31, H 5.36, N 3.15;
found: C 60.15, H 5.39, N 3.14.
Tetrasaccharide 52: From alcohol 22 (150 mg, 0.17 mmol) and imidate 5
(212 mg, 0.24 mmol) as described for the preparation of 6. Flash-silica
chromatography (EtOAc/toluene 3:1, containing 0.1% of NEt3) afforded
the tetrasaccharide 52 (181 mg, 67%) as a white powder. [a]2D0 =+6 (c=1
Pentasaccharide 56: From alcohol 55 (650 mg, 0.59 mmol) and imidate 49
(119 mg, 0.20 mmol) as described for the preparation of 6. Flash-silica
1
in chloroform); H NMR (250 MHz, CDCl3): d=7.33–7.29 (m, 5H; arom.
9574
www.chemeurj.org
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 9561 – 9578

Stereocontrolled Construction of Size-Defined Oligomers
FULL PAPER
chromatography (CH2Cl2/acetone 10:1, containing 0.1% of NEt3; then
toluene/EtOAc 3:2) afforded the pentasaccharide 56 (210 mg, 85%) as a
white powder. M.p. 144–1458C (from 2-propanol/petroleum ether);
[a]2D0 =ꢀ3 (c=1 in chloroform); 1H NMR (400 MHz, CDCl 3): d=8.09–
7.11 (m, 43H; arom. H), 6.88–6.82 (m, 3H; 2 arom. H, GalNI NH), 6.78
(d, J2,NH =7.0 Hz, 1H; GalNIII NH), 6.71 (d, J2,NH =7.0 Hz, 1H; GalNII
NH), 5.81, 5.62 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNI H-4), 5.62 (dd,
CH2N), 3.09 ppm (dd, J6a,6b =12.0 Hz, J5,6b =6.0 Hz, 1H; GalNIII H-6b);
MS: m/z: 2721 [MꢀH]ꢀ (for 35Cl); elemental analysis calcd (%) for
C
121H115Cl9N4O49: C 53.27, H 4.25, N 2.05; found: C 53.08, H 4.26, N 1.91.
Hexasaccharide acetamide 59: From 58 (154 mg, 60 mmol) in N,N-dime-
thylacetamide (2 mL) and benzene (1.5 mL) as described for the prepara-
tion of 39. Flash-silica chromatography (CH2Cl2/MeOH 19:1) afforded
the acetamide 59 (106 mg, 78%) as a white powder. [a]2D0 =+4 (c=1 in
chloroform); m.p. 161–1628C (from CH2Cl2/petroleum ether/diethyl
ether); 1H NMR (400 MHz, CDCl3): d=8.09–6.77 (m, 45H; arom. H),
J
3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNII H-4), 5.51, 5.49 (2dd, J2,3 =J3,4
=
9.0 Hz, 2H; 2GlcA H-3), 5.27–5.02 (m, 3H; 2GlcA H-2, NHCOO), 5.18
(d, J1,2 =8.5 Hz, 1H; GalNI H-1), 5.11 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H;
5.72 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNI H-4), 5.67 (d, J2,NH
=
GalNIII H-4), 5.06 (d, J1,2 =8.5 Hz, 1H; GalNII H-1), 5.11 (dd, J2,3
=
7.0 Hz, 1H; GalNIII NH), 5.54 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNII
H-4), 5.49–5.43 (m, 3H; GalNI NH, GlcAI H-3, GlcAII H-3), 5.38 (d,
11.0 Hz, 1H; GalNIII H-3), 5.01 (s, 2H; CH2Ph), 4.93 (d, J1,2 =7.5 Hz, 1H;
GlcAI H-1), 4.86 (d, J1,2 =8.5 Hz, 1H; GlcAII H-1), 4.85 (d, J1,2 =7.0 Hz,
J
2,NH =7.0 Hz, 1H; GalNII NH), 5.28 (dd, J1,2 =7.0 Hz, J2,3 =9.0 Hz, 1H;
1H; GalNIII H-1), 4.80 (dd, J2,3 =11.0 Hz, 1H; GalNI H-3), 4.67 (dd, J2,3
=
GlcA H-2), 5.22–5.08 (m, 6H; GalNI H-1, GalNIII H-4, GlcAIII H-3, H-4,
GlcA H-2, NHCOO), 5.01 (s, 2H; CH2Ph), 4.94 (d, J1,2 =8.0 Hz, 1H;
GalNII H-1), 4.89–4.81 (m, 3H; GalNII H-3, GlcAII H-2, GlcA H-1), 4.77
(dd, J2,3 =11.0 Hz, 1H; GalNI H-3), 4.76 (d, J1,2 =7.0 Hz, 1H; GlcA H-1),
4.71 (d, J1,2 =8.0 Hz, 1H; GalNIII H-1), 4.60 (d, J1,2 =8.0 Hz, 1H; GlcAIII
1.0 Hz, 1H; GalNII H-3), 4.47 (dd, J6a,6b =12.0 Hz, J5,6a =5.0 Hz, 1H;
GalNI H-6a), 4.39 (dd, J5,6b =7.0 Hz, 1H; GalNI H-6b), 4.32, 4.23 (2dd,
2H; J4,5 =10.0 Hz, 2GlcA H-4), 4.09 (m, 1H; GalNI H-5), 4.05 (d, 2H;
2GlcA H-5), 4.05 (m, 1H; GalNIII H-2), 3.92–3.81 (m, 2H; GalNII H-5,
CH2O), 3.73, 3.61 (2s, 6H; COOCH3), 3.75–3.54 (m, 5H; GalNI H-2,
GalNII H-2, H-6a, GalNIII H-5, CH2O), 3.44–3.20 (m, 4H; GalNII H-6b,
GalNIII H-6a, CH2N), 3.09 (dd, J6a,6b =12.0 Hz, J5,6b =6.0 Hz, 1H; GalNIII
H-6b), 1.95, 1.94, 1.92 ppm (3s, 9H; OCOCH3); MS: m/z: 2447 [MꢀH]ꢀ
(for 35Cl); elemental analysis calcd (%) for C110H101Cl9N4O41: C 53.84, H
4.15, N 2.28; found: C 53.60, H 4.10, N 2.19.
H-1), 4.51–4.45 (m, 2H; GalNI H-6a, GalNIII H-3), 4.33 (dd, J6a,6b
11.5 Hz, J5,6b =7.0 Hz, 1H; GalNI H-6b), 4.22, 4.18 (2dd, J3,4 =9.5 Hz,
=
J
4,5 =10.0 Hz, 2H; GlcAI H-4, GlcAII H-4), 4.09–3.99 (m, 3H; GalNI H-5,
GlcAI H-5, GlcAII H-5), 3.93 (m, 1H; GlcAIII H-5), 3.82–3.59 (m, 3H;
GalNII H-5, GalNIII H-5, CH2O), 3.68, 3.61 (2s, 9H; COOCH3), 3.53 (dd,
J
6a,6b =12.0 Hz, J5,6a =6.0 Hz, 1H; GalNII H-6a), 3.48–3.23 (m, 5H; GalNII
H-6b, GalNIII H-6a, CH2N, CH2O), 3.21–3.08 (m, 4H; 3GalN H-2, GalNIII
H-6b), 2.01, 1.98, 1.97, 1.96, 1.89 (5 s, 15H; OCOCH3), 1.78, 1.40,
1.30 ppm (3s, 9H; NHCOCH3); HRMS: m/z: calcd for C121H124N4O49
[M]2+: 1209.3745; found: 1209.3773.
Pentasaccharide acetamide 57: From pentasaccharide 56 (103 mg,
40 mmol) in benzene (3 mL) and N,N-dimethylacetamide (0.75 mL) as de-
scribed for the preparation of 39. Flash-silica chromatography (CH2Cl2/
acetone 2:1) afforded the acetamide 57 (53 mg, 59%) as a white powder.
M.p. 159–1608C (from petroleum ether/diethyl ether); [a]2D0 =+5.5 (c=1
in chloroform); 1H NMR (400 MHz, CDCl3): d=8.07–6.81 (m, 45H;
arom. H), 5.88 (d, J2,NH =9.0 Hz, 1H; GalNIII NH), 5.82 (dd, J3,4 =3.0 Hz,
Hexasaccharide 60: From alcohol 55 (759 mg, 0.34 mmol) and imidate 19
(705 mg, 0.60 mmol) as described for the preparation of 6. Flash-silica
chromatography (CH2Cl2/acetone 12:1, containing 0.1% of NEt3) afford-
ed the hexasaccharide 60 (754 mg, 66%) as a white powder. M.p. 150–
1558C (from 2-propanol/petroleum ether); [a]2D0 =+5.5 (c=1 in chloro-
form); 1H NMR (400 MHz, CDCl3): d=8.08–7.12 (m, 61H; arom. H),
6.86–6.80 (m, 5H; 4 arom. H; GalNI NH), 6.74 (2d, J2,NH =7.0 Hz, 2H;
GalNII NH, GalNIII NH), 5.81 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNI
H-4), 5.59, 5.55 (2dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 2H; GalNII H-4, GalNIII
H-4), 5.51, 5.48, 5.42 (3dd, J2,3 =J3,4 =9.5 Hz, 3H; 3GlcA H-3), 5.37 (dd,
J
4,5 <1.0 Hz, 1H; GalNI H-4), 5.73 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H;
GalNII H-4), 5.52 (d, J2,NH =9.0 Hz, 1H; GalNI NH), 5.51 (dd, J2,3 =J3,4
=
9.0 Hz, 1H; GlcAI H-3), 5.49–5.44 (m, 2H; GalNII NH, GlcAII H-3), 5.29
(dd, J1,2 =8.0 Hz, 1H; GlcAI H-2), 5.18 (m, 1H; NHCOO), 5.14–5.10 (m,
2H; GalNII H-1, GlcAII H-2), 5.04 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H;
GalNIII H-4), 5.01 (s, 2H; CH2Ph), 4.98 (d, J1,2 =8.0 Hz, 1H; GalNI H-1),
4.92 (dd, J2,3 =11.0 Hz, 1H; GalNIII H-3), 4.91–4.86 (m, 2H; GalNI H-3,
GlcAI H-1), 4.86 (d,
11.0 Hz, 1H; GalNII H-3), 4.51 (d, J1,2 =8.5 Hz, 1H; GalNIII H-1), 4.50–
4.44 (m, 2H; GalNI H-6a, GlcA H-4), 4.31 (dd, J6b,6a =11.0 Hz, J5,6b
J =
1,2 =8.0 Hz, 1H; GlcAII H-1), 4.79 (dd, J2,3
J
4,5 =10.0 Hz, 1H; GlcAIII H-4), 5.18, 5.10 (2d, J1,2 =8.0 Hz, 2H; GalNII
H-1, GalNIII H-1), 5.24–5.09 (m, 4H; 3GlcA H-2, NHCOO), 5.07 (d,
1,2 =8.0 Hz, 1H; GalNI H-1), 5.01 (s, 2H; CH2Ph), 4.91 (d, J1,2 =7.5 Hz,
=
5.0 Hz, 1H; GalNI H-6b), 4.23 (dd, J4,5 =10.0 Hz, 1H; GlcA H-4), 4.16
(dd, 1H; GlcA H-5), 4.09–4.05 (m, 2H; GalNI H-5, GlcA H-5), 3.94 (m,
1H; GalNIII H-2), 3.85–3.75 (m, 2H; GalNII H-5, CH2O), 3.72, 3.67 (2s,
6H; COOCH3), 3.75–3.68 (m, 2H; GalNIII H-5, CH2O), 3.53 (m, 1H;
GalNII H-6a), 3.46–3.40 (m, 1H; GalNII H-6b), 3.44–3.20 (m, 7H; GalNI
H-2, GalNII H-2, GalNIII H-2, H-6a, H-6b, CH2N), 1.96, 1.93, 1.87 (3s,
9H; OCOCH3), 1.76, 1.63, 1.25 ppm (3s, 9H; NHCOCH3); HRMS: m/z:
calcd for C110H110N4O41 [M]2+: 1072.3401; found: 1072.3434.
J
1H; GlcAIII H-1), 4.87, 4.81 (2d, J1,2 =7.5 Hz, 2H; GlcAI H-1, GlcAII H-
1), 4.79 (dd, J2,3 =11.0 Hz, 1H; GalNI H-3), 4.68, 4.65 (2dd, J2,3 =11.0 Hz,
2H; GalNII H-3, GalNIII H-3), 4.47 (dd, J6a,6b =11.5 Hz, J5,6a =5.0 Hz, 1H;
GalNI H-6a), 4.39 (dd, J5,6b =7.0 Hz, 1H; GalNI H-6b), 4.31, 4.25 (2dd,
J
4,5 =9.5 Hz, 2H; GlcAI H-4, GlcAII H-4), 4.10 (d, 1H; GlcAIII H-5),
4.12–4.07 (m, 1H; GalNI H-5), 4.03, 3.96 (2d, 2H; GlcAI H-5, GlcAII H-
5), 3.89–3.82 (m, 3H; GalNII H-5, GalNIII H-5, CH2O), 3.73, 3.64, 3.63
(3s, 9H; COOCH3), 3.74–3.50 (m, 6H; GalNII H-6a, H-6b, 3GalN H-2,
CH2O), 3.42–3.24 (m, 4H; GalNIII H-6a, H-6b, CH2N), 2.60–2.31 (m, 4H;
CH2CO), 2.01 ppm (s, 3H; COCH3); MS: m/z: 1512 [Mꢀ2H]2ꢀ (for 35Cl);
elemental analysis calcd (%) for C144H127Cl9N4O50: C 57.03, H 4.22, N
1.85; found: C 56.95, H 4.25, N 1.73.
Hexasaccharide 58: From alcohol 55 (209 mg, 0.10 mmol) and imidate 5
(162 mg, 0.19 mmol) as described for the preparation of 6. Flash-silica
chromatography (toluene/EtOAc 3:2, containing 0.1% of NEt3) afforded
the hexasaccharide 58 (154 mg, 55%) as a white powder. M.p. 177–1798C
(from 2-propanol/petroleum ether); [a]2D0 =+2.5 (c=1 in chloroform);
1H NMR (250 MHz, CDCl3): d=8.09–6.92 (m, 45H; arom. H), 6.85 (d,
Hexasaccharide acceptor 61: From hexasaccharide 60 (754 mg,
0.25 mmol) as described for the preparation of 16. Flash-silica chroma-
tography (CH2Cl2/acetone 12:1) afforded the alcohol 61 (604 mg, 83%)
as a white powder. M.p. 161–1628C (from 2-propanol); [a]2D0 =+1 (c=1
in chloroform); 1H NMR (400 MHz, CDCl3): d=8.08–7.12 (m, 62H;
arom. H), 6.91 (d, J2,NH =7.0 Hz, 1H; GalNI NH), 6.87–6.76 (m, 5H;
GalNII NH, GalNIII NH, 3 arom. H), 5.81 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz,
1H; GalNI H-4), 5.61, 5.56 (2dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 2H; GalNII H-
4, GalNIII H-4), 5.49, 5.42, 5.38 (3dd, J2,3 =J3,4 =9.5 Hz, 3H; 3GlcA H-3),
5.25–5.09 (m, 6H; GalNII H-1, GalNIII H-1, 3GlcA H-2, NHCOO), 5.06
(d, J1,2 =8.0 Hz, 1H; GalNI H-1), 5.00 (s, 2H; CH2Ph), 4.92, 4.84, 4.81
(3d, J1,2 =7.5 Hz, 3H; 3GlcA H-1), 4.79 (dd, J2,3 =11.0 Hz, 1H; GalNI H-
3), 4.68, 4.63 (2dd, J2,3 =11.0 Hz, 2H; GalNII H-3, GalNIII H-3), 4.45 (dd,
J
2,NH =7.0 Hz, 1H; GalNI NH), 6.84 (d, J2,NH =7.0 Hz, 1H; GalNIII NH),
6.83 (d, J2,NH =7.0 Hz, 1H; GalNII NH), 5.82 (dd, J3,4 =3.0 Hz, J4,5
<
1.0 Hz, 1H; GalNI H-4), 5.59 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNII
H-4), 5.49, 5.47 (2dd, J2,3 =9.0 Hz, J3,4 =10.0 Hz, 2H; GlcAI H-3, GlcAII
H-3), 5.28–5.01 (m, 10H; GalNI H-1, GalNII H-1, GalNIII H-4, GlcAI H-
2, GlcAII H-2, GlcAIII H-3, H-4, CH2PH; NHCOO), 4.94–4.78 (m, 3H;
GlcAI H-1, GlcAII H-1, GlcAIII H-2), 4.81 (dd, J2,3 =11.0 Hz, 1H; GalNI
H-3), 4.89 (d, J1,2 =8.5 Hz, 1H; GalNIII H-1), 4.68 (dd, J2,3 =11.0 Hz, 1H;
GalNII H-3), 4.58 (d, J1,2 =8.0 Hz, 1H; GlcAIII H-1), 4.47–4.31 (m, 2H;
GalNI H-6a, H-6b), 4.30–4.16 (m, 3H; GalNIII H-3, GlcAI H-4, GlcAII H-
4), 4.11 (m, 1H; GalNI H-5), 4.02, 3.97 (2d, J4,5 =10.0 Hz, 2H; GlcAI H-
5, GlcAII H-5), 3.96–3.78 (m, 3H; GalNII H-5, GlcAIII H-5, CH2O), 3.74,
3.71, 3.64 (3s, 9H; COOCH3), 3.79–3.51 (m, 6H; GalNII H-6a, GalNIII H-
5, 3GalN H-2, CH2O), 3.41–3.22 (m, 4H; GalNII H-6b, GalNIII H-6a,
J
6a,6b =11.5 Hz, J5,6a =5.0 Hz, 1H; GalNI H-6a), 4.38 (dd, J5,6b =7.0 Hz,
1H; GalNI H-6b), 4.30, 4.26 (2dd, J3,4 =8.0 Hz, J4,5 =9.5 Hz, 2H; GlcAI
Chem. Eur. J. 2009, 15, 9561 – 9578
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9575

J.-C. Jacquinet et al.
H-4, GlcAII H-4), 4.12–3.95 (m, 5H; GalNI H-5, 3GlcA H-5, GlcAIII H-
4), 3.89–3.81 (m, 3H; GalNII H-5, GalNIII H-5, CH2O), 3.72, 3.67, 3.63
(3s, 9H; COOCH3), 3.72–3.52 (m, 6H; GalNII H-6a, H-6b, 3GalN H-2,
CH2O), 3.40 (d, J4,OH =3.0 Hz, 1H; GlcAIII HO-4), 3.42–3.22 ppm (m,
4H; GalNIII H-6a, H-6b, CH2N); MS: m/z: 2927 [MꢀH]ꢀ, 1463 [MꢀH]2ꢀ
(for 35Cl); elemental analysis calcd (%) for C139H121Cl9N4O48: C 56.89, H
4.16, N 1.91; found: C 56.65, H 4.29, N 1.82.
GalNIII NH), 5.81 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNI H-4), 5.57,
5.54 (2dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 2H; GalNII H-4, GalNIII H-4), 5.47,
5.46, 5.43 (3dd, J2,3 =J3,4 =9.5 Hz, 3H; GlcAI H-3, GlcAII H-3, GlcAIII H-
3), 5.18 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNIV H-4), 5.07 (d, J1,2
=
8.0 Hz, 1H; GalNI H-1), 5.25–5.05 (m, 8H; GalNII H-1, GalNIII H-1,
GlcAI H-2, GlcAII H-2, GlcAIII H-2, GlcAIV H-3, H-4, NHCOO), 5.02 (s,
2H; CH2Ph), 4.91 (d, J1,2 =7.5 Hz, 1H; GlcA H-1), 4.89 (d, J1,2 =8.5 Hz,
1H; GalNIV H-1), 4.87–4.77 (m, 4H; GalNI H-3, GlcAIV H-2, 2GlcA H-
Heptasaccharide 62: From alcohol 61 (250 mg, 85 mmol) and imidate 49
(91 mg, 150 mmol) as described for the preparation of 6. Flash-silica chro-
matography (CH2Cl2/acetone 12:1, containing 0.1% of NEt3) afforded
the heptasaccharide 62 (183 mg, 67%) as a white powder. M.p. 160–
1628C (from 2-propanol); [a]2D0 =ꢀ6 (c=1 in chloroform); 1H NMR
1), 4.65 (2dd, J2,3 =11.0 Hz, 2H; GalNII H-3, GalNIII H-3), 4.57 (d, J1,2
=
8.0 Hz, 1H; GlcAIV H-1), 4.47 (dd, J6a,6b =11.5 Hz, J5,6a =5.0 Hz, 1H;
GalNI H-6a), 4.38 (dd, 1H; J5,6b =7.0 Hz, GalNI H-6b), 4.30, 4.26, 4.22
(3dd, J4,5 =9.5 Hz, 3H; GlcAI H-4, GlcAII H-4, GlcAIII H-4), 4.21 (dd,
J
2,3 =11.0 Hz, 1H; GalNIV H-3), 4.10 (m, 1H; GalNI H-5), 4.03, 3.99, 3.92
(400 MHz, CDCl3): d=8.09–7.55 (m, 63H; arom. H), 7.19 (d, J2,NH
=
(3d, 3H; GlcAI H-5, GlcAII H-5, GlcAIII H-5), 3.96 (d, J4,5 =10.0 Hz, 1H;
GlcAIV H-5), 3.89–3.82 (m, 3H; GalNII H-5, GalNIII H-5, CH2O), 3.73,
3.71, 3.64, 3.62 (4s, 12H; COOCH3), 3.74–3.53 (m, 8H; 4GalN H-2,
GalNII H-6a, GalNIII H-6a, GalNIV H-5, CH2O), 3.36 (dd, J6a,6b =11.0 Hz,
6.5 Hz, 1H; GalNI NH), 6.98–6.95 (m, 2H; arom. H), 6.98–6.95 (m, 3H;
GalNII NH, GalNIII NH, GalNIV NH), 5.82 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz,
1H; GalNI H-4), 5.59, 5.56 (2dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 2H; GalNII H-
4, GalNIII H-4), 5.51, 5.49, 5.45 (3dd, J2,3 =J3,4 =9.0 Hz, 3H; 3GlcA H-3),
5.26–5.19 (m, 2H; 2GlcA H-2), 5.18, 5.13 (2d, J1,2 =8.5 Hz, 2H; GalNII
H-1, GalNIII H-1), 5.15–4.97 (m, 6H; GalNI H-1, GalNIV H-1, GlcA H-2,
CH2Ph, NHCOO), 5.10 (d, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNIV H-4),
4.95, 4.89, 4.86 (3d, J1,2 =7.5 Hz, 3H; 3GlcA H-1), 4.85 (m, 1H; GalNVI
H-3), 4.78 (dd, J2,3 =11.0 Hz, 1H; GalNI H-3), 4.59 (2dd, J2,3 =11.0 Hz,
2H; GalNII H-3, GalNIII H-3), 4.46 (dd, J6a,6b =12.0 Hz, J5,6a =5.0 Hz, 1H;
GalNI H-6a), 4.39 (dd, J5,6b =7.0 Hz, 1H; GalNI H-6b), 4.31, 4.27, 4.24
(3dd, J4,5 =10.0 Hz, 3H; 3GlcA H-4), 4.11–4.03 (m, 2H; GalNI H-5, GlcA
H-5), 4.04, 4.01 (2d, 2H; 2GlcA H-5), 3.98 (m, 1H; GalNIV H-2), 3.90–
3.78 (m, 4H; GalNI H-2, GalNII H-5, GalNIII H-5, CH2O), 3.72, 3.61 (2s,
9H; COOCH3), 3.74–3.52 (m, 6H; GalNII H-2, H-6a, GalNIII H-2, H-6a,
GalNIV H-5, CH2O), 3.48–3.19 (m, 4H; GalNII H-6b, GalNIII H-6b,
CH2N), 3.17 (dd, J6a,6b =11.0 Hz, J5,6a =6.0 Hz, 1H; GalNIV H-6a), 3.13
(dd, J5,6b =5.0 Hz, 1H; GalNIV H-6b), 1.95, 1.93, 1.92 ppm (3s, 9H;
OCOCH3); MS: m/z: 3358 [MꢀH]ꢀ, 1679 [MꢀH]2ꢀ (for 35Cl); elemental
analysis calcd (%) for C153H137Cl12N5O56: C 54.58, H 4.10, N 2.08; found:
C 54.45, H 4.18, N 1.99.
J
5,6a =6.0 Hz, 1H; GalNIV H-6a), 3.29, 3.21 (2dd, J6a,6b =11.0 Hz, J5,6b
=
7.0 Hz, 2H; 2GalN H-6b), 3.40–3.26 (m, 2H; CH2NH), 3.09 (dd, J6a,6b
=
11.0 Hz, J5,6b =7.0 Hz, 1H; GalNIV H-6b), 2.02, 1.99, 1.98, 1.96, 1.82 ppm
(5s, 15H; OCOCH3); MS: m/z: 1815 [Mꢀ2H]2ꢀ; HRMS: m/z: calcd for
C68H108N8O42S [Mꢀ3Na+3H]2ꢀ: 869.3063; found: 869.3078.
Octasaccharide acetamide 65: From 64 (214 mg, 60 mmol) in benzene
(2 mL) and N,N-dimethylacetamide (3 mL) as described for the prepara-
tion of 39. Flash-silica chromatography (CH2Cl2/methanol 19:1) afforded
the acetamide 65 (100 mg, 53%) as a white powder. M.p. 181–1828C
(EtOAc/petroleum ether); [a]2D0 =ꢀ7 (c=1 in chloroform); 1H NMR
(400 MHz, CDCl3): d=8.02–6.74 (m, 65H; arom. H), 5.71 (dd, J3,4
=
3.5 Hz, J4,5 <1.0 Hz, 1H; GalNI H-4), 5.69 (d, J2,NH =6.5 Hz, 1H; GalNI
NH), 5.51, 5.45 (2dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 2H; GalNII H-4, GalNIII
H-4), 5.49–5.41 (m, 4H; GalNIV NH, GlcAI H-3, GlcAII H-3, GlcAIII H-
3), 5.40–5.35 (m, 2H; GalNII NH, GalNIII NH), 5.30–5.11 (m, 6H; GlcAI
H-2, GlcAII H-2, GlcAIII H-2, GlcAIV H-3, H-4, NHCOO), 5.11 (d, J1,2
=
7.5 Hz, 1H; GalNI H-1), 5.09 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNIV
H-4), 5.00 (s, 2H; CH2Ph), 4.91, 4.89 (2d, J1,2 =8.0 Hz, 2H; GalNII H-1,
GalNIII H-1), 4.82, 4.76 (2d, J1,2 =7.5 Hz, 2H; 2GlcA H-1), 4.92–4.74 (m,
Heptasaccharide acetamide 63: From 62 (193 mg, 60 mmol) in benzene
(2 mL) and N,N-dimethylacetamide (3 mL) as described for the prepara-
tion of 39. Flash-silica chromatography (CH2Cl2/methanol 19:1) afforded
the acetamide 63 (130 mg, 77%) as a white powder. M.p. 168–1708C
(from petroleum ether/diethyl ether); [a]2D0 =ꢀ5.5 (c=1 in chloroform);
1H NMR (400 MHz, CDCl3): d=8.02–7.73 (m, 65H; arom. H), 5.88 (d,
4H; GalNI H-3, GalNII H-3, GalNIII H-3, GlcAIV H-2), 4.72 (d, J1,2
=
7.5 Hz, 1H; GlcA H-1), 4.70 (d, J1,2 =8.5 Hz, 1H; GalNIV H-1), 4.58 (d,
1,2 =8.0 Hz, 1H; GlcAIV H-1),4.49–4.43 (m, 2H; GalNI H-6a, GalNIV H-
3), 4.31 (dd, J5,6b =7.0 Hz, 1H; GalNI H-6b), 4.23, 4.16, 4.13 (3dd, J3,4
4,5 =9.5 Hz, 3H; GlcAI H-4, GlcAII H-4, GlcAIII H-4), 4.03 (m, 1H;
J
=
2,NH =9.0 Hz, 1H; GalNIV NH), 5.81 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H;
GalNI H-4), 5.69 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalN H-4), 5.52–5.41
(m, 6H; GalNI NH, GalN NH, GalN H-4, 3GlcA H-3), 5.37 (d, J2,NH
9.0 Hz, 1H; GalN NH), 5.28–5.13 (m, 3H; 2GlcA H-2, NHCOO), 5.10
J
J
GalNI H-5), 4.02, 4.01, 3.93 (3d, 3H; GlcAI H-5, GlcAII H-5, GlcAIII H-
5), 3.92 (d, J4,5 =10.0 Hz, 1H; GlcAIV H-5), 3.81–3.58 (m, 5H; GalNII H-
5, GalNIII H-5, GalNIV H-5, CH2O), 3.69, 3.67, 3.66, 3.61 (4s, 12H;
COOCH3), 3.57–3.06 (m, 12H; GalNII H-6a, H-6b, GalNIII H-6a, H-6b,
GalNIV H-6a, H-6b, 4GalN H-2, CH2N), 2.01, 1.98, 1.97, 1.96, 1.89, 1.78,
1.38, 1.33, 1.29 ppm (9s, 27H; OCOCH3, NHCOCH3); MS: m/z: 1613
=
(d, J1,2 =8.5 Hz, 1H; GalN H-1), 5.10 (dd, 1H; GlcA H-2), 5.02 (dd, J3,4
3.0 Hz, J4,5 <1.0 Hz, 1H; GalNIV H-4), 4.98 (s, 2H; CH2Ph), 4.93 (d, J1,2
=
=
8.0 Hz, 1H; GalNI H-1), 4.91 (d, J1,2 =8.0 Hz, 1H; GalN H-1), 4.77 (dd,
[M+2H]2+
869.3063; found: 869.3078.
;
HRMS: m/z: calcd for C68H108N8O42S [Mꢀ3Na+3H]2ꢀ
:
J
2,3 =11.0 Hz, J3,4 =3.0 Hz, 1H; GalN H-3), 4.92–4.80 (m, 5H; GalNI H-3,
GalNIV H-3, GalN H-3, GlcAI H-1, GlcAII H-1), 4.74 (d, J1,2 =8.0 Hz,
1H; GlcA H-1), 4.51 (d, J1,2 =8.5 Hz, 1H; GalNIV H-1), 4.49–4.45 (m,
2H; GalNI H-6a, GlcA H-4), 4.31 (dd, J6b,6a =11.0 Hz, J5,6b =7.0 Hz, 1H;
GalNI H-6b), 4.21–4.13 (m, 2H; 2GlcA H-4), 4.16 (dd, J4,5 =10.0 Hz, 1H;
GlcA H-5), 4.06–3.90 (m, 2H; GalNI H-5, GalNIV H-2), 4.02, 3.99 (2d,
Disaccharide 66: From disaccharide 48 (80 mg, 25 mmol) as described for
the preparation of 40 to afford the sodium salt 66 (45 mg, 67%) as a
white powder. Rf =0.53 (EtOAc/MeOH/water 4:2:1); [a]2D0 =ꢀ29 (c=1 in
water); 1H NMR (400 MHz, D2O, internal HOD, dH =4.79): d=7.45 (s,
5H; arom. H), 5.14 (s, 2H; CH2Ph), 4.50 (d, J1,2 =8.0 Hz, 1H; GlcA H-
J
4,5 =10.0 Hz, 2H; 2GlcA H-5), 3.81–3.58 (m, 5H; GalNII H-5, GalNIII H-
5, GalNIV H-5, CH2O), 3.68, 3.65, 3.63 (3s, 9H; COOCH3), 3.55–3.39 (m,
4H; GalNII H-6a, H-6b, GalNIII H-6a, H-6b), 3.44–3.20 (m, 7H; GalNI
H-2, GalNII H-2, GalNIII H-2, GalNIV H-6a, H-6b, CH2N), 1.96, 1.93, 1.87
(3s, 9H; OCOCH3), 1.34, 130, 1.25 ppm (3s, 12H; NHCOCH3); elemen-
tal analysis calcd (%) for C153H149N5O56: C 62.21, H 5.08, N 2.37; found:
C 62.01, H 5.18, N 2.11.
1), 4.47 (d, J1,2 =8.5 Hz, 1H; GalN H-1), 4.17 (dd, J3,4 =3.0 Hz, J4,5 <
1.0 Hz, 1H; GalN H-4), 4.01 (dd, J2,3 =11.0 Hz, 1H; GalN H-2), 3.99 (m,
1H; CH2O), 3.91 (dd, 1H; GalN H-3), 3.79–3.68 (m, 2H; GalN H-6a, H-
6b), 3.71 (m, 1H; GalN H-5), 3.70 (d, J4,5 =10.0 Hz, 1H; GlcA H-5), 3.65
(m, 1H; CH2O), 3.53–3.46 (m, 2H; GlcA H-3, H-4), 3.38–3.31 (m, 3H;
GlcA H-2, CH2N), 1.96 ppm (s, 3H; NHCOCH3); MS: m/z: 574
[MꢀNa+H]ꢀ; elemental analysis calcd (%) for C24H33N2NaO12: C 48.32,
H 5.58, N 4.70; found: C 48.13, H 5.59, N 4.38.
Octasaccharide 64: From alcohol 61 (321 mg, 0.11 mmol) and imidate 5
(256 mg, 0.22 mmol) as described for the preparation of 6. Flash-silica
chromatography (toluene/EtOAc 3:2, containing 0.1% of NEt3) afforded
the octasaccharide 64 (260 mg, 65%) as a white powder. M.p. 164–1658C
(from diethyl ether); [a]D20 =ꢀ4.5 (c=1 in chloroform); 1H NMR
Trisaccharide 67: From trisaccharide 51 (37 mg, 20 mmol) as described for
the preparation of 40 to afford the sodium salt 67 (23 mg, 68%) as a
white powder. Rf =0.32 (EtOAc/MeOH/water 4:2:1); [a]2D0 =ꢀ7 (c=1 in
water); 1H NMR (400 MHz, D2O, internal HOD, dH =4.79): d=7.42 (s,
5H; arom. H), 5.12 (s, 2H; CH2Ph), 4.51–4.43 (m, 2H; 2GalN H-1), 4.50
(d, J1,2 =8.0 Hz, 1H; GlcA H-1), 4.11 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H;
(400 MHz, CDCl3): d=8.06–7.12 (m, 43H; arom. H), 6.86 (d, J2,NH
6.5 Hz, 1H; GalNI NH), 6.86–6.77 (m, 2H; arom. H), 6.78 (d, J2,NH
=
=
7.0 Hz, 1H; GalNIV NH), 6.74, 6.71 (2d, J2,NH =7.0 Hz, 2H; GalNII NH,
9576
www.chemeurj.org
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 9561 – 9578

Stereocontrolled Construction of Size-Defined Oligomers
FULL PAPER
GalNI H-4), 4.01 (dd, J1,2 =8.5 Hz, J2,3 =9.5 Hz, 1H; GalNI H-2), 3.95–
3.61 (m, 15H; GalNII H-2, H-4, 2GalN H-3, H-5, H-6a, H-6b, GlcA H-4,
H-5, NHCOO, CH2O), 3.59 (dd, J2,3 =J3,4 =9.5 Hz, 1H; GlcA H-3), 3.38
(dd, 1H; GlcA H-2), 3.35–3.29 (m, 2H; CH2N), 2.06, 1.96 ppm (2s, 6H;
NHCOCH3); HRMS: m/z: calcd for C32H47N3NaO19 [M+H]+: 800.2701;
found: 800.2691.
H-3), 3.51–3.45 (m, 2H; GlcAIV H-3, H-4), 3.41–3.30 (m, 6H; 4GlcA H-
2, CH2N), 2.04, 2.03, 1.96 ppm (3s, 12H; NHCOCH3); HRMS: m/z:
calcd for C66H97N5O47 [Mꢀ4Na+4H]2ꢀ: 854.7599; found: 854.7588.
Biotinylated disaccharide 73: From 48 (19 mg, 30 mmol) as described for
the preparation of 44 to give the intermediate amine. A solution of the
amine and 6-biotinylamidohexanoic acid N-hydroxysuccinimidoyl ester
(58 mg, 130 mmol) in DMF (675 mL), NEt3 (75 mL), and water (750 mL)
was stirred for 1 h at room temperature, then was concentrated. The re-
sulting solid was washed with absolute EtOH, and the residue was eluted
from a column (3ꢅ80 cm) of Sephadex LH-20 with water and lyophilized
to afford the biotinylated disaccharide 73 (17 mg, 67%) as a white
powder. Rf =0.48 (EtOAc/MeOH/water 3:2:1); [a]2D0 =ꢀ0.5 (c=1 in
water); 1H NMR (400 MHz, D2O, internal HOD, dH =4.79): d=4.62
Tetrasaccharide 68: From tetrasaccharide 53 (173 mg, 0.13 mmol) as de-
scribed for the preparation of 40 to afford the sodium salt 68 (78 mg,
62%) as a white powder. Rf =0.64 (EtOAc/MeOH/water 3:2:1); [a]2D0 =+
17 (c=1 in water); 1H NMR (400 MHz, D2O, internal HOD, dH =4.79):
d=7.44 (s, 5H; arom. H), 5.11 (s, 2H; CH2Ph), 4.53 (d, J1,2 =8.5 Hz, 1H;
GalNI H-1), 4.51 (2d, J1,2 =8.0 Hz, 2H; 2GlcA H-1), 4.49 (d, J1,2 =8.5 Hz,
1H; GalNII H-1), 4.17 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNI H-4),
4.11 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNII H-4), 4.02, 3.99 (2dd,
(ddd, Jb,c =8.0 Hz, Jc,d’ =5.0 Hz, Jc,d <1.0 Hz, 1H; H-c), 4.52 (d, J1,2
7.0 Hz, 1H; GalN H-1), 4.51 (d, J1,2 =8.0 Hz, 1H; GlcA H-1), 4.43 (dd,
b,e =5.0 Hz, 1H; H-b), 4.19 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalN H-
=
J
2,3 =11.0 Hz, 2H; 2GalN H-2), 3.92 (m, 1H; CH2O), 3.84–3.68 (m, 11H;
2GalN H-3, H-5, H-6a, H-6b, GlcAI H-4, 2GlcA H-5), 3.60 (dd, J2,3
=
J
8.5 Hz, J3,4 =9.0 Hz, 1H; GlcAI H-3), 3.53–3.46 (m, 2H; GlcAII H-3, H-
4), 3.38 (2dd, 2H; 2GlcA H-2), 3.36–3.30 (m, 2H; CH2N), 2.04, 1.96 ppm
(2s, 6H; NHCOCH3); MS: m/z: 952 [Mꢀ2Na+H]ꢀ; HRMS: m/z: calcd
for C38H55N3NaO25 [MꢀNa+2H]+: 976.3022; found: 976.3004.
4), 4.03 (dd, J2,3 =11.0 Hz, 1H; GalN H-2), 3.93 (m, 1H; CH2-r), 3.84 (dd,
1H; GalN H-3), 3.82–3.67 (m, 6H; GalN H-5, H-6a, H-6b, GlcA H-5,
CH2-r), 3.53–3.46 (m, 2H; GlcA H-3, H-4), 3.40–3.32 (m, 4H; GlcA H-2,
H-e, CH2-q), 3.19 (dd, Jk,k’ =Jk,l =7.0 Hz, 2H; CH2-k), 3.01 (dd, Jd,d’
=
13.0 Hz, 1H; H-d’), 2.80 (dd, 1H; H-d), 2.29–2.23 (m, 4H; CH2-i, CH2-o),
2.03 (s, 3H; NHCOCH3), 1.80–1.30 ppm (m, 12H; CH2-f, CH2-g, CH2-h,
CH2-l, CH2-m, CH2-n); MS: m/z: 778 [MꢀNa]ꢀ; HRMS: m/z: calcd for
C32H53Na N5O15S [M+H]+: 802.3157; found: 802.3149.
Pentasaccharide 69: From pentasaccharide 57 (37 mg, 20 mmol) as de-
scribed for the preparation of 40 to afford the sodium salt 69 (12 mg,
58%) as a white powder. Rf =0.24 (EtOAc/MeOH/water 2:2:1); [a]D20
=
ꢀ9 (c=1 in water); 1H NMR (400 MHz, D2O, internal HOD, dH =4.79):
d=7.41 (s, 5H; arom. H), 5.14 (s, 2H; CH2Ph), 4.54–4.47 (m, 5H; 3GalN
H-1, 2GlcA H-1), 4.13, 4.11 (2dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 2H; GalNI
H-4, GalNII H-4), 4.04–3.98 (m, 2H; GalNI H-2, GalNII H-2), 3.93 (dd,
Biotinylated trisaccharide 74: From 67 (20 mg, 25 mmol) as described for
the preparation of 73 to afford the biotinylated trisaccharide 74 (15 mg,
71%) as a white powder. Rf =0.63 (EtOAc/MeOH/water 1:1:1); [a]D20
+10 (c=1 in water); H NMR (400 MHz, D2O, internal HOD, dH =4.79):
d=4.63 (ddd, Jb,c =8.0 Hz, Jc,d’ =5.0 Hz, Jc,d <1.0 Hz, 1H; H-c), 4.51 (d,
=
3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNIII H-4), 3.92–3.56 (m, 21H; GalNIII
1
J
H-2, 3GalN H-3, H-5, H-6a, H-6b, 2GlcA H-3, H-4, H-5, CH2O), 3.42–
3.31 (m, 4H; 2GlcA H-2, CH2N), 2.06, 2.04, 1.96 ppm (3s, 9H;
NHCOCH3); HRMS: m/z: calcd for C46H68N4O30Na [MꢀNa+2H]+:
J
1,2 =8.0 Hz, 1H; GlcA H-1), 4.50 (d, J1,2 =8.0 Hz, 1H; GalNI H-1), 4.48
(d, J1,2 =8.0 Hz, 1H; GalNII H-1), 4.44 (dd, Jb,e =5.0 Hz, 1H; H-b), 4.13
(dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNI H-4), 4.02 (dd, J2,3 =11.0 Hz,
1H; GalNI H-2), 3.96–3.68 (m, 14H; GalNII H-2, H-4, 2GalN H-3, H-5,
H-6a, H-6b, GlcA H-4, H-5, CH2-r), 3.59 (dd, J2,3 =J3,4 =9.0 Hz, 1H;
1179.3816; found: 1179.3839; calcd for C46H68N4O30 [Mꢀ2Na+2H]2ꢀ
:
577.1181; found: 577.1880.
Octasaccharide 70: From hexasaccharide 59 (41 mg, 20 mmol) as de-
scribed for the preparation of 40 to afford the sodium salt 70 (17 mg,
GlcA H-3), 3.41–3.32 (m, 4H; GlcA H-2, H-e, CH2-q), 3.19 (dd, Jk,k’
=
71%) as a white powder. Rf =0.53 (EtOAc/MeOH/water 2:2:1); [a]D20
=
J
k,l =7.0 Hz, 2H; CH2-k), 3.01 (dd, Jd,d’ =13.0 Hz, 1H; H-d’), 2.80 (dd,
ꢀ21.5 (c=1 in water); 1H NMR (400 MHz, D2O, internal HOD, dH =
4.79): d=7.81–7.30 (m, 5H; arom. H), 5.15 (s, 2H; CH2Ph), 4.52–4.42 (m,
6H; 3GalN H-1, 3GlcA H-1), 4.16 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H;
GalNI H-4), 4.11, 4.10 (2dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 2H; GalNII H-4,
GalNIII H-4), 4.04–3.95 (m, 3H; 3GalN H-2), 3.94–3.54 (m, 22H; 3GalN
H-3, H-5, H-6a, H-6b, GlcAI H-3, H-4, GlcAII H-3, H-4, 3GlcA H-5,
CH2O), 3.50–3.46 (m, 2H; GlcAIII H-3, H-4), 3.39–3.25 (m, 5H; 3GlcA
H-2, CH2N), 2.03, 2.02, 1.95 ppm (3s; NHCOCH3); HRMS: m/z: calcd
for C52H76N4O36 [Mꢀ3Na+3H]2ꢀ: 665.2041; found: 665.2032.
1H; H-d), 2.29–2.23 (m, 4H; CH2-i, CH2-o), 2.06, 2.02 (2s, 6H;
NHCOCH3), 1.80–1.28 ppm (m, 12H; CH2-f, CH2-g, CH2-h, CH2-l, CH2-
m, CH2-n); HRMS: m/z: calcd for C40H66NaN6O20S [M+H]+: 1005.3950;
found: 1005.3953.
Biotinylated tetrasaccharide 75: From 68 (20 mg, 20 mmol) as described
for the preparation of 73 to afford the biotinylated tetrasaccharide 75
(17 mg, 72%) as a white powder. Rf =0.60 (EtOAc/MeOH/water 1:1:1);
[a]2D0 =ꢀ2.5 (c=1 in water); 1H NMR (400 MHz, D2O, internal HOD,
dH =4.79): d=4.63 (ddd, Jb,c =8.0 Hz, Jc,d’ =5.0 Hz, Jc,d <1.0 Hz, 1H; H-c),
4.58–4.48 (m, 4H; 2GalN H-1, 2GlcA H-1), 4.44 (dd, Jb,e =5.0 Hz, 1H;
Heptasaccharide 71: From heptasaccharide 63 (140 mg, 50 mmol) as de-
scribed for the preparation of 40 to afford the sodium salt 71 (71 mg,
H-b), 4.19 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNI H-4), 4.16 (dd, J3,4
3.0 Hz, 1,2 =8.5 Hz, J2,3
4,5 <1.0 Hz, 1H; GalNII H-4), 4.02 (2dd,
=
95%) as a white powder. Rf =0.55 (EtOAc/MeOH/water 1:1:1); [a]D20
=
J
J
=
ꢀ4 (c=1 in water); 1H NMR (400 MHz, D2O, internal HOD, dH =4.79):
d=7.45 (s, 5H; arom. H), 5.13 (s, 2H; CH2Ph), 4.51–4.49 (m, 7H; 4GalN
H-1, 3GlcA H-1), 4.12, 4.11 (2dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 3H; 3GalN
H-4), 4.00, 3.99 (2dd, J1,2 =8.0 Hz, J2,3 =11.0 Hz, 3H; 3GalN H-2), 3.88
(dd, J1,2 =8.0 Hz, J2,3 =11.0 Hz, 1H; GaNIV H-2), 3.94–3.67 (m, 23H;
GalNIV H-4, 4GalN H-3, H-5, H-6a, H-6b, 3GlcA H-4, H-5, CH2O), 3.63
(m, 1H; CH2O), 3.58 (dd, J2,3 =11.0 Hz, J3,4 =3.0 Hz, 3H; 3GlcA H-3),
3.41–3.30 (m, 5H; 3GlcA H-2, CH2N), 2.06, 2.03, 1.96 ppm (3s, 12H;
NHCOCH3); HRMS: m/z: calcd for C153H148N5NaO56 [Mꢀ2Na+3H]+:
1558.4931; found: 1558.4933.
11.0 Hz, 2H; 2GalN H-2), 3.93 (m, 1H; CH2-r), 3.85–3.67 (m, 12H;
2GalN H-3, H-5, H-6a, H-6b, GlcAI H-4, 2GlcA H-5, CH2-r), 3.59 (dd,
J
2,3 =J3,4 =9.0 Hz, 1H; GlcAI H-3), 3.52–3.47 (m, 2H; GlcAII H-3, H-4),
3.41–3.31 (m, 5H; 2GlcA H-2, H-e, CH2-q), 3.19 (dd, 1H; Jk,k’ =Jk,l
=
7.0 Hz, 2H; CH2-k), 3.01 (dd, Jd,d’ =13.0 Hz, 1H; H-d’), 2.80 (dd, 1H; H-
d), 2.29–2.23 (m, 4H; CH2-i, CH2-o), 2.04, 2.02 (2s, 6H; NHCOCH3),
1.81–1.32 ppm (m, 12H; CH2-f, CH2-g, CH2-h, CH2-l, CH2-m, CH2-n);
HRMS: m/z: calcd for C46H74NaN6O26S [MꢀNa+2H]+: 1181.4271;
found: 1181.4279; calcd for C46H74N6O26S [Mꢀ2Na+2H]2ꢀ: 578.2108;
found: 578.2097.
Biotinylated pentasaccharide 76: From 69 (15 mg, 10 mmol) as described
for the preparation of 73 to afford the biotinylated pentasaccharide 76
(10 mg, 75%) as a white powder. Rf =0.78 (EtOAc/MeOH/water 1:1:1);
[a]2D0 =ꢀ2.5 (c=1 in water); 1H NMR (400 MHz, D2O, internal HOD,
dH =4.79): d=4.62 (ddd, Jb,c =8.0 Hz, Jc,d’ =5.0 Hz, Jc,d <1.0 Hz, 1H; H-c),
4.53–4.51 (m, 5H; 3GalN H-1, 2GlcA H-1), 4.43 (dd, Jb,e =5.0 Hz, 1H;
H-b), 4.13, 4.11 (2dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 2H; GalNI H-4, GalNII
H-4), 4.04–3.97 (m, 2H; GalNI H-2, GalNII H-2), 3.95–3.68 (m, 20H; Gal-
NIII H-2, H-4, 3GalN H-3, H-5, H-6a, H-6b, 2GlcA H-4, H-5, CH2-r),
3.59, 3.58 (2dd, J2,3 =J3,4 =9.5 Hz, 2H; 2GlcA H-3), 3.41–3.32 (m, 5H;
Octasaccharide 72: From octasaccharide 65 (80 mg, 25 mmol) as described
for the preparation of 40 to afford the sodium salt 72 (37 mg, 84%) as a
white powder. Rf =0.38 (EtOAc/MeOH/water 1:2:2); [a]2D0 =ꢀ24.5 (c=1
1
in water); H NMR (400 MHz, D2O, internal HOD, dH =4.79): d=7.45 (s,
5H; arom. H), 5.13 (s, 2H; CH2Ph), 4.52–4.45 (m, 8H; 4GalN H-1,
4GlcA H-1), 4.18 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNI H-4),4.12,
4.11 (2dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 3H; 3GalN H-4), 4.04–3.97 (m, 4H;
4GalN H-2), 3.92 (m, 1H; CH2O), 3.83–3.55 (m, 24H; 4GalN H-3, H-5,
H-6a, H-6b, GlcAI H-4, GlcAII H-4, GlcAIII H-4, 4GlcA H-5, CH2O),
3.48 (dd, J2,3 =9.0 Hz, J3,4 =10.0 Hz, 3H; GlcAI H-3, GlcAII H-3, GlcAIII
Chem. Eur. J. 2009, 15, 9561 – 9578
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9577

J.-C. Jacquinet et al.
2GlcA H-2, H-e, CH2-q), 3.19 (dd, Jk,k’ =Jk,l =7.0 Hz, 1H; CH2-k), 3.01
(dd, Jd,d’ =13.0 Hz, 1H; H-d’), 2.80 (dd, 1H; H-d), 2.28–2.21 (m, 4H;
CH2-i, CH2-o), 2.02, 2.02, 2.01 (3s, 9H; NHCOCH3), 1.78–1.22 ppm (m,
12H; CH2-f, CH2-g, CH2-h, CH2-l, CH2-m, CH2-n); HRMS: m/z: calcd
for C54H87N7O31NaS [MꢀNa+2H]+: 1384.5065; found: 1384.5046; calcd
for C54H87N7O31S [Mꢀ2Na+2H]2ꢀ: 679.7505; found: 679.7482.
C-4, GlcAIII C-4), 77.06, 77.04, 76.77, 75.96, 75.63, 75.54, 74.39, 74.35,
73.38, 73.12, 72.46 (17C; 4GalN C-5, GlcAIV C-4, 4GlcA C-2, C-3, C-5),
68.77 (1C; C-r), 68.41, 68.35 (4C; 4GalN C-4), 62.76 (1C; C-b), 61.74
(4C; 4GalN C-6), 60.91 (1C; C-c), 56.06 (1C; C-e), 51.78, 51.69, 51.65
(4C; 4GalN C-2), 40.37 (1C; C-d), 39.93, 39.74 (2C; C-k, C-q), 36.30,
36.19 (2C; C-o, C-i), 28.69, 28.51, 28.35, 26.25, 25.88, 25.66 (6C; C-f, C-g,
C-h, C-l, C-m, C-n), 23.17, 22.94 ppm (4C; NHCOCH3); HRMS: m/z:
calcd for C74H116N8O48S [Mꢀ4Na+4H]2ꢀ: 957.3223; found: 957.3259.
Biotinylated hexasaccharide 77: From 70 (20 mg, 20 mmol) as described
for the preparation of 73 to afford the biotinylated hexasaccharide 77
(17 mg, 72%) as a white powder. Rf =0.88 (EtOAc/MeOH/water1:1:1);
[a]2D0 =ꢀ8.5 (c=1 in water); 1H NMR (400 MHz, D2O, internal HOD,
dH =4.79): d=4.60 (ddd, Jb,c =8.0 Hz, Jc,d’ =5.0 Hz, Jc,d <1.0 Hz, 1H; H-c),
4.53–4.48 (m, 6H; 3GalN H-1, 3GlcA H-1), 4.43 (dd, Jb,e =5.0 Hz, 1H;
H-b), 4.16 (dd, J3,4 =3.0 Hz, J4,5 <11.0 Hz, 1H; GalNI H-4), 4.12, 4.11
(2dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 2H; GalNII H-4, GalNIII H-4), 4.04–3.96
(m, 3H; 3GalN H-2), 3.95–3.67 (20H; 3GalN H-3, H-5, H-6a, H-6b,
Acknowledgements
This research was supported by the Programme ANR-05-BLAN-0198–02
“GT-GAG.” We are grateful for support by the Ministꢃre de l’Enseigne-
ment Supꢀrieur et de le Recherche through a studentship to A.V.
GlcAI H-4, GlcAII H-4, 3GlcA H-5, CH2-r), 3.59 (dd, J3,4 =9.0 Hz, J2,3
=
8.0 Hz, 2H; GlcAI H-3, GlcAII H-3), 3.53–3.45 (m, 2H; GlcAIII H-3, H-
4), 3.39–3.26 (6H; 3GlcA H-2, H-e, CH2-q), 3.18 (dd, Jk,k’ =Jk,l =7.0 Hz,
1H; CH2-k), 3.00 (dd, Jd,d’ =13.0 Hz, 1H; H-d’), 2.79 (dd, 1H; H-d), 2.28–
2.21 (m, 4H; CH2-i, CH2-o), 2.02, 2.02, 2.01 (3s, 9H; NHCOCH3), 1.78–
1.22 ppm (m, 12H; CH2-f, CH2-g, CH2-h, CH2-l, CH2-m, CH2-n); HRMS:
m/z: calcd for C60H94N7O37S [Mꢀ3Na+2H]ꢀ: 1536.5410; found:
1536.5398.
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527.
Biotinylated heptasaccharide 78: From 71 (30 mg, 20 mmol) as described
for the preparation of 73 to afford the biotinylated heptasaccharide 78
(29 mg, 85%) as a white powder. Rf =0.78 (EtOAc/MeOH/water1:1:1);
[a]2D0 =ꢀ12.5 (c=1 in water); 1H NMR (400 MHz, D2O, internal HOD,
dH =4.79): d=4.62 (ddd, Jb,c =8.0 Hz, Jc,d’ =5.0 Hz, Jc,d <1.0 Hz, 1H; H-c),
4.54–4.45 (m, 7H; 4GalN H-1, 3GlcA H-1), 4.43 (dd, Jb,e =5.0 Hz, 1H;
H-b), 4.12, 4.11 (2dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 3H; 3GalN H-4), 4.04–
3.97 (m, 3H; 3GalN H-2), 3.95–3.88 (m, 2H; GalNIV H-2, CH2-r), 3.95–
3.63 (m, 24H; GalNIV H-4, 4GalN H-3, H-5, H-6a, H-6b, 3GlcA H-4, H-
5, CH2-r), 3.59, 3.58 (2dd, J2,3 =J3,4 =9.5 Hz, 3H; 3GlcA H-3), 3.41–3.31
(m, 6H; 3GlcA H-2, H-e, CH2-q), 3.19 (dd, Jk,k’ =Jk,l =7.0 Hz, 1H; CH2-
k), 3.01 (dd, Jd,d’ =13.0 Hz, 1H; H-d’), 2.80 (dd, 1H; H-d), 2.28–2.21 (m,
4H; CH2-i, CH2-o), 2.06, 2.03, 2.02 (3s, 12H; NHCOCH3), 1.78–1.28 ppm
(m, 12H; CH2-f, CH2-g, CH2-h, CH2-l, CH2-m, CH2-n); HRMS: m/z:
calcd for C68H108N8O42S [Mꢀ3Na+3H]2ꢀ: 869.3063; found: 869.3078.
Biotinylated octasaccharide 79: From 72 (36 mg, 20 mmol) as described
for the preparation of 73 to afford the biotinylated octasaccharide 79
(33 mg, 89%) as a white powder. Rf =0.71 (EtOAc/MeOH/water 1:1:1);
[a]2D0 =ꢀ13 (c=1 in water); 1H NMR (400 MHz, D2O, internal HOD,
dH =4.79): d=4.62 (ddd, Jb,c =8.0 Hz, Jc,d’ =5.0 Hz, Jc,d’ <1.0 Hz, 1H; H-
c), 4.54–4.45 (m, 8H; 4GalN H-1, 4GlcA H-1), 4.43 (dd, Jb,e =5.0 Hz, 1H;
H-b), 4.17 (dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 1H; GalNI H-4), 4.12, 4.11
(2dd, J3,4 =3.0 Hz, J4,5 <1.0 Hz, 3H; GalNII H-4, GalNIII H-4, GalNIV H-
4), 4.03–3.94 (m, 4H; 4GalN H-2), 3.91 (m, 1H; CH2-r), 3.85–3.62 (m,
24H; 4GalN H-3, H-5, H-6a, H-6b, 3GlcA H-4, 4GlcA H-5, CH2-r),
3.61–3.55 (m, 3H; GlcAI H-3, GlcAII H-3, GlcAIII H-3), 3.52–3.46 (m,
2H; GlcAIV H-3, H-4), 3.40–3.29 (m, 7H; 4GlcA H-2, H-e, CH2-q), 3.18
(dd, Jk,k’ =Jk,l =7.0 Hz, 1H; CH2-k), 2.99 (dd, Jd,d’ =13.0 Hz, 1H; H-dꢇ),
2.79 (dd, 1H; H-d), 2.28–2.21 (m, 4H; CH2-i, CH2-o), 2.02, 2.01, 2.00 (3s,
12H; NHCOCH3), 1.78–1.28 ppm (m, 12H; CH2-f, CH2-g, CH2-h, CH2-l,
CH2-m, CH2-n); 13C NMR (100 MHz, D2O, internal acetone, dC =30.83):
d=177.60, 177.27, 175.63, 175.35, 175.27, 175.25 (10C; 4GlcA C-6, C=O),
165.99 (1C; C-a), 104.95, 104.77 (4C; 4GlcA C-1), 102.00, 101.47 (4C;
4GalN C-1), 80.96, 80.85, 80.40, 80.35 (7C; 4GalN C-3, GlcAI C-4, GlcAII
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Received: March 23, 2009
Published online: July 2, 2009
9578
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Chem. Eur. J. 2009, 15, 9561 – 9578