Synthesis of complex oligosaccharides by using a mutated (1,3)-β-D-glucan endohydrolase from Barley

Article abstract of DOI:10.1002/chem.200304733

Complex oligosaccharides with newly formed (1,3)-β-glycosidic linkages were obtained in good to excellent yields when substituted or unsubstituted α-laminaribiosyl fluorides, acting as donors, were condensed onto mono- and disaccharide β-D-hexopyranoside

Full text of DOI:10.1002/chem.200304733

FULL PAPER
Synthesis of Complex Oligosaccharides by Using a Mutated (1,3)-b-d-Glucan
Endohydrolase from Barley
Jon K. Fairweather,[a] Maria Hrmova,[b] Simon J. Rutten,[b] Geoffrey B. Fincher,[b] and
Hugues Driguez*[a]
Abstract: Complex
oligosaccharides
branched (1,3)-b-linked oligosacchar-
ides could prove to be important in a
range of medical, pharmaceutical, and
agricultural applications. Furthermore,
the observation that the (1,3)-b-d-glucan
glycosynthase accommodates (1,3)-,
(1,4), - and (1,6)-b-oligosaccharides in
its acceptor subsites suggests novel, yet
unexpected physiological roles for the
wild type (1,3)-b-d-glucan endohydro-
lase from higher plants.
with newly formed (1,3)-b-glycosidic
linkages were obtained in good to ex-
cellent yields when substituted or un-
substituted a-laminaribiosyl fluorides,
acting as donors, were condensed onto
mono- and disaccharide b-d-hexo-
pyranoside acceptors by using a (1,3)-b-
d-glycosynthase. These linear and
Keywords:
carbohydrates
¥
enzymatic synthesis ¥ glycosides ¥
glycosynthase ¥ oligosaccharides
glycosynthase-like conditions.[15] In this study, we report on
the versatility of this (1,3)-b-d-glycosynthase to condense a-
laminaribiosyl fluoride and its derivatives with a variety of b-
d-hexopyranoside acceptors.
Introduction
(1,3)-b-d-Glucans and related polysaccharides have been
implicated in immunomodulating and antitumour activi-
ties.[1, 2] They also exhibit antibacterial and antiviral proper-
ties, stimulate blood coagulation, and accelerate the healing of
wounds.[3 In addition, (1,3)- and (1,3;1,6)-b-d-oligogluco-
6]
Results and Discussion
sides elicit defense processes against microbial attack in
invertebrates and plants.[7, 8] Future medical, pharmaceutical,
and agricultural applications of these compounds will rely on
rapid and specific synthetic procedures, that can be readily
adapted to large-scale production. One approach to the rapid
production of specific oligo- and polysaccharides is through
™glycosynthase∫ technology.
Glycosynthases are generated from retaining glycoside
hydrolases, in which catalytic nucleophiles are replaced with
non-carboxylic amino acid residues. These mutated enzymes,
which catalyze synthetic reactions without hydrolysis, are now
opening new avenues for stereoselective assembly of oligo-
saccharides.[9 14] We have recently produced a (1,3)-b-d-glucan
endohydrolase (Glu231Gly) from barley and demonstrated
that the enzyme polymerizes a-laminaribiosyl fluoride under
Table 1 (entry 1) shows the condensation of a-laminaribiosyl
fluoride (1)[10] and p-nitrophenyl-d-glucoside (2). The identity
of the newly established glycosidic linkage was confirmed by
13C NMR spectroscopy with
a characteristic signal at
ꢀ85 ppm that is representative of C-3 substituted b-d-
glucosyl residues.[16] However, when equimolar concentra-
tions of donor and acceptor were used, only the polymeric
(1,3)-b-d-glucan product arising from the self-condensation of
1 was obtained.[15] The yield of the polymer could be
minimized when the acceptor was used in a high concen-
tration and at a donor/acceptor ratio of 1:5. Under these
conditions, laminaritrioside 3 was obtained in excellent yield
(90%). Reaction of the b-d-xyloside 4 and b-d-galactoside 5
gave equally good yields for the respective trisaccharide
products 6 (82%) and 7 (89%) (entries 2 and 3). Under these
conditions, lack of glycosylation of either p-nitrophenyl b-d-
mannoside or 2-acetylamino-2-deoxy-b-d-glucoside suggest-
ed that both orientation and nature of the substituent at C-2 is
critical for the correct location of the acceptor in subsite ‡1.
In these last cases, only a polymeric (1,3)-b-d-glucan product
was produced.
[a] Dr. H. Driguez, Dr. J. K. Fairweather
¬
Centre de Recherches sur les Macromolecules
¬
¬
Vegetales (CERMAV-CNRS) affiliated with
Universite J. Fourier, B.P. 53
¬
Grenoble Cedex 9, 38041 (France)
Fax : (‡33)476-547-203
E-mail: Hugues.Driguez@cermav.cnrs.fr
[b] Dr. M. Hrmova, S. J. Rutten, Prof. G. B. Fincher
Department of Plant Science, University of Adelaide
Waite Campus, Glen Osmond, 5064 (Australia)
Subsequently, a series of disaccharides were used as
acceptors for the condensation reactions. Entry 4 reveals that
Chem. Eur. J. 2003, 9, 2603 2610
DOI: 10.1002/chem.200304733
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2603

H. Driguez et al.
FULL PAPER
Table 1. Enzymatic syntheses of (1,3)-b-glycosidic bonds by condensation of a-laminaribiosyl fluoride 1, and its analogues 15, 24, and 31, with mono- and
disaccharide acceptors 2, 4, 5, 8, 10, 12, and 14, using (1,3)-b-d-glucanase Glu231Gly.
Entry Donor
Acceptor
Isolated product
Yield
90
1
2
3
1
1
82
89
4
1
70
55
5
6
1
1
R ˆ Me: 45
R ˆ C6H4NO2: 0
7
8
2
81
90
15
14
9
2
2
65
33
10
gentiobioside 8 also serves as an acceptor in the glycosynthase
reaction, and that the branched trisaccharide 9 is obtained as
the sole product (70% yield). It is noteworthy that, despite
having two available C(O)3 acceptor sites for glycosylation
(both glucosyl units of 8 are in the b-d-configuration), only the
residue harboring the aromatic aglycon is glycosylated. This
result, similarly observed with a mutated b-d-mannosidase,[17]
draws attention to the fact that aromatic groups play an
important role in positioning the acceptor molecules in the
active sites of glycosynthases. p-Nitrophenyl b-maltoside was
not an acceptor, but p-nitrophenyl b-cellobioside (10) could
be glycosylated to yield the tetrasaccharide 11 in moderate
yield (55%, entry 5). It is somewhat surprising that the (1,4)-
b-linked disaccharide acceptor can be correctly positioned in
2604
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 2603 2610

Synthesis of Complex Oligosaccharides
2603 2610
the enzyme binding site, which is normally highly specific for
(1,3)-b-d-glucosyl residues.[15] With the relatively short p-
nitrophenyl b-cellobioside acceptor molecule, the glucosyl
unit in the ‡2 subsite has to be upside down relative to the
normal substrate.
required to drive the synthetic reaction, producing the
tetrasaccharide 23 in near quantitative yield (entry 8).
To further investigate the versatility of the barley (1,3)-b-d-
glycosynthase, a-d-glucopyranosyl, a-lactosyl, and a-cello-
biosyl fluorides were tried as glycosyl donors. The failure of
these glycosides to condense onto glucoside 2 is in accordance
with the tight substrate specificity of the wild-type enzyme.
Therefore, the (1,3)-b-d-glycosynthase requires at least a
(1,3)-b-d-linked disaccharide motif in its glycosyl donor
position (subsites À1 and À2).[23] Having observed a toler-
ance of the barley (1,3)-b-d-glycosynthase for a substituted
C-3II OH group on the donor molecule, we investigated the
possibility that a donor with an axial OH group at C-4II would
be viable in the synthetic reaction. The synthesis of the C-4II
modified disaccharide 24 is outlined in Scheme 1. Coupling of
imidate 25[24] with the methyl glycoside (26)[25] gave disac-
charide 27 (85% yield), and subsequently, octaacetate 28 and
fluoride 29 were prepared prior to deacetylation. Condensa-
tion of the donor 24 with the b-d-glucoside 2 and subsequent
acetylation of the reaction products gave rise to trisaccharide
30 in 65% yield (Table 1 entry 9). As observed for the
cellooligosaccharide series,[26] the (1,3)-b-d-oligosaccharide
with a terminal b-d-galactosyl residue could prove to be a
useful compound for oligosaccharide modification by the
galactose oxidase reaction.
The elongation of derivatives of laminaribiose was also
achieved; methyl b-laminaribioside (12)[18] gave the tetrasac-
charide 13 in moderate yield (45%), but the p-nitrophenyl
derivative 14[19] yielded neither the expected product nor a
(1,3)-b-d-glucan polymer (entry 6). It is tempting to speculate
that this is a consequence of competition between the two
disaccharides (1 and 14) for the glycosyl donor position in the
active site. Decreasing the donor/acceptor ratio to 1:1 had no
effect, and when the ratio was altered in favor of the donor 1
(5:1), the polymeric (1,3)-b-d-glucan product was synthesized.
To prevent self-condensation of the donor during equimo-
lar coupling with acceptors, it was necessary to epimerize,
protect, or substitute the 3II-OH of fluoride 1, as was observed
in a (1,4)-b-d-glycosynthase.[11, 12, 20] The 3II-azido fluoride 15
was therefore synthesized as outlined in Scheme 1. The
trichloroacetimidate 16 was prepared in a two-step procedure
from tetra-O-acetyl-3-azido-3-deoxy-d-glucose.[21] Glycosyla-
tion of 16 with methyl glucoside 17[22] yielded the protected
disaccharide 18 in 65% yield. After additional protective
group manipulation, giving firstly the methyl glycoside (19)
and then the heptaacetate 20, the final fluoride (21) was
prepared by treatment with HF in pyridine. Conventional
deacetylation produced the 3II-azido fluoride (15), which was
ready for use as the glycosyl donor in the glycosynthase
reaction. Thus, equimolar condensation of the 3II-azido
fluoride 15 with the b-d-glucoside 2 resulted in the formation
of the trisaccharide 22 in excellent yield (81%) (Table 1
entry 7). This result opens the route to an efficient synthesis of
bifunctionalized (1,3)-b-d-oligosaccharides for the sensitive
assay of (1,3)-b-d-glucan endohydrolases by fluorescence
quenching, as already reported cellulases.[20] However, a
similar ratio could not be employed for the synthesis involving
the disaccharide acceptor 14, because of its inherent inhib-
itory activity. In this case, a fivefold excess of donor was
Finally, we investigated the versatility of the trisaccharide
fluoride 31, which arises from the final hydrolysis product of
cellulase-mediated hydrolysis of (1,3;1,4)-b-d-glucans.[27] The
trisacchaaride was modified by acetylation, fluorination, and
trans-esterification. Entry 10 reveals that while the synthesis
of tetrasaccharide 32 was achieved, its low yield (33%)
suggested that the cellobiosyl unit was not well positioned in
the donor subsites À3 and À2.
In summary, the barley (1,3)-b-d-glycosynthase was used
for the specific synthesis of complex oligosaccharides, includ-
ing (1,3)-b-d-oligoglucosides, C-6 substituted (1,3)-b-d-oligo-
glucosides, (1,3)-b-d-glucoxylosides, (1,3)-b-d-galactogluco-
sides, and linear (1,3;1,4)-b-d-oligoglucosides. While the
(1,3)-b-d-glycosynthase activity depends on forming produc-
OAc
O
AcO
N3
AcO
O
CCl3
NH
OAc
OAc
Ph
O
AcO
O
16
O
a
O
e
O
AcO
N3
b
AcO
O
AcO
R2
N3
15
24
O
O
AcO
AcO
BzO
AcO
OMe
R1
Ph
19 R1 = OMe, R2 = H
20 R1 = R2 = H.OAc
21 R1 = F, R2 = H
O
18
O
c
O
HO
d
RO
OMe
17 R = Bz
26 R = Bn
AcO
AcO
AcO
AcOPh
OAc
O
OAc
O
O
a
e
f
O
O
O
AcO
O
R2
O
AcO
AcO
BnO
AcO
AcO
R1
OMe
AcO
OAc
O
27
28 R1 = R2 = H.OAc
29 R1 = F, R2 = H
AcO
d
AcO
O
CCl3
NH
25
Scheme 1. Syntheses of modified laminaribiosyl fluorides 15 and 24. a) TMSOTf, CH2Cl2; b) 1) NaOMe, MeOH, 2) 60% AcOH, 1008C, 3) Ac2O, pyridine,
N,N-dimethylaminopyridine; c) H2SO4, AcOH, Ac2O (1: 50: 100 v/v; d) HF/pyridine; e) NaOMe, MeOH; f) (1) H2, Pd/C, MeOH, EtOAc, AcOH, 2) H2SO4,
AcOH, Ac2O (1: 25: 50 v/v).
Chem. Eur. J. 2003, 9, 2603 2610
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2605

H. Driguez et al.
FULL PAPER
and 4-nitrophenyl b-d-xyloside (4) yielded the trisaccharide 6 as an
amorphous powder (82%); 1H NMR (D2O): d ˆ 8.24 8.18, 7.24 7.18 (2m,
4H; ArH), 5.20 (d, J(1,2) ˆ 6.9 Hz, 1H; H-1I), 4.79, 4.69 (2d, J(1,2) ˆ
J(1,2) ˆ 8.0 Hz, 2H; H-1II,III), 4.07 4.01, 3.90 3.62, 3.56 3.24 ppm (3m,
18H; H-2I III, H-3I III, H-4I III, H-5I III, H-6I III); 13C NMR (D2O): d ˆ 163.14,
144.31 (2C; ArC), 127.71, 118.13 (4C; ArCH), 104.41, 103.99, 101.43 (C-
1I III), 85.92, 84.86 (C-3I,II), 77.61, 77.22, 77.18, 75.06, 74.80, 73.88, 71.18,
69.78, 69.16 (C-2I III, C-3III, C-4I III, C-5II,III), 66.54 (C-5I), 61.30 ppm (3C;
tive complexes at subsites À2 and À1 with a-laminaribiosyl
fluorides, it displays a greater tolerance for unusual acceptor
molecules at subsites ‡1, ‡2, and beyond. The range and
complexity of oligo- and polysaccharides that are synthesized
by individual glycosynthases could be extended considerably
by the sequential use of the various glycosynthases that are
now available.[28, 29]
‡
C-6I III); MS-FAB: m/z: 618 [M ‡Na].
A
sample of the trisaccharide was treated with Ac2O/pyridine/N,N-
dimethylaminopyridine (12 h) and then subjected to workup (CHCl3) and
Experimental Section
flash chromatography (60 70% EtOAc/petrol ether) to yield p-nitro-
phenyl
(2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl)-(1 ! 3)-(2,4,6-tri-O-
acetyl-b-d-glucopyranosyl)-(1 ! 3)-2,4-di-O-acetyl-b-d-xylopyranoside as
a colorless oil; [a]D ˆ À23.08 (c ˆ 0.4 in CHCl3); 1H NMR (CDCl3) d ˆ
8.22 8.17, 7.18 7.12 (2m, 4H, ArH), 5.45 5.43 (brd, J(1,2) ˆ 2.6 Hz, 1H;
H-1I), 5.13 (t, J(2,3) ˆ J(3,4) ˆ 9.3 Hz, 1H; H-3III), 5.05 (dd, J(1,2) ˆ 8.0,
J(2,3) ˆ 9.7 Hz, 1H; H-2II), 5.04 (t, J(3,4) ˆ J(4,5) ˆ 9.3 Hz, 1H; H-4III),
4.99 4.91 (m, 2H, H-2I,H-4I), 4.88 (dd, J(1,2) ˆ 8.2 Hz, J(2,3) ˆ 9.3 Hz,
1H; H-2III), 4.57, 4.56 (2d, J(1,2) ˆ J(1,2) ˆ 8.0 Hz, 2H; H-1II,III), 4.36 (dd,
J(5,6) ˆ 4.4 Hz, J(6,6) ˆ 12.4 Hz, 1H; H-6II), 4.22 (dd, J(5,6) ˆ 4.9, J(6,6) ˆ
12.4 Hz, 1H; H-6III), 4.17 4.10 (m, 2H; H-5I,H-6II), 4.03 (dd, J(5,6) ˆ
2.2 Hz, J(6,6) ˆ 12.4 Hz, 1H; H-6III), 3.95 3.91 (m, 1H; H-3I), 3.87 (t,
J(2,3) ˆ J(3,4) ˆ 9.5 Hz, 1H; H-3II), 3.73 3.60 (m, 3H; H-5I III), 2.14
Melting points were measured by using a Buchi 535 melting point
apparatus and remain uncorrected. Optical rotations were measured by
means of a Perkin Elmer 341 polarimeter (volume of microcell 1 mL,
10 cm path length) at room temperature. 1H and 13C NMR spectra were
recorded by using a Bruker AC-300 (300 MHz for 1H and 75.5 MHz for 13C)
or a Varian Unity 500 spectrometer (500 MHz for 1H and 125.8 MHz for
13C) either in deuterium oxide (D2O), employing residual H2O (1H, d ˆ
4.78) as internal standard, or in deuteriochloroform (CDCl3) with residual
CHCl3 (1H, d7.26) and CDCl3 (13C, d ˆ 77.0) being employed as internal
standards, at ambient temperatures (298 K) unless specified otherwise.
Where appropriate, analysis of NMR spectra was aided by COSY
(correlated spectroscopy) experiments. High resolution mass spectra
(HRMS) were recorded by using VG ZAB and low resolution MS with a
Nermag R-1010C spectrometer by using the fast atom bombardment
(FAB) technique and employing NaCl as the matrix. The m/z data for the
1.97 ppm (9 s, 27H; C OCH3); 13C NMR (CDCl3): d ˆ 170.68, 170.46,
ˆ
ˆ
170.33, 169.67, 169.35, 169.27, 169.14, 168.69 (9 C; C O), 160.82, 142.87 (2
C; ArC), 125.77, 116.56 (4 C; ArCH), 101.99, 100.99, 99.61 (C-1I III), 78.74
(C-3II), 74.16, 72.93, 72.54, 72.12, 71.78, 71.78, 71.11, 68.97, 68.20, 68.12, 67.72
(C-2I III, C-3I,III, C-4I III, C-5II,III), 62.02, 61.75 (C-6II,III), 59.59 (C-5I), 20.87,
‡
peaks correspond to [M ‡Na]. Microanalyses were performed by the
ˆ
20.83, 20.67, 20.62, 20.51, 20.46, 20.39 ppm (9 C; C OCH3); HRMS-FAB:
™Laboratoire Central d×analyses du CNRS∫ (Vernaisson, France). All
syntheses that involved anhydrous solvents were performed under argon.
Flash chromatography was performed on Merck (Darmstadt, Germany)
silica gel (40 63 mm) under a positive pressure with the specified eluents.
Reversed-phase chromatography was performed on C18 Sep-Pak cartridg-
es (Waters, USA) under a positive pressure by using 5 20% (v/v) MeOH/
H2O as eluent. Progress of the reactions were monitored by thin-layer
chromatography (TLC) with commercially prepared Merck silica gel 60
F254 aluminium plates. Compounds detected on TLC plates were visualized
by charring with 5% (v/v) sulfuric acid in methanol and/or under
ultraviolet light. The term ™work-up∫ refers to dilution with water,
extraction into an organic solvent, sequential washing of the organic
extract with aqueous hydrochloric acid (1m, where appropriate), saturated
aqueous sodium bicarbonate and brine, followed by drying over anhydrous
magnesium sulfate, filtration, evaporation of the solvent by means of a
rotary evaporator at reduced pressure, and drying of the residue at 1 mm
Hg. p-Nitrophenyl glycosides of b-d-glucose, b-d-xylose, b-d-galactose, b-
d-mannose, 2-acetylamino-2-deoxy-b-d-glucose, b-cellobiose, and b-gen-
tiobiose were obtained from Sigma Chemical Co. (St. Louis, USA).
m/z calcd for C41H51NO26Na: 996.2597; found: 996.2597.
p-Nitrophenyl (b-d-glucopyranosyl)-(1! 3)-(b-d-glucopyranosyl)-(1! 3)-
b-d-galactopyranoside (7): Condensation of a-laminaribiosyl fluoride (1)
and p-nitrophenyl b-d-galactoside (5) yielded the trisaccharide (7) as an
amorphous powder (89%); 1H NMR (D2O) d ˆ 8.26 8.18, 7.20 7.18 (2m,
4H; ArH), 5.20 (d, J(1,2) ˆ 7.3 Hz, 1H; H-1I), 4.70 (2d, J(1,2) ˆ J(1,2) ˆ
8.0 Hz, 1H; H-1II,III), 4.22 (d, J(3,4) ˆ 3.0 Hz, J(4,5) ˆ 0.8 Hz, 1H; H-4I),
4.00 3.82, 3.75 3.62, 3.57 3.25 ppm (3m, 17H; H-2I III, H-3I III, H-4II,III
,
H-5I III, H-6I III); 13C NMR (D2O): d ˆ 162.07, 142.96 (2C; ArC), 126.43,
116.83 (4C; ArCH), 103.84, 103.13, 100.06 (C-1I III), 85.58, 82.87 (C-3I,II),
III
76.34, 75.89, 75.78, 75.67, 73.79, 73.42, 69.91, 69.83, 68.41, 68.27 (C-2I
,
C-3III, >C-4I III, C-5I III), 61.04, 60.96, 60.84 ppm (C-6I III); MS-FAB: m/z:
‡
648 [M ‡Na].
A
sample of the trisaccharide was treated with Ac2O/pyridine/N,N-
dimethylaminopyridine (12 h) and then subjected to workup (CHCl3) and
flash chromatography (65 75% EtOAc/petrol ether) to yield p-nitro-
phenyl
(2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl)-(1 ! 3)-(2,4,6-tri-O-
acetyl-b-d-glucopyranosyl)-(1 ! 3)-2,4,6-tri-O-acetyl-b-d-galactopyrano-
1
General procedure for enzymatic synthesis catalyzed by the glycosynthase:
The mutated (1,3)-b-d-glucan endohydrolase Glu231Gly (final concentra-
tion of 0.1 mgmLÀ1) was added to a solution of the fluoride donor
(1.0 equivof 10m m), and the acceptor (5.0 equivof 10m m) was dissolved in
the phosphate buffer (0.25m, pH 7.0). The combined mixture was incubated
at 378C for 12 24 h. The reaction products were separated directly from
the reaction mixture by reversed-phase chromatography. Evaporation and/
or lyophilisation of the solvents first gave the unreacted acceptor
>(4.0 equiv), and subsequently the product of the transglycosylation
reaction.
side as a colorless oil; [a]D À11.98 (c ˆ 0.8 in CHCl3); H NMR (CDCl3):
d ˆ 8.21 8.14, 7.06 7.01 (2m, 4H; ArH), 5.42 (dd, J(1,2) ˆ 8.0 Hz, J(2,3) ˆ
10.0 Hz, 1H; H-2III), 5.42 5.40 (m, 1H; H-4I), 5.11 (t, J(2,3) ˆ J(3,4) ˆ
9.3 Hz, 1H; H-3III), 5.03 (t, J(3,4) ˆ J(4,5) ˆ 9.5 Hz, 1H; H-4III), 5.03 (d,
J(1,2) ˆ 7.9 Hz, 1H; H-1I), 4.96 4.83 (m, 3H; H-2I,II, H-4II), 4.53, 4.45 (2d,
J(1,2) ˆ J(1,2) ˆ 8.0 Hz, 2H; H-1II,III), 4.39 (dd, J(5,6) ˆ 2.9 Hz, J(6,6) ˆ
12.0 Hz, 1H; H-6I), 4.35 (dd, J(5,6) ˆ 4.0 Hz, J(6,6) ˆ 12.2 Hz, 1H; H-6II),
4.14 3.88, 3.87 3.62, 3.69 3.60 (3m, 9H; H-3I,II, H-5I III, H-6I III), 2.12,
ˆ
2.11, 2.10, 2.08, 2.05, 2.04, 2.01, 1.99, 1.98, 1.95 ppm (10 s, 30H; C OCH3);
13C NMR (CDCl3): d ˆ 170.86, 170.46, 170.43, 170.31, 169.72, 169.35, 169.22,
ˆ
169.12, 168.69, 169.41 (10 C; C O), 161.33, 143.17 (2 C; ArC), 125.71,
p-Nitrophenyl
(b-d-glucopyranosyl)-(1 ! 3)-(b-d-glucopyranosyl)-(1 !
116.54 (4 C; ArCH), 100.96, 100.72, 99.36 (C-1I III), 78.37 (C-3II), 75.61,
3)-b-d-glucopyranoside (3): Condensation of a-laminaribiosyl fluoride
(1)[10] and 4-nitrophenyl b-d-glucoside (2) gave trisaccharide 3 as an
amorphous powder (90%); 1H NMR (D2O): d ˆ 8.16 8.12, 7.26 7.22 (2m,
III
72.87, 72.56, 71.95, 71.90, 71.63, 71.01, 70.19, 68.49, 68.31, 68.14 (C-2I
,
C-3I,III, C-4I III, C-5I III), 62.04, 61.74, 61.07 (C-6I III), 20.82, 20.78, 20.66,
4H, ArH), 5.20 (d, J(1,2) ˆ 7.3 Hz, 1H; H-1I), 4.74, 4.66 (2 d, J(1,2) ˆ
ˆ
20.61, 20.50, 20.33 ppm (10C; C OCH3); HRMS-FAB: m/z calcd for
J(1,2) ˆ 8.0 Hz, 2H; H-1II,III), 3.88-3.24 ppm (m, 18H; H-2I III, H-3I
,
III
C44H55NO28Na: 1068.2808; found: 1068.2813.
H-4I III, H-5I III, H-6I III); 13C NMR (D2O) d ˆ 162.04, 143.02 (2C; ArC),
126.47, 116.88 (4 C; ArCH), 103.16, 102.87, 99.63 (C-1I III), 84.66, 84.11 (C-
3I,II), 76.36, 76.28, 75.99, 75.92, 73.83, 73.59, 72.92, 69.94, 68.51, 68.17 (C-
2I III, C-3III, C-4I III, C-5I III), 61.07, 60.79 ppm (3C; C-6I III); HRMS-FAB:
m/z calcd for C24H35NO18Na: 664.1523; found: 664.1525.
p-Nitrophenyl (b-d-glucopyranosyl)-(1! 3)-(b-d-glucopyranosyl)-(1! 3)-
[b-d-glucopyranosyl-(1 ! 6)]-b-d-glucopyranoside (9): Condensation of
a-laminaribiosyl fluoride (1) and p-nitrophenyl b-gentiobioside (8) yielded
the trisaccharide (9) as an amorphous powder (70%); 1H NMR (D2O) d ˆ
8.25 8.19, 7.26 7.20 (2m, 4H; ArH), 5.27 (d, J(1,2) ˆ 7.3 Hz, 1H; H-1I),
4.78, 4.69 (2d, J(1,2) ˆ 8.0 Hz, J(1,2) ˆ 7.7 Hz, 2H; H-1II,III), 4.41 (d,
J(1,2) ˆ 7.7 Hz, 1H; H-1IV), 4.20 4.14, 3.91 3.58, 3.55 3.19 ppm (3m,
p-Nitrophenyl
(b-d-glucopyranosyl)-(1 ! 3)-(b-d-glucopyranosyl)-(1 !
3)-b-d-xylopyranoside (6): Condensation of a-laminaribiosyl fluoride (1)
2606
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 2603 2610

Synthesis of Complex Oligosaccharides
2603 2610
24H; H-2I IV, H-3I IV, H-4I IV, H-5I IV, H-6I IV); 13C NMR (D2O): d ˆ 161.95,
143.10 (2C; ArC), 126.48, 116.99 (4C; ArCH), 103.16, 102.86, 99.51 (4C;
C-1I IV), 84.67, 83.39 (C-3I,II), 76.37, 76.24, 76.02, 75.50, 73.83, 73.60, 73.48,
72.89, 69.98, 68.80, 68.51, 68.12 (15 C; C-2I IV, C-3III,IV, C-4I IV, C-5I IV, C-6I),
2,3,6-Tri-O-acetyl-3-azido-3-deoxy-a-d-glucopyranosyl trichloroacetimi-
date (16): i) Hydrazine acetate (385 mg, 4.2 mmol) was added to a warmed
(508C) solution of 1,2,4,6-tetra-O-acetyl-3-azido-3-deoxy-d-glucose[21]
(1.42 g, 3.8 mmol) in DMF (20 mL), and the combined mixture was stirred
for 30 min. The mixture was poured onto saturated NaCl solution and
extracted (EtOAc). The combined extracts were dried (MgSO4), filtered,
and evaporated, and the residual oil subjected to flash chromatography
(40 45% EtOAc/petrol ether) to yield 2,3,6-tri-O-acetyl-3-azido-3-deoxy-
‡
61.07 ppm (3C; C-6II IV); MS-FAB: m/z: 810 [M ‡Na].
A sample of this trisaccharide was treated with Ac2O/pyridine/N,N-
dimethylaminopyridine (12 h) and then subjected to workup (CHCl3) and
flash chromatography (65 75% EtOAc/petrol ether) to yield p-nitro-
d-glucopyranose as
a colorless oil (1.45 g, 91%; a/b1:1); a-anomer:
phenyl
acetyl-b-d-glucopyranosyl)-(1 ! 3)-[2,3,4,6-tetra-O-acetyl-b-d-glucopyra-
nosyl-(1 ! 6)]-2,4-di-O-acetyl-b-d-glucopyranoside as colorless oil
(2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl)-(1 ! 3)-(2,4,6-tri-O-
13
ˆ
C NMR (CDCl3) d ˆ 170.95, 170.04, 169.43 (C O), 89.50 (C-1), 71.88,
68.45, 67.25 (C-2, C-4, C-5), 62.03 (C-6), 60.57, (C-3), 20.64, 20.57, 20.54 ppm
a
;
13
ˆ
1
(Me); b-anomer: C NMR (CDCl3): d ˆ 170.94, 170.56, 169.31 (C O),
[a]D ˆ À32.38 (c ˆ 1.1 in CHCl3); H NMR (CDCl3): d ˆ 8.29 8.18, 7.05
6.19 (2m, 4H; ArH), 5.22 4.75 (m, 11H; H-1I, H-2I IV, H-3III,IV, H-4I IV),
4.49, 4.48, 4.46 (3 d, J(1,2) ˆ J(1,2) ˆ 7.7 Hz, J(1,2) ˆ 7.8 Hz, 3H; H-1II IV),
4.39 4.19, 4.15 3.75 (m, 10H; H-3I,II, H-6I IV), 3.66 3.53 (m, 4H; H-5I IV),
95.64 (C-1), 73.34, 72.84, 63.85 (C-2, C-4, C-5), 62.03 (C-6), 60.46, (C-3),
‡
ˆ
20.94, 20.64, 20.57 (C OCH3); MS-FAB: m/z: 349 [M ‡NH4].
ii) DBU (50 mL) was added to a solution of the previously described
product (1.1 g, 3.3 mmol) and trichloroacetonitrile (1.0 mL, 10 mmol) in
CH2Cl2 (25 mL), and the combined mixture stirred (08C ! RT, 2 h). The
mixture was evaporated, and the residual oil subjected to flash chroma-
tography (10% EtOAc/petrol ether) to yield the trichloroacetimidate 16 as
a colorless oil (1.30 g, 81%); 1H NMR (CDCl3): d ˆ 8.68 (brs, 1H; NH),
6.49 (d, J(1,2) ˆ 3.5 Hz, 1H; H-1), 5.04 (t, J(3,4) ˆ 10.0 Hz, 1H; H-4), 4.96
(dd, J(1,2) ˆ 3.5 Hz, J(2,3) ˆ 10.6 Hz, 1H; H-2), 4.22 3.99 (m, 4H; H-3,
ˆ
2.13, 2.09, 2.06, 2.05, 2.04, 2.00, 1.99, 1.97, 1.95 ppm (13s, 39H; C OCH3);
13C NMR (CDCl3): d ˆ 170.48, 170.43, 170.25, 170.09, 169.41, 169.38, 169.22,
ˆ
169.15, 169.09, 168.74, 168.68 (13 C; C O), 160.82, 143.26 (2C; ArC),
126.09, 116.42 (4 C; ArCH), 101.05, 100.61, 98.16 (4C; C-1I IV), 78.90, 77.73
(C-3I,II), 74.09, 72.84, 72.61, 72.48, 72.05, 71.85, 71.65, 71.07, 70.92, 68.48,
68.29, 68.19, 68.09 (17 C; C-2I IV, C-3III,IV, C-4I IV, C-5I IV, C-6I), 61.99, 61.71,
61.69 (C-6II IV), 20.90, 20.68, 20.58, 20.52, 20.49, 20.39, 20.34 ppm (13C;
H-5, H-6), 2.11, 2.06, 2.04 ppm (3s, C OCH3); 13C NMR (CDCl3): d ˆ
ˆ
ˆ
C OCH3); HRMS-FAB: m/z calcd for C56H71NO36Na: 1333.3756; found:
ˆ
ˆ
169.51, 169.09 (3C; C O), 160.56 (C NH), 92.51 (2C; C-1, CCl3), 70.45,
70.22, 67.67 (C-2, C-4, C-5), 61.40, 60.95 (C-3, C-6), 20.60, 20.57, 20.94
1333.3659.
p-Nitrophenyl
(b-d-glucopyranosyl)-(1 ! 3)-(b-d-glucopyranosyl)-(1 !
ˆ
(C OCH3); HRMS-FAB: m/z calcd for C14H17Cl3N4O8Na: 497.0010;
3)-(b-d-glucopyranosyl)-(1 ! 4)-b-d-glucopyranoside (11): Condensation
found: 497.0005.
of a-laminaribiosyl fluoride (1) and p-nitrophenyl b-cellobioside (10)
1
yielded the tetrasaccharide 11 as an amorphous powder (55%); H NMR
Methyl (2,4,6-tri-O-acetyl-3-azido-3-deoxy-b-d-glucopyranosyl)-(1 ! 3)-2-
O-benzoyl-4,6-O-benzylidine-a-d-glucopyranoside (18): Freshly activated
molecular sieves (powdered 4 ä, 500 mg) were added to a solution of azide
16 (2.17 g, 4.6 mmol) and alcohol 17[22] (881 mg, 2.3 mmol) in CH2Cl2
(25 mL), and the combined mixture stirred (RT, 15 min). TMSOTf
(50 mL) was introduced with continued stirring (10 min) followed by Et3N
(200 mL). The mixture was filtered and evaporated and the residue
subjected to flash chromatography (30 35% EtOAc/petrol ether) to yield
the title compound as a colorless foam (1.04 g, 65%); [a]D ‡36.08 (c ˆ 1.0 in
(D2O): d ˆ 8.24 8.18, 7.24 7.18 (2m, 4H; ArH), 5.23 (d, J(1,2) ˆ 7.7 Hz,
1H; H-1I), 4.72, 4.68 (2 d, J(1,2) ˆ 8.0 Hz, J(1,2) ˆ 8.2 Hz, 2H; H-1III,IV),
4.68 (d, J(1,2) ˆ 8.0 Hz, 1H; H-1II), 3.99 3.62, 3.55 3.27 ppm (2m, 24H;
H-2I IV, H-3I IV, H-4I IV, H-5I IV, H-6I IV); 13C NMR (D2O): d ˆ 163.28, 144.28
(2C; ArC), 127.73, 118.13 (4C; ArCH), 104.42, 104.10, 103.95, 100.89 (C-
1I IV), 85.90, 85.11 (2C; C-3II,III), 79.81 (C-4I), 77.62, 77.23, 77.18, 76.71, 75.62,
IV
75.07, 74.83, 74.64, 74.15, 71.19, 62.31, 62.20, 61.39 (C-2I IV, C-3I,IV, C-4II
C-5I IV), 62.31, 62.20, 61.39 ppm (4C; C-6I IV).
,
1
CHCl3); H NMR (CDCl3): d ˆ 8.09 8.05, 7.65 7.60, 7.54 7.45, 7.34 7.29
A small sample of the tetrasaccharide was treated with Ac2O/pyridine/N,N-
dimethylaminopyridine (12 h) and then subjected to workup (CHCl3) and
flash chromatography (75% EtOAc/petrol ether) to yield p-nitrophenyl
(2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl)-(1 ! 3)-(2,4,6-tri-O-acetyl-b-
d-glucopyranosyl)-(1 ! 3)-(2,4,6-tri-O-acetyl-b-d-glucopyranosyl)-(1 !
4)-2,3,6-tri-O-acetyl-b-d-glucopyranoside as a colorless oil; [a]D ˆ À30.08
(c ˆ 0.7 in CHCl3); 13C NMR (CDCl3) d ˆ 170.55, 170.47, 170.33, 170.29,
(m, 10H; ArH), 5.11 (dd, J(1,2) ˆ 3.8 Hz, J(2,3) ˆ 9.5 Hz, 1H; H-2I), 4.99
(dd, J(1,2) ˆ 3.8 Hz, 1H; H-1I), 4.92 (t, J(4,5) ˆ 7.8, J(3,4) ˆ 9.9 Hz, 1H;
H-4II), 4.87 (dd, J(1,2) ˆ 7.8 Hz, J(2,3) ˆ 9.9 Hz, 1H; H-2II), 4.69 (d, J(1,2) ˆ
7.8, 1H; H-1II), 4.34 (t, J(3,4) ˆ J(4,5) ˆ 9.3 Hz, 1H; H-4I), 4.27 (d, J(5,6) ˆ
4.0 Hz, J(6,6) ˆ 9.5 Hz, 1H; H-6I), 4.14 (dd, J(5,6) ˆ 4.9 Hz, J(6,6) ˆ
12.3 Hz, 1H; H-6II), 3.99 (dd, J(5,6) ˆ 2.6 Hz, J(6,6) ˆ 12.2 Hz, 1H;
H-6II), 3.95 3.65 (m, 4H; H-3I, H-5I, H-6I, PhCH), 3.54 (ddd, J(5,6) ˆ
ˆ
169.65, 169.41, 169.24, 169.08, 169.03, 168.73, 168.38 (13 C; C O), 161.18,
2.7 Hz, J(5,6) ˆ 4.6 Hz, J(4,5) ˆ 9.9 Hz, 1H; H-5II), 3.36 (s, 3H; OMe), 3.20
143.29 (2C; ArC), 125.85, 116.58 (4C; ArCH), 101.06, 100.74, 100.64, 97.91
II
(4 C; C-1I IV), 78.98, 77.88 (C-3II,III), 75.84, 73.32, 73.01, 72.87, 72.60, 72.34,
ˆ
(t, J(2,3) ˆ J(3,4) ˆ 9.9 Hz, 1H; H-3 ), 2.05, 1.95, 1.67 (3s, 9H; C OCH3);
13
IV
72.12, 71.82, 71.66, 71.07, 70.88, 68.11, 68.00 (14 C; C-2I IV, C-3I,IV, C-4I
,
ˆ
C NMR (CDCl3): d ˆ 170.73, 169.03, 168.91 (C OCH3), 165.61 (C ˆ
C-5I IV), 62.02, 61.94, 61.72 (4 C; C-6I IV), 58.45 (OMe), 20.85, 20.74, 20.68,
OAr), 137.25, 133.60, 129.80, 129.40, 128.96, 128.58, 128.04, 126.03 (12 C;
Ar), 101.14, 101.03 (C-1II, PhCH), 97.59 (C-1I), 79.05 (C-3I), 76.29, 73.62,
72.47, 70.75, 68.76, 68.28 (C-2I,II, C-4I,II, C-5I,II), 64.37 (C-3II), 62.57, 62.06 (C-
ˆ
20.68, 20.50, 20.49, 20.47, 20.43, 20.33ppm (13C; C OCH3); HRMS-FAB:
m/z calcd for C56H71NO36Na: 1356.3653; found: 1356.3638.
6I,II), 55.38 (OMe), 20.63, 20.54, 19.98 ppm (C OCH3); HRMS-FAB: m/z
ˆ
Methyl (2,3,4,6-tetra-O-acetyl-b-d-glucopyranosyl)-(1 ! 3)-(2,4,6-tri-O-
acetyl-b-d-glucopyranosyl)-(1 ! 3)-(2,4,6-tri-O-acetyl-b-d-glucopyrano-
syl)-(1 ! 3)-2,4,6-tri-O-acetyl-b-d-glucopyranoside (13): a-Laminaribiosyl
fluoride (1) and methyl b-laminaribioside (12)[18] were subjected to the
glycosynthase reaction (24 h) and the solution was then lyophilized. The
residue was treated with Ac2O (2 mL), pyridine (3 mL), and N,N-
dimethylaminopyridine (100 mg), and the combined mixture stirred (RT
12 h). The mixture was subjected to workup (CHCl3) and flash chromatog-
raphy (60 80% EtOAc/petrol ether) to yield the tetrasaccharide (13) as a
calcd for C33H37N3O14Na 722.2173; found: 722.2179.
Methyl (2,4,6-tri-O-acetyl-3-azido-3-deoxy-b-d-glucopyranosyl)-(1 ! 3)-
2,4,6-tri-O-acetyl-a-d-glucopyranoside (19): NaOMe (3 mL of 1.0m in
MeOH, 3.0 mmol) was added to a solution of the azide (18) (1.40 g,
2.00 mmol) in MeOH (15 mL), and the mixture stirred (08C ! RT, 2 h).
‡
The solution was treated with Amberlite IR-120 resin (H form) until
neutral and filtered; the solvent was then evaporated. Acetic acid (20 mL of
60% aq.) was added to the residual oil, and the combined mixture heated
(1008C; 20 min) and then evaporated (and co-evaporated with toluene).
Ac2O (6 mL), pyridine (8 mL), and N,N-dimethylaminopyridine (50 mg)
were added to the residual oil, and the combined mixture was stirred (RT,
8 h) before being treated with ice-water (10 mL). The mixture was then
subjected to workup (CH2Cl2) and flash chromatography (45 50%
EtOAc/petrol ether) yielding the hexaacetate (19) as fine needles (1.10 g,
87%); m.p. 153 1548C (EtOH); [a]D ˆ ‡21.98 (c ˆ 1.0 in CHCl3);
1H NMR (CDCl3): d ˆ 4.94 (dd, J(1,2) ˆ 3.6 Hz, J(2,3) ˆ 10.2 Hz, 1H;
H-2I), 4.93-4.81 (m, 3H; H-1I, H-4I,II), 4.77 (dd, J(1,2) ˆ 8.0 Hz, J(2,3) ˆ
10.0 Hz, 1H; H-2II), 4.57 (d, J(1,2) ˆ 8.0 Hz, 1H; H-1II), 4.25 (dd, J(5,6) ˆ
4.6 Hz, J(6,6) ˆ 12.4 Hz, 1H; H-6II), 4.18 4.06 (m, 3H; H-3I, H-6I,II), 4.00
(dd, J(5,6) ˆ 2.4 Hz, J(6,6) ˆ 12.2 Hz, 1H; H-6II), 3.87 (ddd, J(5,6) ˆ 2.6 Hz,
1
colorless oil (45%); [a]D ˆ À23.28 (c ˆ 0.7 in CHCl3); H NMR (CDCl3):
d ˆ 5.12 4.76 (m, 9H; H-2I IV, H-3IV, H-4I IV), 4.48, 4.46 (2d, J(1,2) ˆ
8.0 Hz, J(1,2) ˆ 8.2 Hz, 2H; H-1II,III), 4.39 4.23, 4.18 3.98 (2m, 10H;
H-1I,IV, H-6I IV), 3.85, 3.78, 3.77 (3t, J(2,3) ˆ J(3,4) 9.1 Hz, 3H; H3I III),
3.70 3.62 (m, 4H; H-5I IV), 3.39 (s, 3H; OMe), 2.12, 2.09, 2.06, 2.05, 2.04,
2.01, 2.00, 1.99, 1.98, 1.95 ppm (13s, 39H; C OCH3); 13C NMR (CDCl3):
ˆ
d ˆ 170.73, 170.60, 170.45, 170.22, 169.45, 169.26, 169.18, 169.09, 169.03,
I
168.88, 168.81 (13C; C O), 101.45, 101.04, 100.76, 100.49 (C-1 IV), 78.86,
ˆ
78.22, 78.16 (C-3I III), 72.51, 71.88, 71.63, 70.87, 68.43, 68.27, 68.07 (13 C;
C-2I IV, C-3IV, C-4I IV, C-5I IV), 62.22, 62.13, 61.97, 61.71 (C-6I IV), 56.62
(OMe), 21.07, 20.80, 20.75, 20.69, 20.60, 20.51, 230.49, 20.34 ppm (13C;
‡
ˆ
C OCH3); MS-FAB: m/z: 1249 [M ‡Na].
Chem. Eur. J. 2003, 9, 2603 2610
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2607

H. Driguez et al.
FULL PAPER
J(5,6) ˆ 4.6 Hz, J(4,5) ˆ 10.4 Hz, 1H; H-5I), 3.58 (ddd, J(5,6) ˆ 2.4 Hz,
J(5,6) ˆ 4.6 Hz, J(4,5) ˆ 9.8 Hz, 1H; H-5II), 3.48 (t, J(2,3) ˆ J(3,4) ˆ
10.0 Hz, 1H; H-3II), 3.66 (s, 3H, OMe), 2.15, 2.06, 2.05, 2.03, 2.02,
A small sample of the trisaccharide was treated with Ac2O/pyridine/N,N-
dimethylaminopyridine (12 h) and then subjected to workup (CHCl3) and
flash chromatography (50 70% EtOAc/petrol ether) to yield p-nitro-
phenyl (2,4,6-tri-O-acetyl-3-azido-3-deoxy-b-d-glucopyranosyl)-(1 ! 3)-
(2,4,6-tri-O-acetyl-b-d-glucopyranosyl)-(1 ! 3)-2,4,6-tri-O-acetyl-b-d-glu-
copyranoside as a colorless oil; [a]D ˆ À43.58 (c ˆ 1.0 in CHCl3); 1H NMR
(CDCl3): d ˆ 5.24 (t, J(1,2) ˆ J(2,3) ˆ 8.0 Hz, 1H; H-2III), 5.07 (d, J(1,2) ˆ
7.5 Hz, 1H; H-1I), 4.99-4.87 (m, 4H; H-2II, H-4IÀIII), 4.78 (dd, J(1,2) ˆ
7.8 Hz, J(2,3) ˆ 10.2 Hz, 1H; H-2I), 4.49, 4.45 (2 d, J(1,2) ˆ 8.2 Hz,
J(1,2) ˆ 8.0 Hz, 2H; H-1II,III), 4.35 4.16, 4.10 3.87, 3.82 3.57 (3m, 11H;
H-3II,III, H-5I III, H-6I III), 3.50 (t, J(2,3) ˆ J(3,4) ˆ 10.2 Hz, 1H; H-3I), 2.14,
1.98 ppm (6s, 18H; C OCH3); 13C NMR (CDCl3): d ˆ 170.63, 170.43,
ˆ
II
I
ˆ
169.59, 169.13, 169.04, 168.68 (6C; C O), 100.91 (C-1 ), 96.67 (C-1 ), 76.09
(C-3I), 72.77, 72.49, 70.84, 68.18, 67.99, 67.33 (C-2I,II, C-4I,II, C-5I,II), 64.39 (C-
3II), 62.08, 61.75 (C-6I,II), 55.34 (OMe), 20.87, 20.66, 20.56, 20.51, 20.38,
‡
ˆ
20.31 ppm (6C; C OCH3); MS-FAB: m/z: 656 [M ‡Na]; elemental
analysis calcd (%) for C25H35N3O16 (633.2): C 47.40, H 5.57, N 6.63; found: C
47.47, H 5.59. N 6.75.
(2,4,6-Tri-O-acetyl-3-azido-3-deoxy-b-d-glucopyranosyl)-(1 ! 3)-1,2,4,6-
tetra-O-acetyl-d-glucopyranose (20): H2SO4 (140 mL of 18m) was added to
a solution of the laminaribioside (19) (987 mg, 1.55 mmol), AcOH (7 mL)
and Ac2O (14 mL), and the combined mixtured was stirred (RT, 6 h).
NaOAc (1.0 g) was slowly introduced with continued stirring (15 min), and
the mixture was filtered. The filtrate was subjected to workup (CHCl3) and
flash chromatography (50 60% EtOAc/petrol ether) to yield an anomeric
mixture of the acetates (20) (a/b, 9:1) as a colorless oil (744 mg, 72%); a-
anomer: 1H NMR (CDCl3): d ˆ 6.21 (d, J(1,2) ˆ 3.7 Hz, 1H; H-1I), 5.05
4.92 (m, 3H; H-2I, H-4I,II), 4.78 (dd, J(1,2) ˆ 8.0 Hz, J(2,3) ˆ 10.1 Hz, 1H;
H-2II), 4.58 (d, J(1,2) ˆ 8.0 Hz, 1H; H-1II), 4.32 (dd, J(5,6) ˆ 4.2, J(6,6) ˆ
12.0 Hz, 1H; H-6I), 4.20 4.00 (m, 5H; H-3I, H-5I, H-6I,II), 3.71 3.64 (m,
1H; H-5II), 3.51 (t, J(2,3) ˆ J(3,4) ˆ 10.0 Hz, 1H; H-3II), 2.15, 2.08, 2.07,
ˆ
2.11, 2.10, 2.07, 2.05, 2.04, 2.03, 2.02, 1.99 ppm (9s, 27H; C OCH3);
13C NMR (CDCl3): d ˆ 170.48, 170.41, 169.11, 169.01, 168.70, 168.67 (9C
ˆ
C O), 161.17, 143.22 (2C; ArC), 125.73, 116.52 (4C; ArCH), 101.16, 100.59,
97.94 (C-1I III), 78.63, 77.67 (C-3I,II), 72.82, 72.45, 71.87, 70.60, 68.35, 68.20,
68.17 (9C; C-2I III, C-4I III, C-5I III), 64.33 (C-3III), 62.09, 62.03, 61.77 (C-
III
6I ), 20.91, 20.71, 20.66, 20.61, 20.52, 20.45, 20.44 ppm (9C; C OCH3);
ˆ
HRMS-FAB: m/z calcd for C42H52N4O26Na: 1051.2767; found: 1051.2775.
p-Nitrophenyl (3-azido-3-deoxy-b-d-glucopyranosyl)-(1 ! 3)-(b-d-gluco-
pyranosyl)-(1 ! 3)-(b-d-glucopyranosyl)-(1 ! 3)-b-d-glucopyranoside
(23): Condensation of the fluoride 15 (10 equiv) and p-nitrophenyl b-d-
laminaribioside (14)[19] (1.0 equiv) yielded the trisaccharide 23 (95%) as a
colorless powder; 1H NMR (D2O): d ˆ 8.24 8.18, 7.22 7.17 (2m, 4H;
ArH), 5.23 (d, J(1,2) ˆ 7.7 Hz, 1H; H-1I), 4.77, 4.76, 4.73 (3d, J(1,2) ˆ
2.06, 2.05, 2.03, 2.01 ppm (7s, 21H; C OCH3); 13C NMR (CDCl3): d ˆ
ˆ
IV
7.9 Hz, J(1,2) ˆ 8.1 Hz, 3H; H-1II IV), 3.91 3.28 (m, 24H, H-2I IV, H-3I
,
ˆ
170.63, 170.45, 169.22, 169.20, 168.97, 168.75, 168.62 (7C; C O), 100.92 (C-
H-4I IV, H-5I IV, H-6I IV); 13C NMR (D2O): d ˆ 161.67, 142.67 (2C; ArC),
126.10, 116.52 (4C; ArCH), 102.69, 102.51, 99.25 (4C; C-1I IV), 85.84, 85.53,
84.11 (C-3I III), 76.56, 75.91, 75.62, 73.26, 72.55, 72.15, 68.58, 68.30, 68.09,
1II), 89.11 (C-1I), 76.01 (C-3I), 72.36, 71.23, 70.86, 69.86, 68.18, 67.44 (C-2I,II
C-4I,II, C-5I,II), 64.24 (C-3II), 61.66, 61.65 (C-6I,II), 20.83, 20.63, 20.54, 20.48,
,
ˆ
20.33, 20.29 ppm (7C; C OCH3); HRMS-FAB: m/z calcd for C26H35N3O17-
68.06, 67.79 (13C; C-2I
, ,
C-3IV, C-4I C-5I IV), 61.01, 60.67, 60.47,
IV
IV
Na: 684.4864; found: 684.1859.
60.41 ppm (C-6I IV); HRMS-FAB: m/z calcd for C30H44N4O22Na 835.2345;
(2,4,6-Tri-O-acetyl-3-azido-3-deoxy-b-d-glucopyranosyl)-(1 ! 3)-2,4,6-tri-
O-acetyl-a-d-glucopyranosyl fluoride (21): HF (4 mL of 70% in pyridine)
was added to a cooled (08C) polyethylene vessel containing the heptaa-
cetate (20) (192 mg, 0.29 mmol), and the mixture was stirred (48C; 12 h).
CH2Cl2 (6 mL) was added, and the combined solution poured onto a stirred
suspension of ice in aq. NH3 (60 mL of 3m). The solution was subjected to
workup (CH2Cl2) and flash chromatography (40 60% EtOAc/petrol
ether) to give the fluoride (21) as fine needles (156 mg, 86%); m.p. 175
1768C (EtOH); [a]D ˆ ‡9.98 (c ˆ 0.7 in CHCl3); 1H NMR (CDCl3): d ˆ
5.63 (dd, J(1,2) ˆ 2.7 Hz, J(H,F) 53.2 Hz, 1H; H-1I), 5.03 (t, J(3,4) ˆ
J(4,5) ˆ 9.9 Hz, 1H; H-4II), 4.93 (t, J(3,4) ˆ J(4,5) ˆ 9.9 Hz, 1H; H-4I),
4.89 (ddd, J(1,2) ˆ 2.7 Hz, J(2,3) ˆ 10.2 Hz, J(2,F) ˆ 24.7 Hz, 1H; H-2I),
4.78 (dd, J(1,2) ˆ 8.0 Hz, J(2,3) ˆ 10.2 Hz, 1H; H-2II), 4.57 (d, J(1,2) ˆ
8.0 Hz, 1H; H-1II), 4.29 (dd, J(5,6) ˆ 4.7 Hz, J(6,6) ˆ 12.6 Hz, 1H; H-6II),
3.64 (ddd, J(5,6) ˆ 2.4 Hz, J(5,6) ˆ 4.6 Hz, J(4,5) ˆ 9.9 Hz, 1H; H-5II), 3.52
(t, J(2,3) ˆ J(3,4) ˆ 10.2 Hz, 1H; H-3II), 2.08, 2.05, 2.04, 2.03, 2.01, 1.99 ppm
found: 835.2339.
Methyl (2,3,4,6-tetra-O-acetyl-b-d-galactopyranosyl)-(1 ! 3)-2-O-benzyl-
4,6-O-benzylidine-a-d-glucopyranoside (27): Freshly activated molecular
sieves (powdered 4 ä, 500 mg) was added to a solution of a-d-galactosyl
trichloroacetimidate (25)[24] (1.04 g, 2.1 mmol) and alcohol 26[25] (604 mg,
1.6 mmol) in CH2Cl2 (25 mL), and the combined mixture stirred (RT,
15 min). TMSOTf (30 mL) was added dropwise with continued stirring
(10 min), followed by Et3N (200 mL). The mixture was filtered and
evaporated and the residue was subjected to flash chromatography (30
35% EtOAc/petrol ether) to yield the title compound (27) as powder
(968 mg, 85%); 1H NMR (CDCl3): d7.48 7.43, 7.35 7.28 (2m, 10H; ArH),
5.29 (dd, J(3,4) ˆ 3.2 Hz, J(4,5) ˆ 0.8 Hz, 1H; H-4II), 5.26 (dd, J(1,2) ˆ
8.2 Hz, J(2,3) ˆ 10.6 Hz, 1H; H-2II), 4.94 (dd, J(3,4) ˆ 3.4 Hz, J(2,3) ˆ
10.4 Hz, 1H; H-3II), 4.80 (d, J(1,2) ˆ 8.2 Hz, 1H; H-1II), 4.76, 4.48 (2d,
J(H,H) ˆ 12.0 Hz, 2H; CH2Ph), 4.42 (d, J(1,2) ˆ 3.8 Hz, 1H; H-1I), 4.22
4.01 (m, 3H; H-4II, H-6I,II), 3.83 3.564 (m, 6H; H-3I, H-5I,II, H-6I,II, PhCH),
3.50 (dd, J(1,2) ˆ 3.8 Hz, J(2,3) ˆ 9.3 Hz, 1H; H-2I), 3.32 (s, 3H; OMe),
(6s, 18H; C OCH3); 13C NMR (CDCl3) d ˆ 170.55, 170.43, 169.49, 169.03,
ˆ
I
II
ˆ
168.96, 168.68 (C O), 103.97 (d, J(1,F) ˆ 227 Hz, C-1 ), 101.03 (C-1 ),
75.59, 72.61, 70.86, 68.14, 66.83 (C-2I, C-4I,II, C-5I,II), 72.18 (d, J(2,F) ˆ
24.1 Hz, C-2I), 70.09 (d, J(3,F) ˆ 3.7 Hz, C-3I), 64.33 (C-3II), 61.71, 61.39
2.10, 1.97, 1.94, 1.89 ppm (4s, 12H; C OCH3); 13C NMR (CDCl3): d ˆ
ˆ
ˆ
170.21, 170.13, 170.19, 169.55 (4C; C O), 138.09, 137.24, 129.08, 128.59,
128.48, 128.24, 128.09, 128.00, 127.85, 125.97 (12C; Ar), 101.44, 101.33 (C-
1II, PhCH), 98.97 (C-1I), 80.78, 78.66, 78.60, 74.10, 71.20, 70.63, 69.62, 68.99,
67.00 (C-2I,II, C-3I,II, C-4I,II, C-5I,II, CH2Ph), 61.95, 60.89 (C-6I,II), 55.33
(OMe), 20.79, 20.61, 20.52, 20.50 ppm (4C; C OCH3); HRMS-FAB: m/z
calcd for C35H42O15Na: 725.2421; found: 725.2429.
(C-6I,II), 20.65, 20.57, 20.52, 20.33 ppm (6C; C OCH3); MS: m/z: 644
[M ‡Na]; elemental analysis calcd (%) for C24H32FN3O16 (621.2): C 46.39,
ˆ
‡
H 5.19, N 6.76; found: C 46.45, H 5.19, N 6.85.
ˆ
(3-Azido-3-deoxy-b-d-glucopyranosyl)-(1 ! 3)-a-d-glucopyranosyl fluo-
ride (15): NaOMe (100 mL of 1.0m in MeOH) was added to a solution of
the hexaacetate (21) (317 mg, 0.51 mmol) in MeOH (10 mL), and the
mixture was stirred (08C ! RT, 2 h). The solution was treated with
(2,3,4,6-Tetra-O-acetyl-b-d-galactopyranosyl)-(1 ! 3)-1,2,4,6-tetra-O-ace-
tyl-d-glucopyranose (28): Palladium on charcoal (55 mg of 10%) was
added to a solution of the tetraacetate (27) (550 mg, 0.78 mmol) and glacial
acetic acid (50 mL) in a mixture of MeOH (10 mL) and EtOAc (5 mL), and
the mixture vigorously stirred under an atmosphere of hydrogen (6 12 h).
The mixture was filtered, evaporated, and treated with H2SO4 (200 mL of
18m), AcOH (5 mL), and Ac2O (10 mL), and the combined mixtured
stirred (RT, 12 h). NaOAc (1.0 g) was introduced slowly with continued
stirring (15 min) and the mixture was filtered. The filtrate was subjected to
workup (CHCl3) and flash chromatography (60 70% EtOAc/petrol ether)
to yield an anomeric mixture of the acetate (28) (a/b, 9:1) as a colourless oil
(488 mg, 92%); [a]D ˆ ‡32.08 (c ˆ 1.0 in CHCl3; lit[30] ‡29.08); a-anomer:
13C NMR (CDCl3) d ˆ 170.67, 170.39, 170.18, 170.10, 169.32, 169.00, 168.63
‡
Amberlite IR-120 resin (H form) until neutral, before filtration. The
solvent was evaporated and the residual oil lyophilized (H2O) to yield the
‡
fluoride (18) as a white foam (185 mg, 99%); MS: m/z: 392 [M ‡Na].
p-Nitrophenyl (3-azido-3-deoxy-b-d-glucopyranosyl)-(1 ! 3)-(b-d-gluco-
pyranosyl)-(1 ! 3)-b-d-glucopyranoside (22): Condensation of the fluoride
(15) (1.2 equiv) and p-nitrophenyl b-d-glucoside (2) (1.0 equiv) yielded the
trisaccharide (22) as an amorphous powder (81%); 1H NMR (D2O) d ˆ
8.15 8.09, 7.13 7.09 (2m, 4H; ArH), 5.10 (d, J(1,2) ˆ 7.5 Hz, 1H; H-1I),
4.61, 4.59 (2d, J(1,2) ˆ 8.0 Hz, J(1,2) ˆ 7.6 Hz, 2H; H-1II,III), 3.75 3.13 ppm
(m, 18H; H-2I III, H-3I III, H-4I III, H-5I III, H-6I III); 13C NMR (D2O): d ˆ
161.95, 143.20 (2C; ArC), 127.53, 118.01 (4C; ArCH), 104.20, 103.92, 100.56
(C-1I III), 85.84, 85.53 (C-3I,II), 78.04, 77.31, 77.07, 74.55, 73.82, 73.57, 69.90,
69.80, 69.51, 69.20 (C-2I III, C-3III, C-4I III, C-5I III), 62.07, 61.91, 61.83 ppm
(C-6I III).
II
I
I
ˆ
(8C; C O), 101.05 (C-1 ), 89.17 (C-1 ), 75.64 (C-3 ), 71.23, 70.89, 70.39,
69.92, 69.88, 67.62, 66.68 (C-2I,II, C-3II, C-4I,II, C-5I,II), 61.73, 60.69 (C-6I,II),
ˆ
20.90, 20.68, 20.59, 20.56, 20.48, 20.39 ppm (8C; C OCH3); HRMS-FAB:
m/z calcd for C28H38O19Na: 701.1905; found: 701.1905.
2608
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 2603 2610

Synthesis of Complex Oligosaccharides
2603 2610
(2,3,4,6-Tetra-O-acetyl-b-d-galactopyranosyl)-(1 ! 3)-2,4,6-tri-O-acetyl-a-
d-glucopyranosyl fluoride (29): HF (6 mL of 70% in pyridine) was added to
a cooled (08C) polyethylene vessel containing the heptaacetate (28)
(247 mg, 0.36 mmol), and the mixture was stirred (48C; 12 h). CH2Cl2
(6 mL) was added and the combined solution poured onto a stirred
suspension of ice in aq. NH3 (60 mL of 3m). The solution was subjected to
workup (CH2Cl2) and flash chromatography (40 60% EtOAc/petrol
ether) to give the fluoride 29 as a colorless oil (168 mg, 72%). 1H NMR
(CDCl3): d ˆ 5.63 (dd, J(1,2) ˆ 2.7 Hz, J(1,F) ˆ 53.2 Hz, 1H; H-1I), 5.34
5.32 (m, 1H; H-4II), 5.09 5.01, 4.96 4.82 (2m, 4H; H-2I,II, H-3II, H-4I), 4.59
(d, J(1,2) ˆ 7.9 Hz, 1H; H-1II), 4.22 4.00 (m, 7H; H-3I, H-5I,II, H-6I,II), 2.14,
2II,III, C-3I III, C-4I III, C-5II,III), 72.55 (d, J(2,F) ˆ 24.0 Hz; C-2I), 70.09 (d,
J(3,F) ˆ 3.7 Hz, C-5I), 62.04, 61.57, 61.40 (C-6I III), 20.74, 20.69, 20.64, 20.49,
ˆ
20.47, 20.37, 20.32 ppm (10C; C OCH3); HRMS-FAB: m/z calcd for
C38H51FO25Na 949.2601; found: 949.2586.
ii) NaOMe (50 mL of 1.0m in MeOH) was added to a solution of the
previously described decaacetate synthesized above (87 mg, 94 mmol) in
MeOH (5 mL) and the mixture stirred (08C ! RT, 2 h). The solution was
‡
treated with Amberlite IR-120 resin (H form) until neutral, filtered, the
solvent evaporated and the residue lyophilised (H2O) to yield the title
‡
fluoride (31) as a white foam (47 mg, 99%); MS: m/z: 529 [M ‡Na].
p-Nitrophenyl
(b-d-glucopyranosyl)-(1 ! 4)-(b-d-glucopyranosyl)-(1 !
2.10, 2.06, 2.03, 2.02, 1.94, 1.93 ppm (7s, 21H; C OCH3); 13C NMR
ˆ
3)-(b-d-glucopyranosyl)-(1 ! 3)-b-d-glucopyranoside (32): Condensation
of fluoride 31 and p-nitrophenyl b-d-glucoside (2) yielded the tetrasac-
charide 32 as an amorphous powder (33%); 1H NMR (D2O): d ˆ 8.25
8.19, 7.23 7.16 (2m, 4H; ArH), 5.24 (d, J(1,2) ˆ 7.3 Hz, 1H; H-1I), 4.77, 4.73
(2d, J(1,2) ˆ 8.0 Hz, 2H; H-1II,III), 4.44 (d, J(1,2) ˆ 7.7 Hz, 1H; H-1IV),
3.94 3.22 (m, 24H; H-2I IV, H-3I IV, H-4I IV, H-5I IV, H-6I IV); 13C NMR
(D2O): d ˆ 163.10, 145.23 (2C; ArC), 127.53, 117.95 (4C; ArCH), 103.96,
100.68 (4C; C-1I IV), 85.52, 85.14 (C-3I,II), 80.06 (C-4III), 77.34, 77.04, 76.91,
(CDCl3): d ˆ 170.53, 170.26, 170.12, 170.06, 169.59, 169.01, 168.93 (7C;
I
II
I
ˆ
C O), 103.98 (d, J(1,F) ˆ 227 Hz, C-1 ), 101.07 (C-1 ), 75.17 (C-3 ), 72.10
(d, J(2,F) ˆ 24.1 Hz, C-2I), 70.95, 70.59, 68.91, 67.05, 66.83 (C-2II, C-4I,II
,
C-5II), 70.09 (d, J(3,F) ˆ 3.7 Hz, C-3I), 61.44, 60.92 (C-6I,II), 20.65, 20.52,
ˆ
20.45, 20.36 ppm (7C; C OCH3); HRMS-FAB: m/z calcd for C26H35FO17-
Na: 661.1756; found: 661.1759.
(b-d-Galactopyranosyl)-(1 ! 3)-a-d-glucopyranosyl fluoride (24): NaOMe
(50 mL of 1.0m in MeOH) was added to a solution of the heptaacetate (29)
(85 mg, 0.13 mmol) in MeOH (5 mL), and the mixture stirred (08C ! RT,
76.24, 75.55, 74.66, 74.57, 73.98, 70.88, 69.50, 69.2 (13C; C-2I III, C-3III,IV
,
C-4I,III,IV, C-5I III), 61.07, 60.79 ppm (3C; C-6I III); HRMS-FAB: m/z calcd
‡
2 h). The solution was treated with Amberlite IR-120 resin (H form) until
for C30H45NO23Na 810.2280; found: 810.2266.
neutral, filtered, the solvent evaporated and the residual oil lyophilized
(H2O) to yield the fluoride (24) as a white foam (45 mg, 99%); MS-FAB:
‡
m/z: 367 [M ‡Na].
Acknowledgement
p-Nitrophenyl (2,3,4,6-tri-O-acetyl-b-d-galactopyranosyl)-(1 ! 3)-(2,4,6-
tri-O-acetyl-b-d-glucopyranosyl)-(1 ! 3)-2,4,6-tri-O-acetyl-b-d-glucopyra-
noside (30): The fluoride 24 and p-nitrophenyl b-d-glucoside (2) were
subjected to the glycosynthase reaction (12 h), and the solution was
lyophilized. The residue was treated with Ac2O (2 mL), pyridine (3 mL),
and N,N-dimethylaminopyridine (100 mg), and the combined solution
stirred (RT, 12 h). The mixture was subjected to workup (CHCl3) and flash
chromatography to yield the trisaccharide 30 as a colorless oil (65%);
[a]D ˆ À17.98 (c ˆ 1.1 in CHCl3); 1H NMR (CDCl3): d ˆ 8.20 8.14, 7.08
6.98 (2m, 4H; Ar), 5.33 (dd, J(3,4) ˆ 3.3 Hz, J(4,5) ˆ 0.8 Hz, 1H; H-4III),
5.25 (dd, J(1,2) ˆ 7.7, J(2,3) ˆ 8.6 Hz, 1H; H-2I), 5.07 (d, J(1,2) ˆ 7.9 Hz, 1H;
H-1I), 5.02 (t, J(3,4) ˆ J(4,5) ˆ 7.8 Hz, 1H; H-4II), 4.97 4.90 (m, 3H;
H-2II,III, H-4I), 4.90 (dd, J(3,4) ˆ 3.5 Hz, J(2,3) ˆ 9.1 Hz, 1H; H-3III), 4.49,
4.46 (2d, J(1,2) ˆ 7.7 Hz, J(1,2) ˆ 7.8 Hz, 2H; H-1II,III), 4.30 (dd, J(5,6) ˆ
This work was supported by CNRS, the Australian Research Council, and
the Grains Research and Development Corporation of Australia. Dr A.
Planas is acknowledged for the generous gift of the barley (1,3;1,4)-b-d-
glucan trisaccharide.
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4.4 Hz, J(6,6) ˆ 12.2 Hz, 1H; H-6II), 4.23 4.16, 4.09 3.80 (2m, 9H; H-3I,III
,
H-5II,III, H-6I III), 3.70 3.63 (m, 1H; H-5I), 2.16, 2.12, 2.09, 2.06, 2.04, 2.03,
2.02, 2.00, 1.94 ppm (10s, 30H; C OCH3); 13C NMR (CDCl3): d ˆ 170.09
ˆ
ˆ
168.71 (10C; C O), 161.22, 143.26 (2C; ArC), 125.76, 116.55 (4C; ArCH),
101.25,100.77, 98.03 (C-1I III), 78.38, 77.77 (C-3I,II), 72.69, 72.48, 72.41, 71.88,
70.98, 70.32, 68.51, 68.40, 68.18, 66.63 (C-2I III, C-3III, C-4I III, C-5I III), 62.14,
III
62.05, 60.64 (C-6I ), 20.96, 20.67, 20.57, 20.47 ppm (10C; C OCH3);
ˆ
HRMS-FAB: m/z calcd for C44H55NO28Na: 1068.2808; found: 1068.2780.
(b-d-Glucopyranosyl)-(1 ! 4)-(b-d-glucopyranosyl)-(1 ! 3)-a-d-glucopyr-
anosyl fluoride (31): i) HF (4 mL of 70% in pyridine) was added to a cooled
(08C) polyethylene vessel containing (2,3,4,6-tetra-O-acetyl-b-d-glucopyr-
anosyl)-(1 ! 4)-(2,3,6-tri-O-acetyl-b-d-glucopyranosyl)-(1 ! 3)-1,2,4,6-tet-
ra-O-acetyl-d-glucopyranose[27] (210 mg, 0.21 mmol), and the mixture was
stirred (48C; 12 h). CH2Cl2 (6 mL) was added, and the combined solution
poured onto a stirred suspension of ice in aq. NH3 (60 mL of 3m). The
solution was subjected to workup (CH2Cl2) and flash chromatography
(60% EtOAc/petrol ether) to give the (2,3,4,6-tetra-O-acetyl-b-d-gluco-
pyranosyl)-(1 ! 4)-(2,3,6-tri-O-acetyl-b-d-glucopyranosyl)-(1 ! 3)-1,2,4,6-
tetra-O-acetyl-d-glucopyranosyl fluoride as a white foam (145 mg, 72%);
[a]D ˆ ‡1.18 (c ˆ 0.3 in CHCl3); 1H NMR (CDCl3): d ˆ 5.62 (dd, J(1,2) ˆ
2.7 Hz, J(1,F) ˆ 53.4 Hz, 1H; H-1I), 5.11 (t, J(2,3) ˆ J(3,4) ˆ 9.6 Hz, 1H;
H-3III), 5.09 (t, J(3,4) ˆ J(4,5) ˆ 9.5 Hz, 1H; H-4I), 5.03 (dd, J(3,4) ˆ 9.6 Hz,
J(2,3) ˆ 10.0 Hz, 1H; H-3I), 5.02 (t, J(3,4) ˆ J(4,5) ˆ 9.5 Hz, 1H; H-4III),
4.91 (dd, J(1,2) ˆ 7.9 Hz, J(2,3) ˆ 9.0 Hz, 1H; H-2III), 4.90 (ddd, J(1,2) ˆ
2.6 Hz, J(2,3) ˆ 10.0, J(2,F) ˆ 25.0 Hz, 1H; H-2I), 4.81 (dd, J(1,2) ˆ 8.2 Hz,
J(2,3) ˆ 9.7 Hz, 1H; H-2II), 4.61 (d, J(1,2) ˆ 8.2 Hz, 1H; H-1III), 4.46 (d,
J(1,2) ˆ 7.9 Hz, 1H, H-1II), 4.43 4.01 (m, 8H; H-3I, H-5II, H-6I III), 3.75 (t,
J(3,4) ˆ J(4,5) ˆ 9.0 Hz, 1H; H-4II), 3.65 3.55 (m, 2H; H-5I,III), 2.17, 2.10,
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M. Sh¸lein, S. Cottaz, H. Driguez, J. Am. Chem. Soc. 2000, 122, 5429
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ˆ
2.07, 2.06, 2.01, 1.99, 1.98, 1.97, 1.95, 1.94 ppm (10s, 30H; C OCH3);
13C NMR (CDCl3): d ˆ 170.59, 170.44, 170.17, 170.09, 169.84, 169.76, 169.29,
I
ˆ
169.02, 168.99 (10C; C O), 103.81 (d, J(1,F) ˆ 227 Hz, C-1 ), 100.79 (2C;
C-1II,III), 77.19, 76.21, 75.76, 72.87, 72.79, 72.07, 71.62, 71.39, 67.82, 66.86 (C-
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Received: January 15, 2002 [F4733]
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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