Glycosyltransferase catalyzed assemblage of sialyl-Lewisa-saccharopeptides

Article abstract of DOI:10.1016/S0008-6215(98)00229-8

A series of methyl hexopyranosiduronic acids are coupled to type I disaccharide amines to give 'trisaccharides' which have the natural N-acetyl group of the type I disaccharides replaced by uronic acids (→ saccharopeptides). These saccharopeptides are sur

Full text of DOI:10.1016/S0008-6215(98)00229-8

Carbohydrate Research 312 (1998) 61±72
Glycosyltransferase catalyzed assemblage of
sialyl-Lewisa-saccharopeptides
Gabi Baisch, Reinhold Ohrlein*
Novartis Pharma AG, Postfach, CH-4002 Basle, Switzerland
Received 10 December 1997; accepted in revised form 30 July 1998
Abstract
A series of methyl hexopyranosiduronic acids are coupled to type I disaccharide amines to give
`trisaccharides' which have the natural N-acetyl group of the type I disaccharides replaced by
uronic acids (! saccharopeptides). These saccharopeptides are surprisingly good substrates for ꢀ-
2,3-sialyltransferase and fucosyltransferase III. The enzymes transfer N-acetylneuraminic acid and
fucose, respectively, onto these acceptor substrates, despite the far reaching alterations, regio- and
stereospeci®cally in the expected manner to yield sialyl-Lewisa-saccharopeptides. # 1998 Elsevier
Science Ltd. All rights reserved.
Keywords: ꢀ-2,3-Sialyltransferase; Fucosyltransferase III; Enzymatic glycosylation; Sialyl-Lewisa-saccharopeptide
1. Introduction
altered physiological properties [8,9]. Moreover we
were interested if the glycosyltransferase metho-
dology can be applied to assemble highly altered
oligosaccharide structures predictably.
Oligosaccharides and peptides serve as receptor
determinants in numerous biological reactions and
infectious processes. Consequently, there have been
a number of attempts recently to combine the fea-
tures of both classes of compounds. Sugar amides
(e.g. uronamides) have been designed [1,2] and
integrated into synthetic concepts to serve as oli-
gosaccharide [3±5] or peptide mimetics [6]. These
`peptidosaccharides' or `saccharopeptides' have
also been considered replacements of natural oli-
gonucleotide backbones [7] in the anti-sense ®eld.
Our interest in this quite unusual hybrid-class of
compounds resulted from the search for analogues
of the natural sialyl-Lewisa (SLea)- and sialyl-
Lewisx (SLex)-tetrasaccharides (Scheme 1) with
2. Results and discussion
Enhanced binding of SLea and SLex can e.g. be
achieved by the introduction of hydrophobic
groups [10] or replacements of the N-acetyl group
of the glucosamine moiety with aromatic or het-
eroaromatic amides [9]. There has also been an
intensive search for structurally simpler SLea and
SLex mimetics where one or two of the mono-
saccharide units (Scheme 1) are replaced by various
surrogates [11±13].
The replacement of the N-acetyl group of the N-
acetylglucosamine unit by uronic acids, as descri-
bed in this paper, represents a novel class of SLea
* Corresponding author;
e-mail: reinhold.oehrlein@pharma.novartis.com
0008-6215/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII S0008-6215(98)00229-8

È
G. Baisch, R. Ohrlein/Carbohydrate Research 312 (1998) 61±72
62
Scheme 1. Sialyl-Lewisa- and sialyl-Lewisx-tetrasaccharides (Lem=(CH2)8COOMe).
mimetics. The additional sugar moiety is thought
to a€ect the selectin-ligand interaction [14].
The pathway for the preparation of the SLea-
saccharopeptides is outlined in Scheme 2 and
Scheme 3.
donor substrates regio- and stereospeci®cally onto
a hydroxyl group of a growing oligosaccharide
chain in vivo and in vitro. So, the various sacchar-
opeptides 8 are incubated ®rst with recombinant ꢀ-
2,3-sialyltransferase (ꢀ-2,3-SiaT) [23] and CMP-
Neu5Ac and then with fucosyltransferase III
(FucT III) [24] and GDP-Fuc (Scheme 3 and
Table 1).
Non-commercially available methyl hexopyr-
anosiduronic acids 2 are obtained from their
corresponding ꢀ- or ꢁ-glycosides 1 via TEMPO-
catalyzed oxidation of the CH2OH group in water
containing sodium hypochlorite (Scheme 2) [15,16].
The methyl ꢀ- or ꢁ-glycopyranosiduronic acids 2
are then coupled to the per-O-acetylated aminosu-
gar 7 employing standard peptide coupling proto-
cols [17,18] to give the desired saccharopeptides 8
after a ®nal sodium methanolate treatment in order
to remove the acetyl protection. We did not
improve these couplings as sucient material has
been produced in all cases for the ensuing enzy-
matic glycosylations (Scheme 3).
The amine 7 is obtained from the known
allyloxycarbonyl sugar 3 [19]. Compound 3 is
4,6-O-benzylidenated to give derivative 4 which is
galactosylated with commercial galactosyl bromide
5, promoted by mercury cyanide [20] to give dis-
accharide 6. Subsequent protecting group manip-
ulations according to the protocols of the parent
N-acetyl congener followed by a Pdo-catalyzed
deallylation [21] give the free amine 7.
Surprisingly, the incubations of all the described
non-natural saccharopeptides 8 with SiaT give oli-
1
gosaccharides whose H and 13C NMR spectra
show the required signals for an additional N-
acetylneuraminic acid, e.g, a singulet (3 H) at
about 2.00 ppm for the additional N-acetyl group
and a doublet of doublets (1 H) at about 2.75 ppm
for the equatorial H-3, respectively. The attach-
ment of Neu5Ac is further con®rmed by signals in
the 13C NMR spectrum at about 41.0 ppm (C-3)
and 102.0 ppm (C-2). Extensive NMR investiga-
tions (COSY, TOCSY, HMBC, and HSQC) of the
starting compounds 8 and the sialylated sacchar-
ides 9 con®rmed that the Neu5Ac residue is
attached to the HO-3 group of the terminal Gal
unit in an ꢀ-mode. A down®eld shift of the C-3
signals of Gal in 8 from about 71 ppm to about
77 ppm in 9 is signi®cant in all cases [25] (Table 2)
and veri®es the site of enzymatic sialylation. This is
corroborated by a down®eld shift of about 0.7 ppm
(dd) of the corresponding Gal H-3 signal. No sialyla-
tion of the glycuronic acid part is observed, although
compounds 8a and 8b bear d-GalA residues.
The most convenient way to accomplish further
glycosylations toward the desired SLea-structure
without any tedious protecting group manipula-
tions on the `trisaccharide' 8 is to apply glycosyl-
Finally, the sialylated sugars 9 are incubated
with GDP-Fuc [26] and recombinant FucT III [24].
NMR data of the isolated materials indicate the
transferases [22]. These enzymes transfer
monosaccharide unit from nucleotide-activated
a
1
presence of a Fuc moiety. All H NMR spectra
show additional doublets at about 1.2 ppm (3 H), a
quartet at about 4.9 ppm, and a doublet at 5.1 ppm
(Jꢀ 4.5 Hz, 1 H). These signals are characteristic
for H-6, H-5 and H-1, respectively, of an ꢀ-linked
Fuc unit. This is corroborated by additional 13C
signals in the 13C NMR spectra at about 16.6 ppm
and 99.5 ppm, characteristic for C-6 and C-1 of an
Scheme 2. (1) NaOCl, TEMPO, NaBr, H2O, rt, 50±74%.

È
G. Baisch, R. Ohrlein/Carbohydrate Research 312 (1998) 61±72
63
Scheme 3. (1) THF, PhCH(OMe)2, CSA, 95%; (2) toluene-nitromethane, 4 A MS, Hg(CN)2, 65%; (3) DCM, 90% -TFA, 100%;
(4) Ac2O, pyr, 76%; (5) THF, diethyl malonate, Pd(PPh3)4, 89%; (6) DMF, HBPyU, TEA, glycuronic acids 2; (7) MeOH, MeONa;
(8) 2,3-SiaT, CMP-Neu5Ac; (9) FucT III, GDP-Fuc; yields steps (6)±(9) see Table 1.
ꢀ-linked Fuc moiety. HMBC spectra and addi-
tional ROESY data of the saccharopeptides 10
con®rm the attachment of Fuc to O-4 of the N-
acylglucosamine unit. Also in this case no fucosy-
lation of the glycuronic acid part is observed and
none of the compounds seems to inhibit the trans-
ferase as even acceptors are glycosylated in high
yield, which bear a l-GalA unit (9d and 9e) closely
resembling a fucose moiety.
N-acetylneuraminic acid is exclusively transferred
onto O-3 of the terminal Gal residue in an a-mode
like in the parent N-acetyl compound. Also fuco-
sylations take place in the expected manner at O-4
of the GlcN moiety in an ꢀ-mode, although this
sugar part carries the non-natural bulky urona-
mides close to the OH group which has to be
approached closely by the enzyme in order to
attach Fuc correctly.
These ®ndings and previous ones show that the
scope of the transferase methodology [27] has not
been fully exploited yet. The pronounced ease of
the synthetic use of these catalysts and their high
regio- and stereochemical ®delity make the glyco-
syltransferases a valuable tool for the carbohydrate
chemist. Thus naturally occurring oligosaccharides
and a wealth of non-natural congeners can be pre-
pared eciently reducing the cumbersome manipula-
tion of protecting groups, which is usually necessary
in the classical chemical oligosaccharide synthesis, to
a minimum. This rapid and unambiguous access to
large numbers of carbohydrates makes available
3. Conclusion
In addition to former ®ndings [19,23,27] these
investigations underscoreÐthe previously unex-
pectedÐhigh tolerance of recombinant SiaT and
FucT III with respect to their acceptor substrates.
Both enzymes accept a multitude of aliphatic and
aromatic replacements of the N-acetyl group of
their natural N-acetyl acceptor substrate [9,23] in
vitro. As is shown here, even highly polar and
bulky uronamides are tolerated at this position.

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G. Baisch, R. Ohrlein/Carbohydrate Research 312 (1998) 61±72
64
Table 1
Yields of enzymatic glycosylations; *coupled to the fully de-O-
acetylated amino sugar with HBTU (see Experimental)
TLC was performed on silica gel 60F254 glass
sheets (Merck), and sugars were stained with p-
anisaldehyde/H2SO4
(Pernod-mixture).
Flash
Acid 2a±e
Structure of Couple % Neu5Ac % Fuc %
8a±e
chromatography was carried out with silica gel 60,
0.040±0.063 mm (Merck).
coupled acid
(mg) 9a±e (mg) 10a±e
a ꢁ-d-GlcA
16
70 (24.6) 82 (18.8)
Solvents and chemicals used were of commercial
quality unless otherwise stated. Commercial l-Gal
(Fluka) was used. Bovine serum albumine (BSA)
was obtained from Boehringer (no. 28031). Calf
intestine alkaline phosphatase (E.C. 3.1.3.1)
(CIAP) was purchased from Boehringer (no.
108146, 7500 U/498 ꢂL). Recombinant ꢀ-2,3-SiaT
(ꢀ100 U/L) [23,28] (EMBL accession no. M97754)
and recombinant FucT III (ꢀ11.8 U/L) [24,29]
(EMBL accession no. X53578) were obtained from
transfected insect cells. FABMS spectra were
recorded on a FO25FAB instrument with Cs or Xe
as bombarding gas, and with thioglycerol as matrix.
General procedure A: Preparation of methyl
glycopyranosiduronic acids [16].ÐMethyl ꢀ- or ꢁ-
glycoside 1 (2 g, 10.3 mmol), TEMPO (2,2,6,6-tetra-
methyl-1-piperidinyl oxy radical; 16 mg, 0.1 mmol)
and NaBr (0.53 g, 5.1 mmol) were dissolved at
room temperature in water (30 mL) and the solu-
tion cooled to 0 ꢁC. To this mixture aq 5% NaOCl
(15 mL) was added dropwise with vigorous stirring.
During the addition the pH is kept at 10±11 with
0.5 M NaOH using a pH-dosimeter. When TLC
(6:4:1 CH2Cl2±MeOH±H2O) showed the complete
consumption of the starting sugar (usually 2±7 h),
MeOH (30 mL) was added. The resulting pre-
cipitates were ®ltered o€ and washed with MeOH.
The combined ®ltrates were concentrated, and the
residue chromatographed on silica gel (elute ®rst
with 3:1:0.2 CH2Cl2±MeOH±HOAc to remove
side-products and then with 6:4:1 CH2Cl2±
MeOH±H2O) to give the methyl pyranosiduronic
acids 2.
b ꢁ-d-GalA
c ꢀ-d-GalA
36
36
90 (35.0) 63 (18.8)
93 (29.6) 79 (20.1)
d ꢁ-l-GalA
e ꢀ-l-GalA
15
4*
63 (24.8) 56 (12.2)
74 (10.4)
75 (8.6)
this class of compounds for high-through-put
screens for various biological applications.
4. Experimental
1H NMR and 13C NMR spectra were recorded
on a Bruker AC 250 and/or a Varian Unity 500
spectrometer with multi-probe heads. COSY,
TOCSY, HMBC and HSQC experiments were
1
performed using the manufacturers' softwares. H
NMR shifts were measured in CD3OD and refer-
enced to internal D2O (4.80 ppm) and 13C NMR
shifts were referenced to CD3OD (49.00 ppm)
unless otherwise stated. 1H NMR and 13C NMR
shift assignments are tentative; shifts marked with
an asterik (*) may be interchanged.
General procedure B: Coupling of methyl
glycopyranosiduronic acids (2) to glucosamine
residues (7).ÐAccording to general peptide cou-
pling protocols [18] amine 7 (0.33 mmol), methyl
Table 2
Selected 13C NMR data of saccharopeptides 10
entry
Fuc
Neu5Ac
Gal
GlcN
C-1
MeO
Uronic acid
C-1
C-6
C-2
C-3
C-1
C-3
C-2
OCH3
C-1
C-6
10a
10b
10c
10d
10e
99.6
99.6
99.5
99.5
99.5
16.6
16.6
16.6
16.6
16.6
102.1
101.8
101.4
102.6
101.8
41.4
41.7
41.4
40.6
40.7
103.9
104.2
103.9
103.2
103.2
77.6
77.7
77.5
77.2
77.3
102.4
102.2
101.7
102.6
102.6
56.9
57.7
58.3
56.9
56.8
57.8
58.1
56.6
57.8
56.3
105.4
105.8
102.1
106.0
101.9
172.2
171.6
172.0
171.7
172.3

È
G. Baisch, R. Ohrlein/Carbohydrate Research 312 (1998) 61±72
65
glycopyranosiduronic acid 2 (0.36 mmol) and
HBPyU ([O-(benzotriazol-1-yl)-N,N,N0,N0-bis(tetra-
methylen)-uronium-hexa¯uourophosphate 0.36 mmol)
were dissolved at room temperature in dry DMF
(5 mL). The clear solution was treated with Et3N
(0.36 mmol) and stirred at room temperature for
2±4 days (even after this period the starting amine
was not completely consumed). The mixture was
then concentrated to dryness and the residue
chromatographed over silica gel (10:0.5 CH2Cl2±
MeOH) to give an acetylated saccharopeptide
intermediate. This compound was dissolved in dry
MeOH (4 mL), containing 0.1% NaOMe, and
stirred at room temperature for 1±2 h until TLC
(10:3 CH2Cl2±MeOH) showed complete consump-
tion of the starting material. Evaporation of the
solvent left a solid which was puri®ed over silica
gel (10:3 CH2Cl2±MeOH).
MeOH was concentrated and the resulting residue
was chromatographed on silica gel (10:4:0.8
CH2Cl2±MeOH±H2O) to give the pure saccharides
10 as white powders after lyophilization from water.
Methyl b-d-glucopyranosiduronic acid (2a).Ð
This was available from Sigma as the sodium salt.
Methyl b-d-galactopyranosiduronic acid (2b).Ð
According to general procedure A, methyl ꢁ-d-
galactopyranoside 1b (2 g, 10 mmol) was oxidized
to give 2b (1.04 g, 50%). 1H NMR (CD3OD,
250 MHz): ꢃ 3.40±3.59 (m, 5 H, H-2,3 and OCH3),
3.89 (bs, 1 H, H-5), 4.06±4.18 (m, 2 H, H-1,4); 13C
NMR (CD3OD, 62.9 MHz): ꢃ 58.22 (OCH3), 71.74
(C-4), 71.89 (C-2), 74.51 (C-3), 77.02 (C-5), 105.24
(C-1), 175.72 (C ˆ O). MS Calcd for C7H12O7: 208;
found [M H]- 207.
Methyl a-d-galactopyranosiduronic acid (2c).Ð
According to general procedure A methyl ꢀ-d-
galactopyranoside 1c (2 g, 10 mmol) was oxidized
to give 2c (1.2 g, 57%). 1H NMR (CD3OD,
250 MHz): ꢃ 3.38 (s, 3 H, OCH3), 3.74±3.81 (m, 2
H, H-2,3), 4.11±4.20 (m, 2 H, H-4,5); 13C NMR
(CD3OD, 62.9 MHz): ꢃ 56.21 (OCH3), 69.34 (C-2),
71.11 (C-3), 72.23 (C-4), 72.91 (C-5), 101.08 (C-1),
176.28 (C ˆ O). MS Calcd for C7H12O7: 208; found
[M H]- 207.
General procedure C: Enzymatic sialylation of
type I saccharopeptides (8).ÐFollowing standard
sialylation protocols [23,30] compound
8
(32.2 ꢂmol) and CMP-Neu5Ac (44.6 ꢂmol) [31]
were dissolved in a mixture of 2 mL 0.05 M sodium
cacodylate bu€er (pH 6.5), 2 mL 0.06 M MnCl2
and 1.3 mL deionized water containing 2.1 mg
BSA. The mixture was diluted with Me2SO
ꢁ
(0.5 mL) and incubated at 37 C with CIAP (3 ꢂL)
Methyl b-l-galactopyranosiduronic acid (2d).Ð
According to general procedure A methyl ꢁ-l-
galactopyranoside 1d (0.54 g, 2.8 mmol) was oxi-
dized to give 2d (0.43 g, 74%) after a ®nal passage
and recombinant SiaT (200 ꢂL, 1.4 U) [28]. When
TLC (10:4:0.8 CH2Cl2±MeOH±H2O) showed the
disappearance of the starting acceptorÐusually
overnightÐthe turbid solution was centrifuged and
the supernatant passed over a C-18 reversed phase
column, washed with water and eluted with
MeOH. The MeOH was concentrated and the
resulting residue was chromatographed over silica
gel (10:4:0.8 CH2Cl2±MeOH±H2O) to give the pure
sialylated sugars 9 as white powders after lyophili-
zation from water-dioxane.
1
over a weakly acid Amberlite resin (H+-form). H
NMR (CD3OD, 400 MHz): ꢃ 3.52 (dd, 1 H, J1,2
7.2, J2,3 9.6 Hz, H-2), 3.60 (s, 3 H, OCH3), 3.71
(dd, 1 H, J3,4 4.8, J2,3 9.6 Hz, H-3), 4.06 (d, 1 H,
J4,5 1.2 Hz, H-5), 4.22 (dd, 1 H, J4,5 1.2, J3,4 4.8 Hz,
H-4), 4.31 (d, 1 H, J1,2 7.2 Hz, H-1); 13C NMR
(CD3OD, 100.6 MHz): ꢃ 58.50 (OCH3), 71.63 (C-
4), 71.85 (C-2), 74.30 (C-3), 76.55 (C-5), 105.64 (C-
1), 175.78 (C ˆ O). MS calcd for C7H12O7: 208;
Found [M H]- 207.
General procedure D: Enzymatic fucosylation of
saccharides (9).ÐFollowing general fucosylation
protocols [24,30] compound 9 (22.2 ꢂmol), GDP-
Fuc (34.2 ꢂmol) [26] and BSA (2 mg) were added
to a mixture of 450 ꢂL 0.25 M sodium cacodylate
bu€er (pH 6.5) and 150 ꢂL 0.25 M MnCl2. The
mixture was diluted with deionized water (600 ꢂL)
Methyl a-l-galactopyranosiduronic acid (2e).Ð
According to general procedure A methyl ꢀ-l-
galactopyranside 1e (0.97 g, 5 mmol) was oxidized
to give 2e (0.65 g, 64%) after a ®nal passage over a
1
weakly acid Amberlite resin (H+-form). H NMR
ꢁ
and incubated at 37 C with CIAP (3 ꢂL) and
(CD3OD, 400 MHz): ꢃ 3.55 (s, 3 H, OCH3), 3.95±
4.04 (m, 2 H, H-2,3), 4.39±4.41 (m, 2 H, H-4,5),
5.06 (d, 1 H, J1,2 4.1 Hz, H-1); 13C NMR (CD3OD,
100.6 MHz): ꢃ 56.21 (OCH3), 69.44 (C-2), 71.21 (C-
3), 72.31 (C-4), 72.99 (C-5), 101.21 (C-1), 176.22
(C ˆ O). MS Calcd for C7H12O7: 208; Found
[M H]- 207.
recombinant FucT III (100 ꢂL, 600 mU) [24,29] for
24±36 h until TLC (10:4:0.8 CH2Cl2±MeOH±H2O)
showed the consumption of the starting acceptor 9.
The mixture was then centrifuged and the super-
natant passed over a C-18 reversed phase column,
washed with water and eluted with MeOH. The

È
G. Baisch, R. Ohrlein/Carbohydrate Research 312 (1998) 61±72
66
0
8-Methoxycarbonyloctyl 2-N-allyloxycarbonyl- 6.1, 9.1 Hz, -CHHO-), 3.98 (dd, 1 H, J5 ,6a 6.6,
0
0
0
0
0
4,6-O-benzylidene-2-deoxy-b-d-glucopyranoside
(4).Ð To dry THF (100 mL) were added succes-
J6a ,6b 11.4 Hz, H-6a0), 4.25 (dd, 1 H, J5 ,6b 6.1,
J6a ,6b 11.4 Hz, H-6b0), 4.39 (m, 1 H, H-5), 4.50 (m,
2 H, allylic CH2), 4.59 (d, 1 H, J1,2 7.3 Hz, H-10),
0
0
sively monosaccharide derivative
3
(9.7 g,
0 0 0 0
22.4 mmol) [19], benzaldehyde dimethylacetal
(6 mL, 40 mmol; Fluka) and ‹ camphorsulfonic
acid (0.25 g; Fluka). This mixture was heated to
4.89 (dd, 1 H, J3 ,4 3.6, J2 ,3 10.9 Hz, H-30), 5.05
(m, 1 H, H-50), 5.13 (dd, 1 H, J1 ,2 7.3, J2 ,3
10.9 Hz, H-20), 5.19 (dq, 1 H, J 1.2, 11.5 Hz,
0
0
0
0
ꢁ
0
0
0
0
50 C and stirred overnight until complete con-
ˆ CHH), 5.22 (dd, 1 H, J4 ,3 0.6, J4 ,5 3.6 Hz, H-
40), 5.27 (m, 1 H, ˆ CHH), 5.47 (s, 1 H, -CH(O)2-),
5.86 (m, 1 H, ˆ CH-), 7.30 (m, 3 H, phenyl), 7.40
(m, 2 H, phenyl); 13C NMR (CDCl3, 62.90 MHz): ꢃ
20.52 (2 Ac), 20.62 (2 Ac), 24.80, 25.65, 28.93,
29.00 (2 C), 29.38, and 33.99 (all aglycon -CH2-),
51.44 (OMe), 58.08 (C-2), 60.70, 65.60, 65.87,
66.73, 68.70, 69.06, 70.27, 70.40, 70.97, 76.49, and
78.63 (C-3,4,5,6,20,30,40,50,60, allylic-C, and -CH2O-),
80.18 (-CH(O)2-), 101.01 (C-10), 101.33 (C-1),
117.88 ( ˆ CH2), 126.03 (2 C, phenyl), 128.15 (2 C,
phenyl), 129.14 (phenyl), 137.04 ( ˆ CH1-), 155.43,
169.40, 170.06, 170.11, 170.24, and 174.42 (each
C ˆ O).
sumption of the starting sugar. The mixture was
then cooled to room temperature, treated with
Et3N (0.5 mL) and concentrated to dryness. The
resulting residue was chromatographed on silica
gel (20:1 CH2Cl2±MeOH) to give 4 (11 g, 95%). 1H
NMR (CDCl3, 400.13 MHz): ꢃ 1.23 and 1.51 (m, 8
H and 4 H, -(CH2)6-), 2.23 (t, 2 H, J 7.6 Hz, CH2-
C ˆ O), 3.25±3.50 (m, 6 H, H-1,2,3,6a,6b and -
CHHO-), 3.60 (s, 3 H, OCH3), 3.70 (t, 1 H, J3,4
9.7 Hz, H-4), 3.78 (dt, 1 H, J 4.8, 9.7 Hz, -CHHO-),
4.25 (broad dd, 1 H, J5,6a 4.8, J5,6b 10.9 Hz, H-5),
4.50 (m, 2 H, allylic CH2), 5.12 (m, 1 H, ˆ CH2),
5.23 (dq, J 1.2, 16.3 Hz, 1 H, ˆ CHH), 5.45 (s, 1 H,
-CH(O)2-), 5.84 (m, 1 H, ˆ CH-), 7.30 (m, 3 H,
phenyl), 7.42 (m, 2 H, phenyl); 13C NMR (CDCl3,
100.61 MHz): ꢃ 24.77, 25.63, 28.91, 29.00, 29.35,
and 34.16 (all aglycon -CH2-), 51.46 (OMe), 58.60
(C-2), 65.73, 66.04, 68.59, 70.21, 70.69, and 72.27
(C-3,4,5,6, -CH2-, and allylic CH2), 81.49 (-
CH(O)2-), 101.75 (C-1), 117.60 ( ˆ CH2), 126.21 (2
C, phenyl), 128.23 (2 C, phenyl), 129.17 (phenyl),
132.46 ( ˆ CH1-), 159.16 and 174.53 (each C ˆ O).
8-Methoxycarbonyloctyl (2,3,4,6-tetra-O-acetyl-b-
d-galactopyranosyl)-(1!3)-2-N-allyloxycarbonyl-
4,6-O-benzylidene-2-deoxy-b-d-glucopyranoside
8-Methoxycarbonyloctyl (2,3,4,6-tetra-O-acetyl-
b-d-galactopyranosyl)-(1!3)-4,6-di-O-acetyl-2-
amino-2-deoxy-b-d-glucopyranoside (7).ÐTo a
solution of disaccharide 6 (9.1 g, 10.7 mmol) in
CH2Cl2 (100 mL) at room temperature were added
aq 90% CF3CO2H (5 mL). After 6 h the mixture
was neutralized with satured NaHCO3, diluted
with EtOAc and successively extracted with water
and brine. The organic phase was dried (Na2SO4)
and concentrated to give a syrup which is dissolved
in pyridine (7 mL) and Ac2O (3.5 mL). The mixture
was stirred overnight at room temperature, then
diluted with EtOAc and successively extracted with
4 M HCl, water, and satured NaHCO3. Evapora-
tion of the organic solvent left a yellow syrup
which was chromatographed over silica gel (2:1
petrolether±EtOAc) to yield the N-allylox-
ycarbonyl intermediate (6.9 g, 76%). 1H NMR
(CDCl3, 400.13 MHz): ꢃ 1.22 and 1.51 (m, 8 H and
4 H, -(CH2)6-), 1.93, 1.98, 2.00, 2.01, 2.09, and
2.17 (6 s, each 3 H, 6 Ac), 2.24 (t, 2 H, J 7.6 Hz,
-CH2C ˆ O); 13C NMR (CDCl3, 62.90 MHz): ꢃ
20.50, 20.60 (3 C), 20.67, and 20.79 (each Ac),
24.79, 25.63, 28.91, 28.97, 29.01, 29.30, and 33.99
(aglycon -CH2-), 51.43 (OMe), 58.02 (C-2), 60.98,
62.44, 65.59, 66.76 (2 C), 69.00, 69.15, 70.00,
70.42, 70.95, and 71.65 (C-3,4,5,6,20,30,60,50,60 and
allyl -CH2-), 100.55 (C-10), 101.02 (C-1), 117.91
(C ˆ CH2), 137.50 (C ˆ CH1-), 155.55, 169.15,
169.27, 170.11, 170.19, 170.32, 170.75 and 174.29
(each C ˆ O).
(6).ÐTo 1:1 dry toluene-nitromethane (260 mL),
containing ®nely ground 4 A molecular sieves (ca.
5 g), were added compound 4 (8.7 g, 17 mmol) and
mercury cyanide (5.5 g, 22 mmol). The mixture was
stirred for 30 min at room temperature. Then a
solution of peracetylated galactose 5 (10.3 g,
25 mmol) in 1:1 dry toluene±nitromethane (35 mL)
was dropped to this mixture with vigourous stir-
ꢁ
ring, and the mixture was ®nally heated at 50 C
for 18 h. After complete consumption of com-
pound 4 the mixture was carefully ®ltered over
Celite and concentrated. The residue was chroma-
tographed over silica gel (2:1 hexane±EtOAc) to
yield
6
(9.1 g, 64%). 1H NMR (CDCl3,
400.13 MHz): ꢃ 1.22 and 1.51 (m, 8 H and 4 H,
(CH2)6-), 1.88, 1.89, 1.91, and 2.05 (4 s, each 3 H, 4
Ac), 2.23 (t, 2 H, J 7.6 Hz, CH2-C ˆ O), 3.06 (bs, 1
H, NH), 3.41, 3.59, and 3.71 (m, 2 H, 6 H, 2 H, H-
1,2,3,4,6a,6b, OMe, and -CHHO-), 3.78 (dt, 1 H, J

È
G. Baisch, R. Ohrlein/Carbohydrate Research 312 (1998) 61±72
67
To a solution of this intermediate (4 g, 4.7 mmol)
in dry THF (60 mL) in an argon atmosphere at
room temperature were added malonic acid diethyl
ester (5.6 mL) and tetrakis-(triphenyl phosphino)-
palladium (0.4 g, 0.3 mmol; Fluka). After 1 h the
solvent was removed and the residue chromato-
graphed on silica gel (2:1 petrolether±EtOAc) to
-CH2-), 1.64 (m, 4 H, aglycon -CH2-), 2.33 (t, 2 H,
J 7.5 Hz, -CH2C ˆ O aglycon), 2.80 (bt, 1 H, J1,2
7.4 Hz, H-2), 3.35 (m, 1 H, H-5), 3.44±3.96 (m, 15
H, -CH2O- aglycon, OMe, and H-3,4,6a,6b,20,30,
40,50,6a0,6b0), 4.31 (d, 1 H, J1 ,2 8.6 Hz, H-10), 4.43
(d, 1 H, J1,2 8.6 Hz, H-1).
0
0
8-Methoxycarbonyloctyl b-d-galactopyranosyl-
(1!3)-2-deoxy-2-(methyl b-d-glucopyranosyluron-
amide)-b-d-glucopyranoside (8a).ÐAccording to
general procedure B, from the commercial sodium
(methyl ꢁ-d-glucopyranosid)uronate 2a (83 mg,
0.36 mmol) and compound 7 (250 mg, 0.33 mmol)
was obtained the acetylated intermediate (96.9 mg,
31%) which was deacetylated to give the title
1
give the amine 7 (3.1 g, 89%). H NMR (CDCl3,
250.13 MHz): ꢃ 1.33 and 1.60 (m, 8 H and 4 H,
aglycon -CH2-), 1.99 (s, 3 H, Ac), 2.05 (m, 12 H, 4
Ac), 2.13 (s, 3 H, Ac), 2.29 (t, 2 H, J 7.6 Hz, -
CH2C ˆ O aglycon), 2.92 (dd, 1 H, J1,2 7.6, J2,3
8.2 Hz, H-2), 3.46 (dt, 1 H, J 6.9, 10.3 Hz, -CHHO-
aglycon), 3.58 (m, 1 H, H-5), 3.67 (s, 3 H, OMe),
3.89 (m, 2 H, H-3 and -CHHO- aglycon), 4.14 (m,
6 H, H-6a,6b,20,50,6a0,6b0), 4.73 (d, 1 H, J1,2 7.6 Hz,
H-1), 4.99 (m, 2 H, H-10,30); 5.15 (dd, 1 H, J3,4 7.6,
J4,5 11.7 Hz, H-4), 5.35 (m, 1 H, H-40); 13C NMR
(CDCl3, 62.90 MHz): ꢃ 20.50 (Ac), 20.60 (3 Ac),
20.77 (Ac), 20.81 (Ac), 24.82, 25.83, 28.98, 29.09 (2
C), 29.42, and 33.99 (all aglycon-(CH2)6-), 51.40
(OMe), 57.05 (C-2), 60.91, 62.51, 66.74, 68.70,
69.52, 70.16, 70.58, 70.97, and 72.04 (C-
3,4,5,6,20,30,50,60 and -CH2O- aglycon), 83.53 (C-
40), 101.45 (C-10), 103.12 (C-1), 169.03 (C ˆ O),
169.30 (C ˆ O), 170.13 (C ˆ O), 170.29 (2 C ˆ O),
170.75 (C ˆ O), 174.44 (C ˆ O). MS Calcd for
C34H53NO18: 763; Found [M+H]+ 764.
Alternatively, the fully deacetylated amine 7a [8-
methoxycarbonyloctyl ([2-amino-2-deoxy-ꢁ-d-galac-
topyranosyl)-(1!3)-ꢁ-d-glucopyranoside] has been
used in some cases. It was obtained by stirring the
acetylated intermediate of the above reaction, after
removal of the benzylidene group, at room tem-
perature in dry MeOH, containing 0.1% MeONa,
for 1 h. The mixture was neutralized with Dowex
50Â8 (H+-form), ®ltered, and concentrated. The
resulting residue was chromatographed on silica
gel (9:1 CH2Cl2±MeOH) to give the deacetylated
N-allyloxy compound which was fully deprotected
as follows: to the N-protected disaccharide (4.08 g,
6.8 mmol), dissolved in a mixture of dry THF
(160 mL) and dry MeOH (60 mL) were added at
room temperature 1,4-bis(diphenylphosphino)butane
(0.50 g, 1.2 mmol), sodium thiophenolate (1.5 g,
11.3 mmol), and tris(dibenzylideneacetone)-dipalla-
dium-0 adduct (0.3 g, 0.3 mmol; Aldrich). The
mixture was stirred overnight in an argon atmo-
sphere, then concentrated and chromatographed
on silica gel (5:1 CH2Cl2±MeOH) to give the fully
deacetylated amine 7a (2.2 g, 64%). 1H NMR
(CD3OD±CDCl3, 400.13 Hz): ꢃ 1.35 (m, 8 H, aglycon
1
compound (35 mg, 50%). H NMR (Me2SO-d6,
500.13 MHz): GlcN (from H-1 to H-6a,6b): ꢃ 4.12
(d, 1 H, J1,2 ꢀ 7.6 Hz), 3.34, 3.60, 3.50, 3.67, 3.16,
3.33; Gal (from H-1 to H-6a,6b): ꢃ 4.07 (d, 1 H, J1,2
& 7.4 Hz), 2.97, 3.27, 3.56, 3.15, 3.47, 3.68; GlcA
(from H-1 to H-5, OCH3): ꢃ 4.45 (d, 1 H, J1,2
&
8.0 Hz), 3.23, 3.12, 3.30, 3.21, 3.39; aglycon: ꢃ 2.25,
1.48, 1.20, 1.42, 3.44, 3.69, 3.60; 13C NMR
(Me2SO-d6): GlcN (from C-1 to C-6): ꢃ 103.6, 55.9,
83.7, 75.0, 75.5, 60.7; Gal (from C-1 to C-6): ꢃ
104.0, 72.9, 70.4, 68.1, 75.9, 60.4; GlcA (from C-1
to C-6, OCH3): ꢃ 100.3, 72.9, 76.4, 71.5, 73.0, 169.2,
56.0; aglycon: ꢃ 173.4, 33.1, 24.2, 28.3, 25.0, 28.8,
68.3, 53.6. MS calcd for C29H51NO18: 701; found
[M+Na]+ 724. Anal. Calcd for C29H51NO18: C,
46.64; H, 7.33. Found: C, 46.53; H, 7.48.
8-Methoxycarbonyloctyl
b-d-galactopyranosyl-
(1!3)-2-deoxy-2-(methyl b-d-galactopyranosylur-
onamide)-b-d-glucopyranoside (8b).ÐAccording to
general procedure B, from methyl ꢁ-d-galactopyr-
anosiduronate 2b (75 mg, 0.36 mmol) and com-
pound 7 (250 mg, 0.33 mmol) was obtained the
acetylated intermediate (230 mg, 74%) which was
deacetylated to give the title compound (82.7 mg,
1
49%). H NMR (Me2SO-d6, 500.13 MHz): GlcN
(from H-1 to H-6a,6b): ꢃ 4.77 (d, 1 H, J1,2
&
7.6 Hz), 3.41, 3.76, 3.37, 3.14, 3.67; Gal (from H-1
to H-6a,6b): ꢃ 4.14 (d, 1 H, J1,2 & 7.4 Hz), 3.25,
3.14, 3.56, 3.24, 3.46; GalA (from H-1 to H-5,
OCH3): ꢃ 4.05 (d, 1 H, J1,2 & 8.0 Hz), 3.30, 3.30,
3.91, 3.85, 3.45; aglycon: ꢃ 2.26, 1.48, 1.21, 1.43,
3.37, 3.68, 3.55; 13C NMR (Me2SO-d6): GlcN
(from C-1 to C-6): ꢃ 99.6, 54.8, 83.4, 75.4, 76.6,
60.7; Gal (from C-1 to C-6): ꢃ 103.8, 73.0, 68.7,
68.1, 70.5, 60.5; GalA (from C-1 to C-6, OCH3): ꢃ
104.2, 69.9, 73.0, 68.9, 74.7, 168.5, 56.0; aglycon: ꢃ
173.3, 33.1, 24.3, 28.4, 25.1, 28.9, 68.4, 51.0. MS
calcd for C29H51NO18: 701; found [M+Na]+ 724.

È
G. Baisch, R. Ohrlein/Carbohydrate Research 312 (1998) 61±72
68
Anal. Calcd for C29H51NO18: C, 46.64; H, 7.33.
Found: C, 46.71; H, 7.58.
onamide)-b-d-glucopyranoside (8e).ÐTo a solu-
tion of amine 7a (0.2 g, 0.39 mmol) in DMF (5 mL)
at room temperature was added sugar acid 2e (0.1 g,
0.47mmol), O-(1H-benzotriazole-1-yl)-N,N,N0,N0-
tetramethyluronium hexa¯uorophosphate (0.18 g,
0.47 mmol; Fluka), and Et3N (65 ꢂL). This hetero-
genous mixture was stirred for 5 days then worked
up according general procedure B to give com-
8-Methoxycarbonyloctyl
b-d-galactopyranosyl-
(1!3)-2-deoxy-2-(methyl a-d-galactopyranosylur-
onamide)-b-d-glucopyranoside (8c).ÐAccording to
general procedure B, from methyl ꢀ-d-galactopyr-
anosiduronate 2c (75 mg, 0.36 mmol) and com-
pound 7 (250 mg, 0.33 mmol) was obtained the
acetylated intermediate (131 mg, 42%) which was
deacetylated to give the title compound (86 mg,
1
pound 8e (10.8 mg, 4%). H NMR (Me2SO-d6,
400.13 MHz): GlcN (from H-1 to H-6a,6b): ꢃ 4.52
(d, 1 H, J1,2 & 7.6 Hz), 4.00, 3.80, 3.50, 3.40, 3.78,
3.91; Gal (from H-1 to H-6a,6b): ꢃ 4.45 (d, 1 H, J1,2
& 7.4 Hz), 3.52, 3.50, 3.82, 3.65, 3.71, 3.74; GalA
1
86%). H NMR (Me2SO-d6, 500.13 MHz): GlcN
(from H-1 to H-6a,6b): ꢃ 4.76 (d, 1 H, J1,2
&
7.6 Hz), 3.41, 3.69, 3.38, 3.42, 3.65; Gal (from H-1
to H-6a,6b): ꢃ 4.11 (d, 1 H, J1,2 & 7.4 Hz), 3.24,
3.24, 3.12, 3.11, 3.44, 3.48; GalA (from H-1 to H-5,
OCH3): 4.67 (d, 1 H, J1,2 & 4.9 Hz), 3.55, 3.52,
3.99, 3.97, 3.28; aglycon: ꢃ 2.25, 1.47, 1.20, 1.41,
3.35, 3.67, 3.55; 13C NMR (Me2SO-d6): GlcN
(from C-1 to C-6): ꢃ 99.5, 54.7, 84.2, 75.5, 79.9,
60.7; Gal (from C-1 to C-6): 103.8, 72.8*, 70.7*,
68.7, 76.4, 60.4; GalA (from C-1 to C-6, OCH3): ꢃ
104.2, 69.9, 73.0, 68.9, 74.7, 168.5, 56.0; aglycon: ꢃ
173.3, 33.1, 24.3, 28.4, 25.1, 28.9, 68.4, 51.0. MS
calcd for C29H51NO18: 701; found [M H]- 700.
Anal. Calcd for C29H51NO18: C, 46.64; H, 7.33.
Found: C, 46.67; H, 7.56.
(from H-1 to H-5, OCH3): 4.93 (d, 1 H, J1,2
&
4.0 Hz), 3.86, 3.83, 4.30, 4.22, 3.45; aglycon: ꢃ 2.38,
1.60, 1.31, 1.53, 3.49, 3.89, 3.69; 13C NMR
(Me2SO-d6, 100.61 MHz): GlcN (from C-1 to C-6):
ꢃ 102.3, 55.4, 83.9, 73.8, 77.0, 62.3; Gal (from C-1
to C-6): ꢃ 104.7, 72.5, 70.5, 70.1, 76.9, 62.5; GalA
(from C-1 to C-6, OCH3): ꢃ 101.4, 69.4, 71.1, 71.3,
72.8, C-6 not resolved, 56.4; aglycon: ꢃ C ˆ O not
resolved, 34.8, 25.8, 30.0, 26.8, 30.4, 70.0, 52.5. MS
calcd for C29H51NO18: 701; found [M+Na]+ 724.
Anal. Calcd for C29H51NO18: C, 46.64; H, 7.33.
Found: C, 46.73; H, 7.58.
8-Methoxycarbonyloctyl 5-N-acetyl-a-neuraminyl-
(2!3)-b-d-galactopyranosyl-(1!3)-2-deoxy-2-
(methyl b-d-gluco pyranosyluronamide)-b-d-gluco-
pyranoside (9a).ÐAccording to general procedure
C, compound 8a (25 mg, 35.6 ꢂmol) was incubated
with CMP-Neu5Ac (33.1 mg, 50.2 ꢂmol) for 1 day
8-Methoxycarbonyloctyl
b-d-galactopyranosyl-
(1!3)-2-deoxy-2-(methyl b-l-galactopyranosylur-
onamide)-b-d-glucopyranoside (8d).ÐAccording to
general procedure B, from methyl ꢁ-l-galactopyr-
anosiduronate 2d (75 mg, 0.36 mmol) and com-
pound 7 (250 mg, 0.33 mmol) was obtained the
acetylated intermediate (104 mg, 33%) which was
deacetylated to give the title compound (34.1 mg,
1
to give the title compound (24.6 mg, 70%). H
NMR (CD3OD, 500.13 MHz): GlcN (from H-1 to
H-6a,6b): 4.54 (d, 1 H, J1,2 & 7.6 Hz), 3.85, 3.81,
3.44, 3.32, 3.74, 3.89; Gal (from H-1 to H-6a,6b): ꢃ
4.44 (d, 1 H, J1,2 & 7.4 Hz), 3.58, 3.98, 3.93, 3.56,
3.64, 3.71; Neu5Ac (from H-3ax,3eq to H-9a,9b,
HNCOCH3): ꢃ 1.83 (t, 1 H, J3ax,4 & 12.1 Hz), 2.77
(dd, 1 H, J3eq,4 & 4.2, J3ax,3eq & 12.1 Hz), 3.74, 3.71,
3.54, 3.51, 3.85, 3.63, 3.84, 2.02; GlcA (from H-1 to
H-5, OCH3): ꢃ 4.28 (d, 1 H, J1,2 & 8.0 Hz), 3.28,
3.46, 3.57, 3.73, 3.55; aglycon: ꢃ 2.30, 1.58, 1.29,
1.31, 1.53, 3.47, 3.86, 3.64; 13C NMR (CD3OD,
100.61 MHz): GlcN (from C-1 to C-6): 102.2, 55.8,
83.9, 70.6, 77.5, 62.7; Gal (from C-1 to C-6): ꢃ 104.6,
70.7, 77.1, 69.5, 76.8, 62.7; Neu5Ac (from C-1 to C-
9, HNCOCH3): ꢃ C-1 not resolved, C-2 not resolved,
41.1, 69.4, 53.9, 74.8, 70.1, 72.8, 64.4, 175.5, 22.5;
GlcA (from C-1 to C-6, OCH3): ꢃ 105.3, 74.3, 77.3,
73.2, 76.8, 172.2, 57.5; aglycon: ꢃ C ˆ O not resolved,
34.6, 25.9, 30.1, 26.8, 30.4, 70.6, 51.9. MS Calcd for
C40H68N2O26: 992; found [M H]- 991.
1
45%). H NMR (Me2SO-d6, 500.13 MHz): GlcN
(from H-1 to H-6a,6b): ꢃ 4.53 (d, 1 H, J1,2
&
7.6 Hz), 3.70, 3.70, 3.16, 3.10, 3.66; Gal (from H-1
to H-6a,6b): ꢃ 4.16 (d, 1 H, J1,2 & 7.4 Hz), 3.27,
3.27, 3.57, 3.39, 3.48; GalA (from H-1 to H-5,
OCH3): ꢃ 4.04 (d, 1 H, J1,2 & 7.4 Hz), 3.31, 3.34,
3.92, 3.81, 3.44; aglycon: ꢃ 2.26, 1.45, 1.19, 1.40,
3.34, 3.65, 3.55; 13C NMR (Me2SO-d6): GlcN
(from C-1 to C-6): ꢃ 100.3, 53.4, 83.8, 69.0, 76.2,
60.7; Gal (from C-1 to C-6): ꢃ 103.5, 72.7*, 70.6*,
68.1, 75.5, 60.5; GalA (from C-1 to C-6, OCH3): ꢃ
104.2, 70.0, 72.6, 69.1, 75.3, C-6 not resolved, 56.1;
aglycon: ꢃ 173.3, 33.1, 24.2, 28.4, 25.0, 28.6, 68.5,
51.0. MS calcd for C29H51NO18: 701; found
[M+Na]+ 724. Anal. Calcd for C29H51NO18: C,
46.64; H, 7.33. Found: C, 46.31; H, 6.99.
8-Methoxycarbonyloctyl b-d-galactopyranosyl-
(1!3)-2-deoxy-2-(methyl a-l-galactopyranosylur-

È
G. Baisch, R. Ohrlein/Carbohydrate Research 312 (1998) 61±72
69
8-Methoxycarbonyloctyl-N-acetyl-a-neuraminyl-
(2!3)-b-d-galacto-pyranosyl-(1!3)-2-deoxy-2-
(methyl b-d-galactopyranosyluronamide)-b-d-glu-
copyranoside (9b).ÐAccording to general proce-
dure C, compound 8b (27.5 mg, 39.2 ꢂmol) was
incubated with CMP-Neu5Ac (34.8 mg, 52.8 ꢂmol)
for one day to give the title compound (35 mg,
90%). 1H NMR (CD3OD, 500.13 MHz): GlcN
GalA (from C-1 to C-6, OCH3): ꢃ 100.6, 69.4, 71.0,
71.6, 72.3, 171.5, 56.4; aglycon: ꢃ 176.3, 34.6, 25.9,
30.2, 26.9, 30.4, 70.5, 51.5. MS Calcd for
C40H68N2O26: 992; Found [M H]- 991.
8-Methoxycarbonyloctyl 5-N-acetyl-a-neuraminyl-
(2!3)-b-d-galactopyranosyl-(1!3)-2-deoxy-2-
(methyl b-l-galactopyranosyluronamide)-b-d-gluco-
pyranoside (9d).ÐAccording to general procedure
C, compound 8d (28 mg, 39.9 ꢂmol) was incubated
with CMP-Neu5Ac (36.1 mg, 54.8ꢂmol) for 1 day
(from H-1 to H-6a,6b): ꢃ 5.01 (d, 1 H, J1,2
&
7.6 Hz), 3.42, 3.99, 3.38, 3.33, 3.67, 3.87; Gal (from
H-1 to H-6a,6b): ꢃ 4.47 (d, 1 H, J1,2 & 7.4 Hz),
3.55, 3.95, 3.94, 3.59, 3.67, 3.74; Neu5Ac (from H-
3ax,3eq to H-9a,9b, HNCOCH3): ꢃ 1.92 (t, 1 H,
J3ax,4 & 12.1 Hz), 2.75 (dd, 1 H, J3eq,4 & 4.2,
J3ax,3eq & 12.1 Hz), 3.79, 3.70, 3.48, 3.49, 3.79,
3.60, 3.82, 2.00; GalA (from H-1 to H-5, OCH3): ꢃ
4.24 (d, 1 H, J1,2 & 8.0 Hz), 3.54, 3.56, 4.18, 4.08,
3.60; aglycon: ꢃ 2.30, 1.58, 1.30, 1.55, 3.51, 3.85,
3.64; 13C NMR (CD3OD, 100.61 MHz): GlcN
(from C-1 to C-6): ꢃ 100.9, 57.9, 84.3, 70.7, 77.5,
62.7; Gal (from C-1 to C-6): ꢃ 105.1, 71.7, 76.8,
70.4, 76.5, 62.6; Neu5Ac (from C-1 to C-9,
HNCOCH3): ꢃ 175.3, 102.5, 40.5, 69.7, 54.0, 74.7,
70.1, 72.8, 64.4, 176.7, 22.5; GalA (from C-1 to C-
6, OCH3): ꢃ 105.8, 71.0, 74.6, 71.1, 76.4, 171.2,
57.7; aglycon: ꢃ 176.0, 34.6, 25.8, 30.2, 26.8, 30.4,
70.6, 51.9. MS Calcd for C40H68N2O26: 992; Found
[M H]- 991.
8-Methoxycarbonyloctyl 5-N-acetyl-a-neuraminyl-
(2!3)-b-d-galactopyranosyl-(1!3)-2-deoxy-2-
(methyl a-d-galactopyranosyluronamide)-b-d-gluco-
pyranoside (9c).ÐAccording to general procedure
C, compound 8c (22.6 mg, 32.2 ꢂmol) was incu-
bated with CMP-Neu5Ac (29.4 mg, 44.6 ꢂmol) for
one day to give the title compound (29.6 mg, 93%).
1H NMR (CD3OD, 500.13 MHz): GlcN (from H-1
to H-6a,6b): ꢃ 4.88 (d, 1 H, J1,2 & 7.6 Hz), 3.34,
4.02, 3.36, 3.35, 3.68, 3.89*; Gal (from H-1 to H-
6a,6b): ꢃ 4.48 (d, 1 H, J1,2 & 7.4 Hz), 3.57, 3.95,
3.95, 3.62, 3.75, 3.88*; Neu5Ac (from H-3ax,3eq to
1
to give the title compound (24.8 mg, 63%). H
NMR (CD3OD, 500.13 MHz): GlcN (from H-1 to
H-6a,6b): ꢃ 4.51 (d, 1 H, J1,2 & 7.6 Hz), 3.96, 3.86,
3.42, 3.34, 3.72, 3.90; Gal (from H-1 to H-6a,6b):
4.53 (d, 1 H, J1,2 & 7.4 Hz), 3.57, 3.99, 3.94, 3.60,
3.65, 3.75; Neu5Ac (from H-3ax,3eq to H-9a,9b,
HNCOCH3): ꢃ 1.88 (t, 1 H, J3ax,4 & 12.1 Hz), 2.73
(dd, 1 H, J3eq,4 & 4.2, J3ax,3eq & 12.1 Hz), 3.74,
3.72, 3.51, 3.50, 3.83, 3.63, 3.83, 2.00; GalA (from
H-1 to H-5, OCH3): ꢃ 4.20 (d, 1 H, J1,2 & 7.4 Hz),
3.59*, 3.56*, 4.21, 3.99, 3.59; aglycon: ꢃ 2.30, 1.58,
1.29, 1.31, 1.52, 3.46, 3.85, 3.64; 13C NMR
(CD3OD, 100.61 MHz): GlcN (from C-1 to C-6): ꢃ
102.4, 55.7, 83.4, 70.5, 77.4, 62.7; Gal (from C-1 to
C-6): ꢃ 104.1, 70.8*, 76.7*, 69.9, 76.7, 62.8;
Neu5Ac (from C-1 to C-9, HNCOCH3): ꢃ C-1 not
resolved, C-2 not resolved, 40.8, 69.5, 53.9, 74.7,
70.0, 72.8, 64.4, 175.5, 22.4; GalA-unit (from C-1
to C-6, OCH3): ꢃ 105.8, 72.0*, 74.5, 70.8, 77.1*,
171.5, 57.1; aglycon: ꢃ 175.9, 34.6, 25.4, 30.2, 26.9,
30.4, 70.7, 51.9. MS Calcd for C40H68N2O26: 992;
Found [M H]- 991.
8-Methoxycarbonyloctyl 5-N-acetyl-a-neuraminyl-
(2!3)-b-d-galacto-pyranosyl-(1!3)-2-deoxy-2-
(methyl a-l-galactopyranosyluronamide)-b-d-gluco-
pyranoside (9e).ÐAccording to general procedure
C, compound 8e (10 mg, 14.3 ꢂmol) was incubated
with CMP-Neu5Ac (14.8 mg, 22.5 ꢂmol) for 1 day
1
to give the title compound (10.4 mg, 74%). H
NMR (CD3OD, 400.13 MHz): GlcN (from H-1 to
H-6a,6b): ꢃ 4.49 (d, 1 H, J1,2 & 7.6 Hz), 3.97, 3.85,
3.44, 3.34, 3.72, 3.91; Gal (from H-1 to H-6a,6b): ꢃ
4.54 (d, 1 H, J1,2 & 7.4 Hz), 3.56, 4.01, 3.94, 3.58,
3.65, 3.78; Neu5Ac (from H-3ax,3eq to H-9a,9b,
HNCOCH3): ꢃ 1.88 (t, 1 H, J3ax,4 & 12.1 Hz), 2.79
(dd, 1 H, J3eq,4 & 4.2, J3ax,3eq & 12.1 Hz), 3.75,
3.72, 3.52, 3.50, 3.85, 3.64, 3.87, 2.02; GalA (from
H-1 to H-5, OCH3): ꢃ 4.88 (d, 1 H, J1,2 & 4.0 Hz),
3.90, 3.77, 4.29, 4.20, 3.43; aglycon: ꢃ 2.30, 1.60,
1.31, 1.55, 3.47, 3.89, 3.66. 13C NMR (CD3OD,
100.61 MHz): GlcN (from C-1 to C-6): ꢃ 101.3,
54.3, 82.2, 70.0, 76.5, 61.5*; Gal (from C-1 to C-6):
H-9a,9b, HNCOCH3): ꢃ 1.95 (t, 1 H, J3ax,4
12.1 Hz), 2.72 (dd, 1 H, J3eq,4 & 4.2, J3ax,3eq
&
&
12.1 Hz), 3.81, 3.73, 3.51, 3.51, 3.78, 3.62, 3.81,
2.00; GalA (from H-1 to H-5, OCH3): ꢃ 5.11 (d, 1
H, J1,2 & 4.9 Hz), 3.79, 3.78, 4.26, 4.23, 3.45;
aglycon: ꢃ 2.29, 1.58, 1.30, 1.32, 1.53, 3.50, 3.87,
3.64; 13C NMR (CD3OD, 100.61 MHz): GlcN
(from C-1 to C-6): ꢃ 101.7, 56.4, 84.3, 70.8, 77.5,
62.6; Gal (from C-1 to C-6): ꢃ 105.1, 71.1, 76.6,
70.6, 76.4, 62.6; Neu5Ac (from C-1 to C-9,
HNCOCH3): ꢃ C-1 not resolved, C-2 not resolved,
40.2, 69.4, 53.8, 74.8, 70.0, 72.7, 64.4, 175.5, 22.6;

È
G. Baisch, R. Ohrlein/Carbohydrate Research 312 (1998) 61±72
70
ꢃ 103.8, 70.0, 75.5, 68.5, 75.5, 62.2*; Neu5Ac (from
C-1 to C-9, HNCOCH3): ꢃ C-1 not resolved, C-2
not resolved, 40.4, 68.8, 52.9, 73.7, 69.4, 72.0, 63.7,
C ˆ O not resolved, 21.4; GalA (from C-1 to C-6,
OCH3): ꢃ 100.9, 68.8, 70.3, 70.6, 72.3, C-6 not
resolved, 55.2; aglycon: ꢃ C ˆ O not resolved, 34.1,
25.2, 29.1, 26.1, 29.6, 69.4, 51.2. MS Calcd for
C40H68N2O26: 992; Found [M H]- 991.
3.74, 3.42, 3.91; Gal (from H-1 to H-6a,6b): ꢃ 4.61
(d, 1 H, J1,2 & 7.4 Hz), 3.52, 3.89, 3.90, 3.41, 3.63,
3.76; Neu5Ac (from H-3ax,3eq to H-9a,9b,
HNCOCH3): ꢃ 1.87 (t, 1 H, J3ax,4 & 12.1 Hz), 2.72
(dd, 1 H, J3eq,4 & Hz, J3ax,3eq & 12.1 Hz), 3.72*,
3.74, 3.65, 3.56, 3.74, 3.65, 3.87, 2.02; Fuc (from H-
1 to H-6): ꢃ 5.05 (d, 1 H, J1,2 & 3.8 Hz), 3.73*,
3.88, 3.66, 4.88, 1.17 (d, 3 H, J5,6 & 6.4 Hz); GalA
8-Methoxycarbonyloctyl 5-N-acetyl-a-neuraminyl-
(2!3)-b-d-galacto-pyranosyl-(1!3)-[a-l-fucopy-
ranosyl-(1!4)]-2-deoxy-2-(methyl b-d-glucopyr-
(from H-1 to H-5, OCH3): ꢃ 4.35 (d, 1 H, J1,2 &
8.0 Hz), 3.59, 3.65, 4.18, 4.16, 3.57; aglycon: ꢃ 2.31,
1.62, 1.31, 1.58, 3.51, 3.81, 3.59; 13C NMR
(CD3OD, 100.61 MHz): GlcN (from C-1 to C-6): ꢃ
102.2, 57.7, 78.0, 77.4, 77.4, 61.4; Gal (from C-1 to
C-6): ꢃ 104.2, 71.0, 77.7, 69.3, 76.8, 63.2; Neu5Ac
(from C-1 to C-9, HNCOCH3): ꢃ 175.0, 101.8,
41.7, 69.6*, 53.9, 74.9, 70.0, 73.0, 64.6, 176.0, 22.6;
Fuc (from C-1 to C-6): 99.6, 70.0*, 71.1, 73.7, 67.6,
16.6; GlcA (from C-1 to C-6, OCH3): ꢃ 105.8, 72.0,
74.5, 71.1, 76.4, 171.6, 58.1; aglycon: ꢃ 175.4, 34.8,
26.0, 30.3, 26.9, 30.6, 70.2, 52.0. MS calcd for
C46H78N2O30: 1138; found [M H]- 1137. Anal.
Calcd for C46H78N2O30: C, 48.50; H, 6.90. Found:
C, 48.31; H, 6.68.
anosyluronamide)-b-d-glucopyranoside
(10a).Ð
According to general procedure D, compound 9a
(20 mg, 20.1 ꢂmol) was incubated with GDP-Fuc
(18.4 mg, 29 ꢂmol) for 1 day to give the title com-
pound (18.8 mg, 82%). 1H NMR (CD3OD,
500.13 MHz): GlcN (from H-1 to H-6a,6b): ꢃ 4.50
(d, 1 H, J1,2 & 7.6 Hz), 4.01, 4.09, 3.77, 3.42, 3.65*,
3.88; Gal (from H-1 to H-6a,6b): ꢃ 4.59 (d, 1 H, J1,2
& 7.4 Hz), 3.52, 3.88, 3.91, 3.54, 3.64*, 3.78;
Neu5Ac (from H-3ax,3eq to H-9a,9b HNCOCH3):
ꢃ 1.91 (t, 1 H, J3ax,4 & 12.1 Hz), 2.80 (dd, 1 H,
J3eq,4 & 4.2, J3ax,3eq & 12.1 Hz), 3.72, 3.74, 3.64,
3.52, 3.83, 3.65, 3.88, 2.03; Fuc (from H-1 to H-6):
ꢃ 5.06 (d, 1 H, J1,2 & 3.8 Hz), 3.74, 3.86, 3.65, 4.85,
1.18 (d, 3 H, J5,6 & 6.4 Hz); GlcA (from H-1 to H-
5, OCH3): ꢃ 4.36 (d, 1 H, J1,2 & 8.0 Hz), 3.28, 3.50,
3.58, 3.68, 3.59; aglycon: ꢃ 2.35, 1.62, 1.32, 1.38,
1.58, 3.47, 3.85, 3.68; 13C NMR (CD3OD,
100.61 MHz): GlcN (from C-1 to C-6): ꢃ 102.4,
56.9, 77.6, 77.3, 77.1, 63.3; Gal (from C-1 to C-6): ꢃ
103.9, 70.9, 77.6, 69.4, 77.4, 61.3; Neu5Ac (from C-
1 to C-9, HNCOCH3): ꢃ 175.2, 102.1, 41.4, 70.0,
54.0, 73.9, 70.9, 72.9, 64.6, 175.5, 22.7; Fuc (from
C-1 to C-6): ꢃ 99.6, 69.6, 71.1, 73.6, 67.6, 16.6;
GlcA (from C-1 to C-6, OCH3): ꢃ 105.4, 74.6, 77.4,
73.5, 74.4, 172.2, 57.8; aglycon: ꢃ 176.1, 34.8, 26.0,
30.2, 26.9, 30.6, 70.7, 52.0. MS Calcd for
C46H78N2O30: 1138; Found [M H]- 1137. Anal.
Calcd for C46H78N2O30: C, 48.50; H, 6.90. Found:
C, 48.15; H, 7.21.
8-Methoxycarbonyloctyl 5-N-acetyl-a-neuraminyl-
(2!3)-b-d-galacto-pyranosyl-(1!3)-[a-l-fucopy-
ranosyl-(1!4)]-2-deoxy-2-(methyl a-d-galactopy-
ranosyluronamide)-b-d-glucopyranoside
(10c).Ð
According to general procedure D, compound 9c
(22.0 mg, 22.2 ꢂmol) was incubated with GDP-Fuc
(21.7 mg, 34.2 ꢂmol) for 1 day to give the title
1
compound (20.1 mg, 79%). H NMR (CD3OD,
400.13 MHz): GlcN (from H-1 to H-6a,6b): ꢃ 4.84
(d, 1 H, J1,2 & 7.6 Hz), 3.71, 3.89, 3.75, 3.42, 3.91;
Gal (from H-1 to H-6a,6b): ꢃ 4.68 (d, 1 H, J1,2
7.4 Hz), 3.52, 3.91*, 3.92*, 3.50, 3.64, 3.75; Neu5Ac
&
(from H-3ax,3eq to H-9a,9b, HNCOCH3): ꢃ 1.88
(t, 1 H, J3ax,4 & 12.1 Hz), 2.79 (dd, 1 H, J3eq,4
&
4.2, J3ax,3eq & 12.1 Hz), 3.74, 3.76, 3.57, 3.50, 3.83,
3.65, 3.88, 2.01; Fuc (from H-1 to H-6): ꢃ 5.06 (d, 1
H, J1,2 & 3.8 Hz), 3.73, 3.86, 3.65, 4.86, 1.17 (d, 3
H, J5,6 & 6.4 Hz); GalA (from H-1 to H-5, OCH3):
ꢃ 4.91 (d, 1 H, J1,2 & 4.9 Hz), 3.92, 3.83, 4.29, 4.25,
3.50; aglycon: ꢃ 2.34, 1.60, 1.32, 1.57, 3.50, 3.83,
3.69; 13C NMR (CD3OD, 100.61 MHz): GlcN
(from C-1 to C-6): ꢃ 101.7, 58.3, 77.4, 77.2, 76.1,
61.4; Gal (from C-1 to C-6): ꢃ 103.9, 71.0, 77.5,
69.9, 77.2, 63.1; Neu5Ac (from C-1 to C-9,
HNCOCH3): ꢃ 175.2, 101.4, 41.4, 69.4*, 53.8, 74.7,
69.9, 72.8, 64.4, 175.6, 22.7; Fuc (from C-1 to C-6):
ꢃ 99.5, 69.5*, 70.9, 73.6, 67.7, 16.6; GalA (from C-1
to C-6, OCH3): 102.1, 69.2, 72.8, 71.2, 72.6, 172.0,
56.6; aglycon: ꢃ 176.4, 34.8, 26.0, 30.1, 30.2, 26.9,
8-Methoxycarbonyloctyl 5-N-acetyl-a-neuraminyl-
(2!3)-b-d-galacto-pyranosyl-(1!3)-[a-l-fucopy-
ranosyl-(1!4)]-2-deoxy-2-(methyl b-d-galactopy-
ranosyluronamide)-b-d-glucopyranoside
(10b).Ð
According to general procedure D, compound 9b
(26 mg, 26.2 ꢂmol) was incubated with GDP-Fuc
(22.6 mg, 35.6 ꢂmol) for one day to give the title
compound (18.8 mg, 63%). A TLC of the crude
mixture still showed some starting sugar. 1H NMR
(CD3OD, 400.13 MHz): GlcN (from H-1 to H-
6a,6b): ꢃ 4.70 (d, 1 H, J1,2 & 7.6 Hz), 3.85, 3.89,

È
G. Baisch, R. Ohrlein/Carbohydrate Research 312 (1998) 61±72
71
30.6, 70.9, 52.2. MS Calcd for C46H78N2O30: 1138;
Found [M H]- 1137. Anal. Calcd for
C46H78N2O30: C, 48.50; H, 6.90. Found: C, 48.78;
H, 6.69.
(t, 1 H, J3ax,4 & 12.1 Hz), 2.71 (dd, 1 H, J3eq,4
&
4.2, J3ax,3eq & 12.1 Hz), 3.71, 3.72, 3.48, 3.44, 3.83,
3.63, 3.87, 2.01; Fuc (from H-1 to H-6): 5.04 (d, 1
H, J1,2 & 3.8 Hz), 3.74, 3.88, 3.72, 4.86, 1.19 (d, 3
H, J5,6 & 6.4 Hz); GalA (from H-1 to H-5, OCH3):
ꢃ 4.91 (d, 1 H, J & 4.0 Hz), 3.94, 3.76, 4.36, 4.17,
3.52; aglycon: ꢃ 2.32, 1.59, 1.29, 1.59, 3.47, 3.87,
3.68. 13C NMR (CD3OD, 100.61 MHz): GlcN
(from C-1 to C-6): ꢃ 102.6, 56.5, 76.9, 73.4, 76.0,
61.1; Gal (from C-1 to C-6): 103.8, 70.6, 76.6, 70.6,
76.1, 63.0; Neu5Ac (from C-1 to C-9, HNCOCH3):
ꢃ C-1 not resolved, C-2 not resolved, 40.2, 69.5,
52.7, 75.7, 70.6, 72.8, 64.4, C ˆ O not resolved,
22.7; Fuc (from C-1 to C-6): 99.5, 69.5, 70.6, 74.4,
67.6, 16.6; GalA (from C-1 to C-6, OCH3): 101.8,
69.6, 71.1, 71.1, 72.8, 172.0, 55.8; aglycon: ꢃ 176.5,
34.8, 25.9, 30.0, 30.2, 27.0, 30.6, 70.7, 52.1. MS
Calcd for C46H78N2O30: 1138; found [M H]- 1137.
Anal. Calcd for C46H78N2O30: C, 48.50; H, 6.90.
Found: C, 48.87; H, 7.19.
8-Methoxycarbonyloctyl 5-N-acetyl-a-neuraminyl-
(2!3)-b-d-galacto-pyranosyl-(1!3)-[a-l-fucopy-
ranosyl-(1!4)]-2-deoxy-2-(methyl b-l-galactopy-
ranosyluronamide)-b-d-glucopyranoside
(10d).Ð
According to general procedure D, compound 9d
(19 mg, 19.1 ꢂmol) was incubated with GDP-Fuc
(16.8 mg, 26.4 ꢂmol) for 4 days to give the title
compound (12.2 mg, 56%). The crude mixture still
1
showed some starting sugar. H NMR (CD3OD,
400.13 MHz): GlcN (from H-1 to H-6a,6b): ꢃ 4.46
(d, 1 H, J1,2 & 7.6 Hz), 4.11, 4.12, 3.78, 3.40, 3.92;
Gal (from H-1 to H-6a,6b): ꢃ 4.74 (d, 1 H, J1,2
7.4 Hz), 3.52, 3.93, 3.91, 3.54, 3.64, 3.77; Neu5Ac
&
(from H-3ax,3eq to H-9a,9b, HNCOCH3): ꢃ 1.85
(t, 1 H, J3ax,4 & 12.1 Hz), 2.72 (dd, 1 H, J3eq,4
&
4.2, J3ax,3eq & 12.1 Hz), 3.72, 3.74, 3.61, 3.47, 3.83,
3.62, 3.85, 2.02; Fuc (from H-1 to H-6): ꢃ 5.06 (d, 1
H, J1,2 & 3.8 Hz), 3.73, 3.88, 3.63, 4.89, 1.19 (d, 3
H, J5,6 & 6.4 Hz); GalA (from H-1 to H-5, OCH3):
ꢃ 4.20 (d, 1 H, J1,2 & 7.4 Hz), 3.61, 3.55, 4.29, 3.98,
3.62; aglycon: ꢃ 2.33, 1.60, 1.31, 1.55, 3.47, 3.85,
3.68. 13C NMR (CD3OD, 100.61 MHz): GlcN
(from C-1 to C-6): ꢃ 102.6, 56.9, 77.4*, 74.6, 77.4,
61.4; Gal (from C-1 to C-6): ꢃ 103.2, 71.0, 77.2,
70.0, 76.1, 63.3; Neu5Ac (from C-1 to C-9,
HNCOCH3): ꢃ 173.7, 102.6, 40.6, 70.2, 54.0, 74.7,
70.9, 73.7, 64.7, 175.6, 22.6; Fuc (from C-1 to C-6):
ꢃ 99.5, 69.8, 71.9, 73.7, 67.7, 16.6; GalA (from C-1
to C-6, OCH3): ꢃ 106.0, 71.9, 74.7, 70.9, 77.3*,
171.7, 57.8; aglycon: ꢃ 176.0, 34.8, 26.0, 30.2, 30.4,
27.0, 30.6, 70.9, 52.0. MS calcd for C46H78N2O30:
1138; found [M H]- 1137. Anal. Calcd for
C46H78N2O30: C, 48.50; H, 6.90. Found: C, 48.24;
H, 6.61.
Acknowledgements
The authors thank Drs. M. Strei€, A. Katopodis
and F. Kolbinger and their teams for cloning and
overexpression of the transferases and T. Winkler
and his team for the NMR work.
References
[1] E. Graf von Roedern, E. Lohof, G. Hessler, M.
Ho€mann, and H. Kessler, J. Am. Chem. Soc., 118
(1996) 10156±10167.
[2] H.P. Wessel, C. Mitchell, C.M. Lobato, and G.
Schmid, Angew. Chem. Int. Ed. Engl., 34 (1995)
2712±2713.
[3] C. Muller, E. Kitas, and H.P. Wessel, J. Chem.
Soc. Chem. Commun., (1995) 2425±2426.
[4] Y. Suhara, J.E.K. Hildreth, and Y. Ichikawa,
Tetrahedron Lett., 37 (1996) 1575±1578.
[5] P. Fugedi, C.J. Peto, and L. Wang, 9th Eur.
Carbohydr. Symp., Utrecht, The Netherlands
(1997) abstract A113 and A114.
[6] M. Du and O. Hindsgaul, Synlett, (1997) 395±397.
[7] R.A. Goodnow, A.-R. Richou, and S. Tam,
Tetrahedron Lett., 38 (1997) 3195±3198.
[8] G. Baisch and R. Ohrlein, Angew. Chem. Int. Ed.
Engl., 35 (1996) 1812±1815.
[9] G. Baisch, R. Ohrlein, A. Katopodis, and B. Ernst,
Bioorg. Med. Chem. Lett., 6 (1996) 759±762.
[10] J.Y. Ramphal, M. Hiroshige, B. Lou, J.J.
Gaudino, M. Hayashi, S.M. Chen, L.C. Chiang,
8-Methoxycarbonyloctyl 5-N-acetyl-(-neuraminyl-
(2!3)-b-d-galacto-pyranosyl-(1!3)-[a-l-fucopy-
ranosyl-(1!4)]-2-deoxy-2-(methyl a-l-galactopy-
ranosyluronamide)-b-d-glucopyranoside
(10e).Ð
According to general procedure D, compound 9e
(10 mg, 10.0 ꢂmol) is incubated with GDP-Fuc
(14.5 mg, 22.8 ꢂmol) for 5 days to give the title
compound (8.6 mg, 75%). The crude mixture still
1
showed some starting sugar. H NMR (CD3OD,
400.13 MHz): GlcN (from H-1 to H-6a,6b): ꢃ 4.45
(d, 1 H, J1,2 & 7.6 Hz), 4.11, 4.11, 3.75, 3.40, 3.93;
Gal (from H-1 to H-6a,6b): ꢃ 4.73 (d, 1 H, J1,2
7.4 Hz), 3.52, 3.92, 3.89, 3.52, 3.68, 3.79; Neu5Ac
&
(from H-3ax,3eq to H-9a,9b, HNCOCH3): ꢃ 2.04

È
G. Baisch, R. Ohrlein/Carbohydrate Research 312 (1998) 61±72
72
F.C.A. Gaeta, and S.A.De Frees, J. Med. Chem.,
39 (1996) 1357±1360.
[22] H.J.M. Gijsen, L. Qiao, W. Fitz, and C.-H, Wong,
Chem. Rev., 96 (1996) 443±473.
[11] G. Kretzschmar, A. Toepfer, C. Huls, and M.
Krause, Tetrahedron, 53 (1997) 2485±2594.
[12] S.-H. Wu, M. Shimazaki, C.-C. Lin, L. Qiao, W.J.
Moree, G. Weitz-Schmidt, and C.-H. Wong,
Angew. Chem. Int. Ed. Engl., 35 (1996) 88±90.
[13] H. Huang and C.-H. Wong, J. Am. Chem. Soc., 60
(1995) 3100±3106.
[14] N. Kojima, K. Handa, W. Newmann, and S.-i.
Hakomori, Biochem. Biophys. Res. Commun., 189
(1992) 1686±1694.
[23] G. Baisch, R. Ohrlein, M. Strei€, and B. Ernst,
Bioorg. Med. Chem. Lett., 6 (1996) 755±758.
[24] G. Baisch, R. Ohrlein, and A. Katopodis, Bioorg.
Med. Chem. Lett., 6 (1996) 2953±2956.
[25] Y. Ichikawa, Y.-C. Lin, D.P. Dumas, G.-J. Shen, E.
Garcia-Junceda, M.A. Williams, R. Bayer, C.
Ketcham, L.E. Walker, J.C. Paulson, and C.-H.
Wong, Confer the data of the parent N-acetyl com-
pounds: J. Am. Chem. Soc., 114 (1992) 9283±9298.
[26] G. Baisch and R. Ohrlein, Bioorg. Med. Chem., 5
(1997) 383±391.
[15] N.J. Davis and S.L. Flitsch, Tetrahedron Lett., 34
(1993) 1181±1184.
[16] K. Li and R.F. Helm, Carbohydr. Res., 273 (1995)
249±253.
[27] G. Baisch, R. Ohrlein, A. Katopodis, M. Strei€,
and F. Kolbinger, Bioorg. Med. Chem. Lett., 7
(1997) 2447±2450.
[17] L.A. Carpino and A. El-Faham, J. Org. Chem., 59
(1994) 695±698.
[18] V. Dourtoglou, B. Gross, V. Lampropoulou, and
C. Zioudrou, Synthesis, (1984) 572±574.
[19] G. Baisch, R. Ohrlein, and B. Ernst, Bioorg. Med.
Chem. Lett., 6 (1996) 749±754.
[28] D.X. Wen, B.D. Livingston, K.F. Medziharaszky,
S. Kelm, A.L. Burlingame, and J.C. Paulson, J.
Biol. Chem., 267 (1992) 21011±21019.
[29] K. Sasaki, K. Kurata, K. Funayama, M. Nagata,
E. Watanabe, S. Ohta, N. Hanai, and T. Nishi, J.
Biol. Chem., 269 (1994) 14730±14737.
[20] R.U. Lemieux, D.R. Bundle, and D.A. Baker, J.
Am. Chem. Soc., 97 (1975) 4076±4083.
[21] P. Boulanger, J. Banoub, and G. Descotes, Can. J.
Chem., 65 (1987) 1343±1348.
[30] M.M. Palcic, Methods Enzymol., 230 (1994) 300±316.
[31] M. Kittelmann, T. Klein, U. Kragl, C. Wandrey,
and O. Ghisalba, Appl. Microbiol. Biotechnol., 44
(1995) 59±67.