Synthesis of Pseudomonas aeruginosa lipopolysaccharide core antigens containing 7-O-carbamoyl-L-glycero-α-D-manno-heptopyranosyl residues

Article abstract of DOI:10.1016/S0008-6215(99)00059-2

The monosaccharide allyl 7-O-carbamoyl-L-glycero-α-D-manno-heptopyranoside, the reducing disaccharide 7-O-carbamoyl-L-glycero-α-D-manno-heptopyranosyl-(1→3)-L-glycero-D-manno-heptopyranose and the disaccharides allyl 7-O-carbamoyl-L-glycero-α-D-manno-hept

Full text of DOI:10.1016/S0008-6215(99)00059-2

www.elsevier.nl/locate/carres
Carbohydrate Research 317 (1999) 39–52
Synthesis of Pseudomonas aeruginosa lipopolysaccharide
core antigens containing
7-O-carbamoyl-
L
-glycero-a-
D
-manno-heptopyranosyl
residuesꢀ
Andreas Reiter, Alla Zamyatina, Heidemarie Schindl, Andreas Hofinger, Paul Kosma *
Institute of Chemistry, Uni6ersity of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria
Received 4 January 1999; accepted 25 February 1999
Abstract
The monosaccharide allyl 7-O-carbamoyl-
carbamoyl- -glycero-a- -manno-heptopyranosyl-(1“3)-
lyl 7-O-carbamoyl- -glycero-a- -manno-heptopyranosyl-(1“3)-
L
-glycero-a-
D
-manno-heptopyranoside, the reducing disaccharide 7-O-
-glycero- -manno-heptopyranose and the disaccharides al-
-glycero-b- and a- -manno-heptopyranoside were
L
D
L
D
L
D
L
D
prepared in good yields. The 7-O-carbamoyl substituent was regioselectively introduced via NH3–NH4HCO3
treatment of a 6,7-O-carbonate group. Glycosylation steps were carried out using Me3SiOTf or BF3·Et2O promoted
coupling of allyl alcohol with trichloroacetimidate or fluoride glycosyl donors, respectively. The deprotected allyl
glycosides were reacted with cysteamine to afford spacer glycosides which were subsequently linked to bovine serum
albumin. The artificial antigens which are related to the dephosphorylated heptose region of the lipopolysaccharide
core region from Pseudomonas aeruginosa classified into RNA group I may be used for the characterization of
monoclonal antibodies directed against inner core epitopes of human-pathogenic Pseudomonas species. © 1999
Elsevier Science Ltd. All rights reserved.
Keywords: Pseudomonas aeruginosa; Lipopolysaccharide; Heptose; Carbamate; Neoglycoprotein
has emerged as a common bacterial nosocomial
1. Introduction
pathogen with increasing resistance to antibi-
otic treatment, thus causing 10–20% of infec-
tions in hospitals at a high mortality rate [2].
LPS of Pseudomonas differ from enterobacter-
ial LPS with respect to the reduced chain
length of the fatty acids of lipid A, the high
number and different types of phosphate
Lipopolysaccharides (LPS)—also termed
endotoxins—are structurally complex am-
phipathic and microheterogeneous glycolipids
located in the external membrane of the outer
cell wall of Gram-negative bacteria [1]. LPS are
highly potent toxins leading to multiple organ
failure as a consequence of Gram-negative
sepsis. In this context, Pseudomonas aeruginosa
residues and the presence of -alanine amide-
L
linked to galactosamine in the outer core region
[3,4]. The structural elucidation of the phos-
phate substitution pattern and the nature of the
individual phosphate substituents, which most
likely include triphosphates, diphosphates,
ꢀ Dedicated to Professor Aleksander Zamojski on the occa-
sion of his 70th birthday.
* Corresponding author.
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00059-2

40
A. Reiter et al. / Carbohydrate Research 317 (1999) 39–52
2-aminoethyl-diphosphate and phosphate mo-
noesters, respectively, has been partially ac-
intermediates. The reduced stereoselectivity of
the Me3SiOTf-mediated glycosylation might
be due to enhanced acid-catalyzed transester-
ification of the 2-O-acetyl group with allyl
alcohol, thereby eliminating the neighboring
group participation for the transition state
[16]. Alternatively, the anomeric fluoride
donors 3 and 4 were synthesized in 58 and
33% yield, respectively, by treatment of 1 with
diethylaminosulfur trifluoride [17]. Under pro-
motion of the glycosylation reaction with
BF3·Et2O using either fluoride donor, the allyl
a-glycoside derivative 5 was produced in a
similar yield (85%); however, the formation of
the b isomer 6 could not be observed by TLC.
Zemple´n O-deacetylation of the protected
monosaccharide derivatives 5 and 6 furnished
the crystalline allyl a-glycoside 7 and the b
isomer 8 in high yields. Measurement of the
heteronuclear coupling constant JC-1,H-1 for C-
1 of compound 8 (162.6 Hz) confirmed the
assignment of the anomeric configuration
(Scheme 1).
complished
[5].
In
addition,
recent
investigations revealed the occurrence of a
7-O-carbamoyl residue within the heptose re-
gion [6], which is present in rough mutants as
well as smooth-type LPS of Pseudomonas spp.
classified into RNA group I, thereby consti-
tuting a taxonomic marker [7]. Furthermore,
antibodies against the core and lipid A region
have been raised, their immunodominant epi-
topes have been defined [8,9] and a cross-reac-
tive monoclonal antibody mAb (7-4)—which
still retains its reactivity in the presence of
O-chains—has been expressed in an E. coli
system [10]. Consequently, these core-determi-
nants may serve as candidate antigens for the
production of vaccines [11]. As a first part of
synthetic studies on inner-core LPS determi-
nants of Pseudomonas species, we report on
the synthesis of the dephosphorylated heptose
region comprising 7-O-carbamoyl-substituted
L
-glycero- -manno-heptopyranosyl monosac-
D
As a suitable precursor for the carbamate
moiety, the 6,7-O-carbonate group was cho-
sen, which could be used as a temporary
protecting group of the side chain diol as well.
Thus, by using trichloromethyl chloroform-
ate–sym-collidine–oxolane under carefully
controlled conditions (slow addition of
reagent at −30 °C), the O-deacetylated
derivative 7 was converted in a regioselective
fashion into the 6,7-O-carbonate derivative 9
in 76% yield [18]. The formation of the cyclic
diester was evident from the downfield shift of
H-6 (5.28 ppm) and the geminal protons at
C-7 (4.70 and 4.58 ppm, respectively) and
after subsequent O-acetylation of the remain-
ing hydroxy groups with acetic anhydride–
pyridine to give the 2,3,4-tri-O-acetyl
heptopyranoside derivative 10 in quantitative
yield. The formation of the 7-O-carbamate
was achieved in excellent yield (84%) and in
high selectivity by treatment of 9 with NH3–
NH4HCO3–oxolane to give the crystalline al-
charide and a-(1“3)-linked disaccharide allyl
glycosides. In continuation of previous investi-
gations directed toward the characterization
of epitope specificities of monoclonal antibod-
ies against LPS core antigens, the allyl gly-
cosides were converted into neoglycoproteins
following introduction of a cysteamine spacer
group [12,13].
2. Results and discussion
For the synthesis of the monosaccharide
allyl 7-O-carbamoyl-
L
-glycero-a- -manno-he-
D
ptapyranoside (11) and the disaccharides allyl
7-O-carbamoyl- -glycero-a- -manno-hepta-
pyranoside-(1“3)- -glycero-b- and a- -hep-
tapyranoside (29 and 30, respectively), the pre-
viously described [14] allyl -glycero-a-
L
D
L
D
L
D-
manno-heptopyranoside derivative 5 was em-
ployed as the starting material. The reaction
of the trichloroacetimidate donor 2—pre-
pared from the reducing heptopyranose
derivative 1 [15]—with allyl alcohol in the
presence of trimethylsilyl trifluoromethanesul-
fonate afforded the allyl a-glycoside 5 in 70%
yield and the b isomer 6 in 11% yield, isolated
after reacetylation of partially O-deacetylated
lyl 7-O-carbamoyl-
L
-glycero-a- -manno-hep-
D
topyranoside (11) and a small proportion of
the 6-O-carbamoyl derivative 12 (9%). In ad-
dition, the feasibility of the selective carba-
mate formation with concomitant hydrolysis
of the O-acetyl protecting groups was demon-
strated for the 2,3,4-tri-O-acetyl derivative 10

A. Reiter et al. / Carbohydrate Research 317 (1999) 39–52
41
as well, which gave 11 in 89% yield and a
small proportion (9%) of the 6-O-substituted
isomer 12. The structural assignment of 11
was based on the downfield shift observed for
C-7 (66.41 ppm) in the 13C NMR spectrum
(Table 1). The carbamoyl group at O-7 was
prone to slow acyl migration to O-6 also
under neutral conditions. Thus, upon standing
of an aqueous solution of 11 for 2 weeks at
room temperature (rt), formation of com-
pound 12 (in ꢀ10% yield) was evident from
the characteristic downfield shift of H-6 (to
5.13 ppm) in the 300 MHz 1H NMR
spectrum.
The allyl glycoside 11 was transformed in
high yield into the 3-(2-aminoethylthio)propyl
spacer derivative 13 by radical addition of
cysteamine hydrochloride under UV-irradia-
tion [13]. The spacer compound 13, which was
isolated as the acetate salt by silica gel chro-
matography, was subsequently activated with
thiophosgene and coupled to the e-amino
groups of lysine residues of bovine serum al-
bumin (BSA) to give the neoglycoconjugate
14.
tive 15 was subjected to O-deacetylation with
sodium methoxide giving 16, which was con-
verted regioselectively into the 6%,7%-O-car-
bonyl derivative 17 in 95% overall yield
similar to the preparation of 9. Since H-6% and
the geminal protons at C-7 did not display a
significant downfield shift upon carbonate for-
mation, probably due to the presence of the
benzyl groups of the heptofuranosyl unit, the
remaining hydroxy groups of the pyranose
ring were O-acetylated with acetic anhydride–
pyridine–N,N-dimethylaminopyridine, which
afforded the 2%,3%,4%-tri-O-acetyl derivative 18
in 98% yield. The observed downfield shift of
1
the H NMR signals of H-2%, H-3% and H-4%
(5.27–5.35 ppm) confirmed the structural as-
signments. Treatment of 17 with NH3–
NH4HCO3 in methanol afforded the
7%-O-carbamoyl derivative 19 in 66% yield,
which was isolated by HPLC following re-
moval of compound 16, originating from hy-
drolysis of the carbonate ester group. Finally,
hydrogenolysis of the benzyl protecting
groups of 19 furnished the reducing disacchar-
ide 20 as a 3:1 a/b mixture in quantitative
yield. The 13C NMR data of compound 20
revealed a downfield shift for C-7% (to 66.22
ppm for the a and 66.12 ppm for the b
For the synthesis of the reducing disaccha-
ride derivative 20 (Scheme 2), the previously
reported [19] a-benzyl heptofuranoside deriva-
Scheme 1.

42
A. Reiter et al. / Carbohydrate Research 317 (1999) 39–52
Table 1
13C NMR dataa for mono- and disaccharides 7, 8, 11, 20, 29 and 30
Residue
Carbon
l (ppm)
7
8
11
20a
20b
29
30
a-Hepp
1
2
3
4
5
6
7
1
2
3
4
5
6
7
1
2
3
100.02
70.88
71.67
67.02
71.88
66.74
66.41
103.18
70.86
71.29
66.75
72.02
67.09
66.22
94.87
71.47
78.10
66..83
72.41
69.50
63.82
102.78
70.86
71.29
66.75
72.02
67.01
66.12
94.60
71.92
79.95
66.66
75.22
69.37
63.54
102.95
70.88
71.37
66.78
72.39
66.98
66.05
99.72
71.07
80.19
66.67
75.24
69.59
63.70
70.96
134.46
119.17
159.84
103.11
70.84
71.32
67.04
72.40
66.71
66.05
99.77
70.84
78.14
66.71
72.40
69.04
63.80
69.49
134.29
118.88
159.75
Hepp
99.90
70.87
71.69
66.90
72.14
69.59
63.77
68.87
134.04
119.26
99.86
71.40
74.07
66.78
75.41
69.69
63.67
70.99
Allyl
68.95
134.05
119.11
160.02
134.38
119.23
CONH2
159.93
159.93
a Spectra (75.47 MHz) were recorded at 297 K and referenced to 1,4-dioxane (67.40). Assignments are based on HMQC-spectra.
isomer), an upfield shift of ꢀ2 ppm for C-6
as well as the characteristic signal of the car-
bamoyl carbon at ꢀ159.9 ppm (Table 1).
Next, the synthesis of a heptosyl donor with
the carbonate group present in the side chain,
which will be used for further glycosylation of
the Kdo region, was elaborated. Compound
17 was hydrogenolyzed with 10% Pd–C to
remove the benzyl groups followed by O-
acetylation (acetic anhydride–pyridine) to
give the acetylated heptobiose derivative 21 in
91% yield as a 5:1 a/b mixture. The anomeric
acetyl group was selectively cleaved by the
action of hydrazine acetate [20] to afford 22 in
91% yield, which was subsequently trans-
formed with K2CO3–trichloroacetonitrile into
the a-trichloroacetimidate disaccharide donor
23 in 86% yield. Reaction of 23 with 2 equiva-
lents of allyl alcohol promoted by 0.5 equiva-
lents of trimethylsilyl trifluoromethane-
sulfonate in dichloromethane proceeded with
low stereoselectivity to give the disaccharide
allyl a-glycoside 25 in 23% yield. The
anomeric configuration of the disaccharide 25
was unambiguously established on the basis of
the heteronuclear coupling constant JC-1,H-1
(172.0 Hz) for C-1 of compound 25. In addi-
tion, the partially O-deacetylated products 26
and 27 were formed in 30 and 33% yield,
respectively. The structural assignments of 26
and 27 were based upon the high-field shift
displayed by the signal attributable to H-2
(4.14 and 4.13 ppm), which upon O-acetyla-
tion (acetic anhydride–pyridine)—to afford
the 2-O-acetyl derivatives 25 and 28—experi-
enced a downfield shift to 5.22 and 5.41 ppm,
respectively. By using a smaller amount of
Me3SiOTf (0.05 equivalents) the formation of
exo/endo orthoester derivatives (ꢀ3:1 ratio)
1
was observed (based on H NMR data of
exo/endo CH3 signals). By employing boron
trifluoride etherate as the promoter of the
glycosylation step, the anomeric selectivity
was improved (a/b ratio 4:1).
As an alternative to the trichloroacetimidate
donor 23, the disaccharide fluoride donor 24
was prepared in 90% yield by treatment of the
reducing disaccharide 22 with diethyl-
aminosulfur trifluoride. Reaction of the a-
fluoride donor 24 with allyl alcohol in the
presence of boron trifluoride etherate in
dichloromethane afforded the disaccharides in
an a/b ratio of 10:1.

A. Reiter et al. / Carbohydrate Research 317 (1999) 39–52
43
reaction of cysteamine hydrochloride with the
allyl glycoside 30 gave compound 31 in high
yield, which was isolated by ion-exchange
chromatography. Subsequent coupling of the
isothiocyanate-activated ligand to BSA fur-
nished the neoglycoprotein 32. Immunochemi-
cal results obtained with the glycoconjugates
will be published elsewhere.
O-Deacetylation of the b-allyl glycoside 28
with simultaneous formation of the 7%-O-car-
bamate was accomplished with NH3–
NH4HCO3 in 1:1 methanol–oxolane, which
gave the disaccharide derivative 29 in 63%
yield after final purification on Sephadex LH-
20.
Deprotection of both a-anomeric disacchar-
ide derivatives 25 and 26 was performed un-
der similar conditions to afford the
6%-O-carbamoyl derivative (20% by integration
3. Experimental
1
of H NMR signals) and the target disacchar-
ide allyl glycoside 30 isolated by silica gel
chromatography in 64 and 63% yield, respec-
tively. Introduction of the spacer group by
General methods.—Melting points were de-
termined with a Kofler hot stage and are
uncorrected. Optical rotations were measured
Scheme 2.

44
A. Reiter et al. / Carbohydrate Research 317 (1999) 39–52
with a Perkin–Elmer 243 B polarimeter. [h]D20
values are given in units of 10−1 deg cm2/g.
1H NMR spectra were recorded at 297 K with
a Bruker DPX instrument operating at 300
l 5.58 (ddd, 1 H, J1,2 1.2, J2,3 4.0, J2,F 5.5 Hz,
H-2), 5.45 (dd, 1 H, J1,F 46.6 Hz, H-1), 5.34
(dd, 1 H, J3,4 =J4,5 9.8 Hz, H-4), 5.32 (ddd, 1
H, J5,6 2.3 Hz, H-6), 5.11 (dd, 1 H, J3,FB1
Hz, H-3), 4.38 (dd, 1 H, J6,7a 5.1, J7a,7b 11.8
Hz, H-7a), 4.26 (dd, 1 H, J6,7b 7.3 Hz, H-7b),
3.83 (dd, 1 H, H-5), 2.23, 2.16, 2.08, 2.05, and
1
MHz for H using CDCl3 as solvent and
Me4Si as standard, unless stated otherwise.
13C NMR spectra were measured at 75.47
MHz and referenced to 1,4-dioxane (l 67.40).
Homo- and heteronuclear 2D NMR spec-
troscopy was performed with Bruker standard
software. TLC was performed on E. Merck
precoated plates (5×10 cm, layer thickness
0.25 mm, Silica Gel 60F254); spots were de-
tected by spraying with anisaldehyde–H2SO4.
For column chromatography, silica gel
(0.040–0.063 mm, 10 mm for HPLC) was
used. Concentration of solutions was per-
formed at reduced pressure at temperatures
B40 °C. Elemental analyses were provided by
Dr J. Theiner, Mikroanalytisches Laborato-
rium, Institut fu¨r Physikalische Chemie, Uni-
versita¨t Wien.
13
2.03 (5 s, each 3 H, 5 Ac); C NMR: l 105.3
(C-1, J1,F 260 Hz), 73.2 (C-5), 70.4 (C-3), 67.8
(C-2), 66.2 (C-6), 65.0 (C-4), 62.6 (C-7), 20.0–
21.0 (5 Ac).
Allyl 2,3,4,6,7-penta-O-acetyl-
-manno-heptopyranoside (5).—Solutions of 3
L
-glycero-h-
D
(19.2 mg, 0.045 mmol) and 4 (15.5 mg, 0.037
mmol) in dry CH2Cl2 (3 mL) containing allyl
alcohol (9.5 and 7.5 mL, respectively) were
,
stirred with 4 A molecular sieves (200 mg) for
1 h at rt under N2. Boron trifluoride etherate
(40 mL, 0.18 mmol) was added in four por-
tions over a period of 3 h to each batch. After
1 h, the conversion of 4 into 3 was observed
using TLC. After stirring for 5 h at rt, the
reaction was stopped by addition of triethyl-
amine (0.1 mL). The solutions were combined,
filtered over a pad of Celite and concentrated
to give a syrup which was treated with Ac2O
(0.1 mL) in pyridine (2 mL) for 20 h at rt. The
solution was concentrated and purified on sil-
ica gel (3:1“0:1 hexane–Et2O), followed by
chromatography on Sephadex LH-20 (2:1
CH2Cl2–MeOH) which gave 5 as a syrup.
2,3,4,6,7-Penta-O-acetyl-
no-heptopyranosyl fluoride (3) and 2,3,4,6,7-
penta-O-acetyl- -glycero-i- -manno-hepto-
L
-glycero-h- -man-
D
L
D
pyranosyl fluoride (4).—DAST (37 mL, 0.28
mL) was added to a solution of 1 (58.8 mg,
0.14 mmol) in dry CH2Cl2 (3 mL) at −30 °C
under Ar. The solution was warmed to rt and
stirred for 4 h. Methanol (0.5 mL) was added
at −30 °C, and the solution was diluted with
CH2Cl2 (30 mL), washed with aq NaHCO3,
dried (MgSO4) and concentrated. Purification
of the residue on silica gel (2:1“0:1 hexane–
Et2O) afforded 3 (34 mg, 58%) as a syrup;
1
Yield: 32 mg (85%). H NMR data were in
agreement with published data [14].
Allyl 2,3,4,6,7-penta-O-acetyl- -glycero-i-
L
1
[h]2D0 −8° (c 1.0, CHCl3); H NMR: l 5.59
D
-manno-heptopyranoside (6).—A solution of
2 (420 mg, 0.74 mol) in dry CH2Cl2 (10 mL)
(dd, 1 H, J1,2 1.8, J1,F 48.3 Hz, H-1), 5.42 (m,
1 H, H-2), 5.36 (dd, 1 H, J3,4 =J4,5 9.8 Hz,
H-4), 5.34 (dd, 1 H, J2,3 3.5 Hz, H-3), 5.32
(ddd, 1 H, J5,6 2.0 Hz, H-6), 4.34 (dd, 1 H,
J6,7a 5.3, J7a,7b 11.5 Hz, H-7a), 4.20 (dd, 1 H,
J6,7b 7.7 Hz, H-7b), 4.22 (dd, 1 H, H-5), 2.20,
2.14, 2.06, 2.04 and 2.01 (5 s, each 3 H, 5 Ac);
13C NMR: l 105.2 (C-1, J1,F 243 Hz), 71.2
(C-5), 68.7 (C-3), 68.0 (C-2), 67.0 (C-6), 64.5
(C-4), 62.4 (C-7), 21.2–20.0 (5 Ac). Anal.
Calcd for C17H23FO11: C, 48.35; H, 5.49.
Found: C, 48.12; H, 5.46.
containing allyl alcohol (0.1 mL, 1.47 mmol)
,
was stirred with 4 A molecular sieves (200 mg)
for 60 min at rt under Ar, then Me3SiOTf (15
mL) was added. The suspension was stirred for
15 h, diluted with CH2Cl2 (20 mL) and filtered
over Celite. The filtrate was concentrated and
treated with Ac2O (0.1 mL) as described be-
fore. Purification on silica gel (1:1 EtOAc–
toluene) afforded 5 (240 mg, 70%), followed
by 6 as the less-mobile isomer. Yield for 6: 37
1
mg (11%); [h]2D0 −45° (c 0.6, CHCl3); H
NMR: l 5.87 (m, 1 H, –CHꢀ), 5.50 (dd, 1 H,
J1,2 1.1, J2,3 3.5 Hz, H-2), 5.30 (t, 1 H, J3,4 =
J4,5 10.1 Hz, H-4), 5.28 (dq, 1 H, ꢀCH2 trans),
Further elution afforded a mixture of 3 and
4 (4 mg, 6%), then 4 (19.5 mg, 33%) as a
1
syrup; [h]2D0 −21° (c 1.0, CHCl3); H NMR:

A. Reiter et al. / Carbohydrate Research 317 (1999) 39–52
45
H, J7a,7b =J7a,6 8.5 Hz, H-7a), 4.58 (dd, 1 H,
J7b,6 6.0 Hz, H-7b), 4.22 and 4.10 (m, 2 H,
OCH2), 3.98 (dd, 1 H, J2,3 3.3 Hz, H-2), 3.83
(t, 1 H J3,4 =J4,5 =9.6 Hz, H-4), 3.78 (dd, 1
H, H-3) and 3.72 (dd, 1 H, J5,6 2.2 Hz, H-5).
Anal. Calcd for C11H16O8·0.5H2O: C, 46.31;
H, 5.99. Found: C, 46.46; H, 5.81.
5.27 (ddd, 1 H, J5,6 2.3, J6,7a 5.1, J6,7b 7.5 Hz,
H-6), 5.25 (dq, 1 H, ꢀCH2 cis), 5.04 (dd, 1 H,
H-3), 4.67 (d, 1 H, H-1), 4.44 (t, 1 H, J7a,7b
11.2 Hz, H-7a), 4.58 (dd, 1 H, H-7b), 4.35 and
4.12 (m, 2 H, OCH2), 4.18 (dd, 1 H, H-7b),
3.66 (dd, 1 H, H-5), 2.22, 2.13, 2.06, 2.01 and
1.98 (5 s, each 3 H, 5 Ac). Anal. Calcd for
C20H28O12: C, 52.17; H, 6.13. Found: C, 52.46;
H, 5.98.
Allyl 2,3,4-tri-O-acetyl-6,7-O-carbonyl-
L-
glycero-h- -manno-heptopyranoside (10).—A
D
Allyl
L
-glycero-i- -manno-heptopyranoside
D
solution of 9 (20 mg, 0.072 mmol) in pyridine
(8 mL) was stirred with Ac2O (0.25 mL) and a
catalytic amount of DMAP for 4 h at rt.
Methanol (0.5 mL) was added, and the solu-
tion was coevaporated three times with
toluene and concentrated. Purification of the
residue on silica gel (1:1 toluene–EtOAc) gave
10 as a syrup (29 mg, ꢀquant.); [h]2D0 +117°
(8).—A solution of 6 (6.4 mg, 0.014 mmol) in
dry MeOH (2 mL) was stirred with 0.1 M
methanolic NaOMe (0.2 mL) for 2 h at rt.
The solution was deionized by adding Dowex
50 (H+) resin, filtered and evaporated to dry-
ness to give 8 as a colorless syrup. Yield: 3.1
1
mg (89%); [h]2D0 −45° (c 0.3, H2O); H NMR
(D2O): l 5.98 (m, 1 H, –CHꢀ), 5.35 (dq, 1 H,
ꢀCH2 trans), 5.28 (dq, 1 H, ꢀCH2 cis), 4.70 (d, 1
H, J1,2 1.0 Hz, H-1), 4.34 and 4.20 (m, 2 H,
OCH2), 4.02 (ddd, 1 H, J5,6 1.7, J6,7a 6.4, J6,7b
7.0 Hz, H-6), 3.99 (dd, 1 H, J2,3 3.4 Hz, H-2),
3.83 (t, 1 H, J3,4=J4,5 10.0 Hz, H-4), 3.73 (dd,
1 H, H-7a), 3.73 (dd, 1 H, H-7b), 3.64 (dd, 1
H, H-3) and 3.31 (dd, 1 H, H-5).
1
(c 1.0, CHCl3); H NMR: l=5.89 (m, 1 H,
–CHꢀ), 5.50 (t, 1 H, J3,4=J4,5 =9.9 Hz, H-4),
5.39 (dd, 1 H, J2,3 3.3 Hz, H-3), 5.32 (dq, 1 H,
ꢀCH2 trans), 5.27 (dq, 2 H, ꢀCH2 cis, H-6), 5.22
(dd, 1 H, J1,2 1.9 Hz, H-2), 4.93 (d, 1 H, H-1),
4.79 (ddd, 1 H, J7a,6 8.3, J6,5 1.3, J7b,6 5.5 Hz,
H-6), 4.52 (t, 1 H, J7a,7b 8.3 Hz, H-7a), 4.40
(dd, 1 H, H-7b), 4.18 and 4.07 (m, 2 H,
OCH2), 3.84 (dd, 1 H, H-5), 2.17, 2.07 and
2.02 (3 s, each 3 H, 3 Ac). Anal. Calcd for
C17H22O11: C, 50.75; H, 5.51. Found: C, 50.50;
H, 5.24.
Allyl 6,7-O-carbonyl-
L
-glycero-h- -manno-
D
heptopyranoside (9).—A solution of 5 (175
mg, 0.38 mmol) in dry MeOH (5 mL) was
stirred with 0.1 M methanolic NaOMe (1 mL)
for 2 h at rt. The pH of the solution was
neutralized by addition of Dowex 50 (H+)
resin, the resin was filtered off and the filtrate
Allyl 7-O-carbamoyl-
L
-glycero-h- -manno-
D
heptopyranoside (11)
Method A. To a solution of 9 (38 mg, 0.138
mmol) in THF (3 mL) solid NH4HCO3 (250
mg) was added, followed by addition of two
portions of 25% aq NH3 (0.5 mL each) during
2 h at rt. The solution was concentrated and
coevaporated three times with MeOH. The
residue was crystallized from EtOH to give 11
as colorless needles. Yield: 23 mg (57%), mp
was evaporated to give allyl
L
-glycero-a- -
D
manno-heptopyranoside (7) as colorless
needles; mp 130 °C (EtOH). NMR data were
in agreement with published data [14]. A solu-
tion of 7 (92 mg, 0.37 mmol) and sym-col-
lidine (3.8 mmol, 0.5 mL) in dry THF (10 mL)
was cooled to −30 °C under argon. A solu-
tion of diphosgene (40 mL, 0.33 mmol) in
THF (4 mL) was added during 5 h. The
reaction was quenched by addition of MeOH
(0.5 mL), the suspension was diluted with
THF (5 mL), centrifuged, and the supernatant
was concentrated. Purification of the residue
on silica gel (4:4:1 toluene–EtOAc–EtOH) af-
forded 9 as colorless syrup. Yield: 78 mg
1
154 °C (EtOH); [h]2D0 +56° (c 0.5, H2O); H
NMR (D2O): l 5.94 (m, 1 H, –CHꢀ), 5.32
(dq, 1 H, ꢀCH2 trans), 5.24 (dq, 1 H, ꢀCH2 cis),
4.89 (d, 1 H, J1,2 1.7 Hz, H-1), 4.19 (ddd, 1 H,
J7a,6 8.0, J6,5 1.5, J7b,6 5.5 Hz, H-6), 4.18–4.13
(m, 1 H, OCH2), 4.14–4.12 (m, 2 H, H-7a,
7b), 3.91 (dd, 1 H, J2,3 3.3 Hz, H-2), 3.84 (t, 1
H, J3,4 =J4,5 9.6 Hz, H-4), 3.77 (dd, 1 H, H-3)
and 3.59 (dd, 1 H, H-5). Anal. Calcd for
C11H19NO8: C, 45.05; H, 6.53; N, 4.78. Found:
C, 44.82; H, 6.30; N, 4.61.
1
(76%); [h]2D0 +103° (c 0.7, H2O); H NMR
(CD3OD–CDCl3): l 5.95 (m, 1 H, –CHꢀ),
5.32 (dq, 1 H, ꢀCH2 trans), 5.28 (dq, 2 H, ꢀCH2 cis
,
H-6), 4.95 (d, 1 H, J1,2 1.8 Hz, H-1), 4.70 (t, 1

46
A. Reiter et al. / Carbohydrate Research 317 (1999) 39–52
Benzyl (6,7-O-carbonyl-
no - heptopyranosyl) - (1“3) - 2,5,6,7 - tetra - O-
benzyl- -glycero-h- -manno-heptofuranoside
L
-glycero-h- -man-
D
The mother liquor was evaporated to dry-
ness giving 15 mg (37%) of a 3:1 mixture of 11
1
L
D
and 12. H NMR (D2O) for 12: l 5.13 (ddd, 1
(17).—A solution of 15 (120 mg, 0.11 mmol)
in dry MeOH (3 mL) was stirred with 0.1 M
methanolic MeONa (0.3 mL) for 2 h at rt.
The reaction mixture was neutralized with
Dowex 50 (H+) resin, filtered and concen-
trated under reduced pressure to give crude
H, J7a,6 ꢀJ7b,6 ꢀ5.8, J6,5 1.6 Hz, H-6), 4.90 (d,
1 H, J1,2 1.3 Hz, H-1), 3.68 (dd, 1 H, J4,5 9.6
Hz, H-5); 13C NMR: l 72.78 (C-6), 71.49
(C-3), 70.62 (C-2), 69.09 (OCH2), 66.91 (C-4),
61.47 (C-7).
Method B. A solution of 10 (11.4 mg, 0.028
mmol) in dry THF (3 mL) was stirred with
NH4HCO3 (160 mg) and 25% aq NH3 (1.6
mL) for 24 h at ambient temperature. Addi-
tional NH4HCO3 (150 mg) and 25% aq NH3
(1.5 mL) were added and stirring was contin-
ued for 24 h. The solution was concentrated
and the residue was extracted with MeOH.
Evaporation of the methanolic solution af-
forded 8.2 mg (98%) of a 10:1 mixture of 11
and 12.
benzyl
L
-glycero-a-
D
-manno-heptopyranosyl-
-glycero-a- -
(1“3)-2,5,6,7-tetra-O-benzyl-
L
D
manno-heptofuranoside (16). The residue was
coevaporated with dry toluene (10 mL) and
redissolved in dry CH2Cl2 (5 mL). sym-Col-
lidine (182 mg, 0.19 mL, 1.5 mmol) was
added, and the solution was cooled to
−40 °C under N2. A solution of trichloro-
methyl chloroformate (0.09 mmol, 11 mL) in
CH2Cl2 (8 mL) was added dropwise under
vigorous stirring at −40 °C under N2 during
4 h at a rate of 2 mL/h. The reaction was
stopped by addition of dry MeOH (0.2 mL),
and the reaction mixture was concentrated to
dryness. Purification of the residue by chro-
matography on silica gel (99:1“9:1 CH2Cl2–
MeOH) gave 17 as a colorless syrup. Yield 93
3-(2-Ammoniumethylthio)propyl7-O-carbam-
oyl-
L
-glycero-h- -manno-heptopyranoside ac-
D
etate (13).—A solution of 11 (7.5 mg, 0.026
mmol) and cysteamine hydrochloride (8.4 mg,
0.074 mmol, recrystallized from MeOH) in
water (0.1 mL) was placed in a quartz vial and
irradiated at 254 nm for 90 min at rt. The
solution was purified by chromatography on
silica gel (7:2:1 EtOAc–HOAc–H2O) to re-
move excess of reagent followed by elution
with 3:2:1 EtOAc–HOAc–H2O to give the
spacer compound 13, which was further
purified on Sephadex LH-20 (MeOH). Yield:
mg (95%); [h]2D0 +44.5° (c 1.0, CHCl3); H
1
NMR: l 7.39–7.22 (m, 25 H, 5 Ph), 5.24 (d, 1
H, J1,2 4.6 Hz, H-1), 4.85 (s, 1 H, J1%,2% 1.1 Hz,
H-1%), 4.77 (d, 1 H, J 11.2 Hz, CHPh), 4.75 (d,
1 H, J 11.8 Hz, CHPh), 4.68 (d, 1 H, J 12.1
Hz, CHPh), 4.66 (d, 1 H, J 11.6 Hz, CHPh),
4.62 (d, 1 H, J 11.8 Hz, CHPh), 4.55 (d, 1 H,
J 12.1 Hz, CHPh), 4.53 (s, 1 H, CHPh), 4.53
(d, 1 H, J 11.9 Hz, CHPh), 4.52 (s, 1 H,
CHPh), 4.50 (ddd, 1 H, J5%,6% 2.2, J6%,7a% 6.2,
J6%,7b% 8.5 Hz, H-6%), 4.47 (dd, 1 H, J3,4 2.1, J4,5
8.2 Hz, H-4), 4.40 (d, 1 H, J 1.1 Hz, CHPh),
4.24 (dd, 1 H, J2,3 4.6 Hz, H-3), 4.05–3.96 (m,
5 H, H-2, 5, 6, 5%, 7a%), 3.82 (m, 1 H, H-2%),
3.76 (d, 2 H, H-7a, 7b), 3.70 (m, 2 H, H-3%, 4%),
3.51 (dd, 1 H, H-7b%); 13C NMR (CDCl3): l
155.05 (CO), 105.07 (C-1%), 100.92 (C-1). Anal.
Calcd for C50H54O14·0.5H2O: C, 67.63; H,
6.24. Found: C, 67.82; H, 6.22.
11.5 mg (ꢀquant.), colorless syrup; [h]D20
+
1
29° (c 0.3, H2O); H NMR (D2O): l 4.87 (d, 1
H, H-1), 4.25–4.20 (m, 2 H, H-6, 7a), 4.15
(dd, 1 H, J7a,7b 11.9, J7b,6 8.7 Hz, H-7b), 3.92
(dd, 1 H, J1,2 1.7, J2,3 3.3 Hz, H-2), 3.87 (t, 1
H, J3,4=J4,5 9.5 Hz, H-4), 3.78 (dd, 1 H, H-3),
3.74 (t, 1 H, OCH2), 3.59 (dd, 1 H, H-5), 3.59
(t, 1 H, OCH2), 3.21 (t, 2 H, NCH2), 2.85 (t, 2
H, SCH2), 2.67 (dt, 2 H, SCH2), 1.95 (s, 3 H,
13
Ac) and 1.92 (m, 2 H, CH2); C NMR (D2O):
l 182.32 (CO), 160.06 (CONH2), 100.65 (C-1),
72.01, 71.66 (C-5,3), 70.85 (C-2), 67.08 (C-4),
66.84 (C-6), 66.68 (C-7, OCH2), 39.10
(NCH2), 28.95, 28.22 (2 SCH2), 27.83 (CH2)
Benzyl (2,3,4-tri-O-acetyl-6,7-O-carbonyl-
glycero-h- -manno-heptopyranosyl)-(1“3)-
2,5,6,7-tetra-O-benzyl- -glycero-h- -manno-
L-
D
and
24.05
(Ac).
Anal.
Calcd
for
L
D
C13H26N2O8S·CH3COOH·0.5H2O: C, 40.99;
H, 7.11; N, 6.37. Found: C, 40.48; H, 6.96; N,
6.14.
heptofuranoside (18).—A solution of 17 (10.5
mg, 0.012 mmol), 4-dimethylaminopyridine (2
mg, 0.01 mmol), and Ac2O (10 mL) in dry

A. Reiter et al. / Carbohydrate Research 317 (1999) 39–52
47
(ddd, 1 H, J5,6 2.3, J6,7a=J6,7b 6.1 Hz, H-6),
4.01 (dd, 1 H, H-5), 4.00 (dd, 1 H, J2,3 4.5
Hz, H-2), 3.91–3.84 (m, 4 H, H-2%, 3%, 5%, 6%),
3.79 (m, 2 H, H-4%, 7b%), 3.70 (m, 2 H, H-7a,
7b); 13C NMR (CDCl3): l 157.40 (CONH2),
104.59 (C-1%), 99.92 (C-1). Anal. Calcd for
C50H57NO14·H2O: C, 65.70; H, 6.51; N, 1.53.
Found: C, 66.24; H, 6.62; N, 1.54.
pyridine (2 mL) was stirred for 20 h at rt.
The reaction mixture was concentrated, co-
evaporated with toluene (15 mL) and the
residue was purified on silica gel (93:7“
85:15 toluene–EtOAc) affording 18 as a
syrup. Yield 11.8 mg (98%); [h]2D0 +60° (c
1
1.0, CHCl3); H NMR: l 7.37–7.15 (m, 25
H, 5 Ph), 5.35 (d, 1 H, J1,2 4.8 Hz, H-1),
5.35–5.27 (m, 2 H, H-2%, 3%), 5.27 (dd, 1 H,
J3%,4%ꢀJ4%,5% 9.1 Hz, H-4%), 4.83 (d, 1 H, J 11.5
Hz, CHPh), 4.79 (d, 1 H, J 11.6 Hz, CHPh),
4.78 (s, 1 H, H-1%), 4.73 (d, 1 H, J 10.6 Hz,
CHPh), 4.71 (d, 1 H, J 12.1 Hz, CHPh), 4.68
(d, 1 H, J 11.5 Hz, CHPh), 4.57 (d, 1 H, J
11.7 Hz, CHPh), 4.56 (s, 3 H, 3 CHPh), 4.48
(dd, 1 H, H-4), 4.36 (d, 1 H, J 10.6 Hz,
CHPh), 4.30 (dd, 1 H, J4%,5% 9.1 Hz, H-5%),
4.25 (dd, 1 H, J3,4 2.0 Hz, H-3), 4.11 (dd, 1
H, J1,2 4.7, J2,3 4.5 Hz, H-2), 4.07 (ddd, 1 H,
J6,7a 6.0, J6,7b 8.3 Hz, H-6), 3.99 (dd, 1 H, J4,5
8.7, J5,6 2.0 Hz, H-5), 3.98 (dd, 1 H, H-7a%),
3.88 (ddd, 1 H, J6%,7a% 6.0, J6%,7b% 8.5, J6%,5% 1.4
Hz, H-6%), 3.81–3.79 (m, 2 H, H-7a, 7b), 3.42
(t, 1 H, J7a%,7b% 8.4 Hz, H-7b%), 2.05, 2.02, and
1.99 (3 s, each 3 H, 3 Ac). Anal. Calcd for
C56H60O17: C, 66.92; H, 6.02. Found: C,
66.83; H, 5.86.
7-O-Carbamoyl-
topyranosyl-(1“3)-
L
-glycero-h-
-glycero-
D
-manno-hep-
L
D
-manno-hep-
topyranose (20).—Compound 19 (14 mg,
0.016 mmol) was dissolved in MeOH (10 mL)
and hydrogenated over 10% Pd–C (0.05 g)
under H2 at atmospheric pressure at ambient
temperature for 16 h. The solution was
filtered and concentrated under reduced pres-
sure. The residue was taken up in water and
purified on a Bio-gel P-2 column (2.5×100
cm, 1% aq NaCl, 1% aq NH4HCO3) to af-
ford 20 as a white solid after lyophiliza-
tion.Yield 6.8 mg (98%); [h]2D0 +17° (c 0.4,
1
H2O); H NMR (D2O) for the a anomer: l
5.20 (d, 1 H, H-1%), 5.13 (d, 1 H, J1,2 1.7 Hz,
H-1), 4.21 (m, 1 H, H-6%), 4.18 (dd, 1 H,
J6%,7a% 6.7 Hz, H-7a%), 4.11 (dd, 1 H, J6%,7b% 7.9
Hz, H-7b%), 4.07 (dd, 1 H, J1%,2% 1.7 Hz, H-2%),
4.01 (ddd, 1 H, H-6), 3.97 (dd, 1 H, J1,2 1.7
Hz, H-2), 3.93 (dd, 1 H, H-4), 3.92 (dd, 1 H,
J2,3 3.5, J3,4 10.5 Hz, H-3), 3.91 (dd, 1 H, J4%,5%
6.0 Hz, H-4%), 3.90 (dd, 1 H, J2%,3% 2.3, J3%,4% 6.8
Hz, H-3%), 3.77 (dd, 1 H, J5%,6% 1.1 Hz, H-5%),
3.71 (dd, 1 H, J4,5 10.2, J5,6 1.3 Hz, H-5),
3.69 (dd, 1 H, J6,7a 7.2 Hz, H-7a), 3.67 (dd, 1
Benzyl (7-O-carbamoyl-
no - heptopyranosyl) - (1“3) - 2,5,6,7 - tetra - O-
benzyl- -glycero-h- -manno-heptofuranoside
L
-glycero-h- -man-
D
L
D
(19).—To a stirred solution of 17 (32.8 mg,
0.037 mmol) in MeOH (5 mL), ammonium
hydrogenocarbonate (300 mg) and 25% aq
NH3 (1 mL) were added. The suspension was
vigorously stirred for 24 h at rt, the reaction
mixture was concentrated under reduced
pressure. Purification of the residue on silica
gel (99:1“93:7 CH2Cl2–MeOH), followed by
a second purification by HPLC (10:1 CHCl3–
MeOH), afforded 19 as a solid. Yield: 22 mg
1
H, J7a,7b 10.8, J6,7b 5.8 Hz, H-7b); H NMR
(D2O) for the b anomer: l 5.22 (d, 0.3 H,
H-1%), 4.87 (d, 0.3 H, J1,2 1.0 Hz, H-1), 3.35
(dd, 0.3 H, J4,5 9.8, J5,6 1.8 Hz, H-5). Anal.
Calcd for C15H27NO14·2H2O: C, 37.43; H,
6.49; N, 2.91. Found: C, 37.42; H, 6.28; N,
2.78.
1
(66%); [h]2D0 +38° (c 1.0, CHCl3); H NMR
(2,3,4-Tri-O-acetyl-6,7-O-carbonyl-
ero-h- -manno-heptopyranosyl)-(1“3)-1,2,4,-
6,7-penta-O-acetyl- -glycero- -manno-hepto-
L
-glyc-
(CDCl3 –CD3OD): l 7.34–7.24 (m, 25 H, 5
Ph); 5.14 (d, 1 H, J1,2 3.4 Hz, H-1), 4.95 (d, 1
D
L
D
H, J1% 1.6 Hz, H-1%), 4.75 (d, 1 H, J 11.3
2%
pyranose (21).—Compound 17 (88 mg, 0.1
mmol) was dissolved in MeOH (10 mL) and
hydrogenated under atmospheric pressure of
H2 over 10% Pd–C (0.1 g) for 16 h at 20 °C.
The solution containing crude (6,7-O-car-
Hz, CHPh), 4.68 (d, 1 H, J 12.2 Hz, CHPh),
4.66 (d, 1 H, J 11.0 Hz), 4.63 (s, 3 H, 3
CHPh), 4.62 (s, 1 H, CHPh), 4.50 (s, 1 H,
CHPh), 4.49 (s, 1 H, CHPh), 4.50 (dd, 1 H,
J3,4 3.5, J4,5 8.2 Hz, H-4), 4.44 (d, 1 H, J 12.0
Hz, CHPh), 4.42 (m, 1 H, H-3), 4.13 (dd, 1
H, J6%,7a% 8.9, J7a%,7b% 12.3 Hz, H-7a%), 4.04
bonyl-
L
-glycero-a-
D
-manno-heptopyranosyl)-
(1“3)-
L
-glycero- -manno-heptopyranose was
D

48
A. Reiter et al. / Carbohydrate Research 317 (1999) 39–52
1
CHCl3); H NMR: l 8.85 (s, 1 H, NH), 6.25
(d, 1 H, J1,2 1.5 Hz, H-1), 5.50 (dd, 1 H,
J3%,4%=J4%,5% 9.8 Hz, H-4%), 5.46 (dd, 1 H, J3,4 =
J4,5 10.0 Hz, H-4), 5.42 (m, 1 H, H-2), 5.25–
5.20 (m, 2 H, H-3%, H-6), 5.11 (d, 1 H, J1%,2% 1.8
Hz, H-1%), 4.94 (dd, 1 H, J2%,3% 3.2 Hz, H-2%),
4.77 (ddd, 1 H, J5%,6% 1.8, J6%,7b% 5.3 Hz, H-6%),
4.50 (dd, 1 H, J6%,7a% 8.5, J7a%,7b% 8.5 Hz, H-7a%),
4.36 (dd, 1 H, H-7b%), 4.29-4.14 (m, 4 H,
H-3,5,7a,7b), 4.02 (dd, 1 H, H-5%), 2.32 (s, 3
H), 2.17 (s, 3 H), 2.16 (s, 6 H), 2.09 (s, 3 H)
and 2.03 (s, 6 H, total 7 Ac).
filtered over Celite and concentrated under
reduced pressure. The residue was coevapo-
rated with dry toluene (5 mL), redissolved in
pyridine (2 mL) and treated with Ac2O (0.2
mL) overnight at rt. The reaction mixture was
concentrated, and coevaporated with toluene
(10 mL). Purification of the residue on silica
gel (1:1“2:3 toluene–EtOAc) afforded 21 as
a 5:1 a/b mixture. Yield 68 mg (91%);
1H NMR for the a isomer: l 5.97 (d, 1 H, J1,2
1.7 Hz, H-1), 5.38 (dd, 1 H, J3%,4%=J4%,5% 9.8 Hz,
H-4%), 5.27 (dd, 1 H, J3,4 =J4,5 10.0 Hz, H-4),
5.14 (dd, 1 H, H-2), 5.11 (dd, 1 H, J2%,3% 3.5 Hz,
H-3%), 5.10 (ddd, 1 H, J5,6 1.9, J6,7a 5.5, J6,7b
7.0 Hz, H-6), 4.99 (d, 1 H, J1%,2% 2.0, H-1%), 4.84
(dd, 1 H, H-2%), 4.67 (ddd, 1 H, J5%,6% 1.8, J6%,7b%
5.3, J6%,7a% 8.3 Hz, H-6%), 4.38 (dd, 1 H, J7a%,7b%
8.5 Hz, H-7a%), 4.28 (dd, 1 H, H-7b%), 4.18 (dd,
1 H, J7a,7b 11.5 Hz, H-7a), 4.08 (dd, 1 H,
H-7b), 4.07 (dd, 1 H, J2,3 3.5 Hz, H-3), 3.95
(dd, 1 H, H-5), 3.87 (dd, 1 H, H-5%), 2.27,
2.19, 2.16, 2.15, 2.13, 2.08, 2.04, and 2.01 (8 s,
each 3 H, 8 Ac). Anal. Calcd for C31H40O22:
C, 48.70; H, 5.27. Found: C, 48.98; H, 5.09.
(2,3,4-Tri-O-acetyl-6,7-O-carbonyl-
cero-h- -manno-heptopyranosyl)-(1“3)-2,4,-
6,7-tetra-O-acetyl- -glycero-h- -manno-hep-
L
-gly-
D
L
D
topyranose fluoride (24).—To a solution of 22
(22 mg, 0.03 mmol) in CH2Cl2 (3 mL) under
nitrogen, diethylaminosulfur trifluoride (20
mL, 0.15 mmol) was added at −30 °C. After
stirring for 3 h at 20 °C, the mixture was
cooled (−30 °C), excess DAST was quenched
with MeOH (0.1 mL), and the mixture was
concentrated under reduced pressure. The re-
sulting oil was taken up in CH2Cl2 (10 mL),
washed with H2O, 10% aq NaHCO3, H2O,
dried over MgSO4, filtered and concentrated
to give an oil. Purification on silica gel (1:1“
0:1 hexane–Et2O) gave 24 as a syrup (13 mg,
59%), a 1:1 mixture of 24 and a-fluoride (6
mg, 27%) and b-fluoride (1 mg, 4%); [h]D20
(2,3,4-Tri-O-acetyl-6,7-O-carbonyl-
ro-h- -manno-heptopyranosyl)-(1“3)-2,4,6,-
7-tetra-O-acetyl- -glycero-h- -manno-hepto-
L
-glyce-
D
L
D
pyranose trichloroacetimidate (23).—A solu-
tion of 21 (50 mg, 0.065 mmol) and hydrazine
acetate (6.4 mg. 0.07 mmol) in dry DMF (2
mL) was stirred for 4 h. The reaction mixture
was dissolved in CH2Cl2, and was sequentially
washed with water, 10% aq NaHCO3, and
water. The organic layer was dried over
MgSO4 and concentrated under reduced pres-
sure. The product was purified by silica gel
column chromatography (3:2“7:3 EtOAc–
toluene), which afforded (2,3,4-tri-O-acetyl-6,7-
1
+42° (c 1.0, CHCl3); H NMR: l=5.57 (dd,
1 H, J1,2 1.0, J1,F 49.2 Hz, H-1), 5.49 (dd, 1 H,
J3%,4%=J4%,5% 9.8 Hz, H-4%), 5.40 (dd, 1 H, J3,4 =
J4,5 10.2 Hz, H-4), 5.36 (m, 1 H, H-2), 5.26
(ddd, 1 H, J5,6 1.9 Hz, H-6), 5.21 (dd, 1 H,
J3%,4% 10, J2%,3% 3.2 Hz, H-3%), 5.07 (d, 1 H, J1%,2%
1.7, H-1%), 4.91 (dd, 1 H, H-2%), 4.77 (ddd, 1
H, J5%,6% 1.3 Hz, H-6%), 4.55 (dd, 1 H, J6%,7a%
=
O-carbonyl-
anosyl)-(1“3)-2,4,6,7-tetra-O-acetyl-
cero- -manno-heptopyranose (22) as a syrup.
L
-glycero-a-
D
-manno-heptopyr-
J7a%,7b% 8.5 Hz, H-7a%), 4.40 (dd, 1 H, J6%,7b% 5.2
Hz, H-7b%), 4.32 (dd, 1 H, J6,7a 5.6, J7a,7b 11.4
Hz, H-7a), 4.19 (dd, 1 H, J6,7b 7.6 Hz, H-7b),
4.14 (dd, 1 H, H-5), 4.13 (dd, 1 H, H-3), 3.95
(dd, 1 H, H-5%), 2.36, 2.28, 2.15, 2.14, 2.08,
2.07 and 2.01 (7 s, each 3 H, 7 Ac); 13C NMR:
l 170.43, 170.38, 170.12, 170.11, 169.65,
169.58, 169.32 (7 CO, COCH3), 154.32 (CO),
105.14 (C-1, JC-1,F 223 Hz), 99.10 (C-1%), 74.56
(C-3), 72.94 (C-6%), 71.16 (C-5), 70.52 (C-5%),
69.59 (C-2%), 68.69 (C-2, JC-2,F 40.7 Hz), 67.65
(C-3%), 66.37 (C-6), 65.75 (C-4%), 65.40 (C-7%),
65.30 (C-4), 61.80 (C-7); 1H NMR data for the
L
-gly-
D
Yield: 43 mg (91%). A suspension of 22 (40
mg, 0.055 mmol), K2CO3 (70 mg) and
trichloroacetonitrile (70 mL) in dry CH2Cl2 (5
mL) was stirred for 20 h at ambient tempera-
ture. The reaction mixture was diluted with
toluene (5 mL), filtered and the filtrate was
concentrated. The residue was purified by
flash column chromatography on silica gel
(1:1“2:3 toluene–EtOAc) to give 23 as a
syrup. Yield: 41 mg (86%); [h]2D0 +57° (c 1.0,

A. Reiter et al. / Carbohydrate Research 317 (1999) 39–52
49
[h]2D0 −0.5° (c 0.7, CHCl3); H NMR: l 5.88
(m, 1 H, –CHꢀ), 5.49 (dd, 1 H, J3%,4%=J4%,5% 9.9
Hz, H-4%), 5.37 (dd, 1 H, J3,4 9.9, J4,5 9.8 Hz,
H-4), 5.32 (dq, 1 H, ꢀCH2 trans), 5.26 (dq, 1 H,
ꢀCH2 cis), 5.22 (dd, 1 H, J1,2 1.6 Hz, H-2),
5.25–5.20 (m, 2 H, H-6, H-3%), 5.06 (d, 1 H,
J1%,2% 1.9 Hz, H-1%), 4.93 (dd, 1 H, J2%,3% 3.2 Hz,
H-2%), 4.87 (d, 1 H, H-1), 4.78 (ddd, 1 H, J5%,6%
1.3 Hz, H-6%), 4.55 (dd, 1 H, J6%,7a% 8.5, J7a%,7b%
8.4 Hz, H-7a%), 4.44 (dd, 1 H, J6%,7b% 5.3 Hz,
H-7b%), 4.30 (dd, 1 H, J6,7a 6.2, J7a,7b 11.2 Hz,
H-7a), 4.23 (dd, 1 H, J6,7b 7.4 Hz, H-7b), 4.22
(dd, 1 H, J2,3 3.3 Hz, H-3), 4.18–4.15 (m, 1 H,
OCH2), 4.02 (dd, 1 H, J5,6 1.7 Hz, H-5),
4.02–3.96 (m, 1 H, OCH2), 3.96 (dd, 1 H,
H-5%), 2.26, 2.17, 2.16, 2.14, 2.09, 2.07, and
2.02 (7 s, each 3 H, 7 Ac). ‘Gated decoupled’
13C NMR: l 99.50 (1JC-1%,H-1%ꢀ172.0 Hz),
97.20 (1JC-1,H-1 ꢀ171.0 Hz); 13C NMR: l
170.8–169.0 (7 COCH3), 160.0 (CO), 133.50
(–CHꢀ), 119.0 (ꢀCH2), 99.48 (C-1%), 97.22 (C-
1), 75.82 (C-3), 73.30 (C-6%), 71.06 (C-2), 73.50
(C-5), 70.08 (C-2%), 68.24 (C-5%), 68.04 (OCH2),
68.0 (C-3), 67.55 (C-6), 66.48 (C-4), 66.05
(C-4%), 65.85 (C-7%), 61.82 (C-7), 21.15–20.05
(7 CH3). Anal. Calcd for C32H42O21: C, 50.40;
H, 5.55. Found: C, 50.65; H, 5.31.
1
the b-fluoride: l 5.20 (m, 1 H, H-2), 5.47 (dd,
1 H, J3%,4%=J4%,5% 9.9 Hz, H-4%), 5.42 (dd, 1 H,
J1,2 1.0, J1,F 47.7 Hz, H-1), 5.35 (dd, 1 H,
J3,4 =J4,5 10.1 Hz, H-4), 5.26 (ddd, 1 H, J5,6
1.9 Hz, H-6), 5.22 (dd, 1 H, J2%,3% 3.2 Hz, H-3%),
5.03 (d, 1 H, J1%,2% 2.0 Hz, H-1%), 4.93 (dd, 1 H,
H-2%), 4.77 (ddd, 1 H, J5%,6% 1.9 Hz, H-6%), 4.56
(dd, 1 H, J6%,7a%=J7a%,7b% 8.5 Hz, H-7a%), 4.48
(dd, 1 H, J6%,7b% 5.4 Hz, H-7b%), 4.37 (dd, 1 H,
J6,7a 5.3, J7a,7b 11.7 Hz, H-7a), 4.26 (dd, 1 H,
J6,7b 6.9 Hz, H-7b), 3.96 (dd, 1 H, H-5%), 4.13
(dd, 1 H, J2,3 4.0, H-3), 3.73 (dd, 1 H, H-5),
2.31 (s, 3 H), 2.17 (s, 6 H), 2.14, 2.09, 2.08,
and 2.02 (3 s, each 3 H, total 7 Ac).
Allyl (2,3,4-tri-O-acetyl-6,7-O-carbonyl-
glycero-h- -manno-heptopyranosyl)-(1“3)-
2,4,6,7-tetra-O-acetyl- -glycero-h- -manno-
heptopyranoside (25), allyl (2,3,4-tri-O-acetyl-
6,7-O-carbonyl- -glycero-h- -manno-hepto-
pyranosyl)-(1“3)-4,6,7-tri-O-acetyl- -gly-
cero-h- -manno-heptopyranoside (26) and allyl
(2,3,4-tri-O-acetyl-6,7-O-carbonyl- -glycero-h-
-manno-heptopyranosyl)-(1“3)-4,6,7-tri-O-
L
-
D
L
D
L
D
L
D
L
D
acetyl-
(27)
L
-glycero-i- -manno-heptopyranoside
D
Method A. A solution of glycosyl donor 23
(35 mg, 0.04 mmol) and allyl alcohol (4.8 mg,
5.6 mL, 0.08 mmol) in dry CH2Cl2 (5 mL) was
stirred at ambient temperature with powdered
Final purification of crude 26 by chro-
matography (99:1“95:5 CH2Cl2–MeOH) fur-
nished pure 26 as a syrup. Yield: 8.5 mg
,
4 A molecular sieves for 1 h under N2. A
solution of Me3SiOTf (2.1 mmol, 0.47 mg, 0.4
mL) in CH2Cl2 (100 mL) was added to the
mixture, followed by addition of the same
amount every 30 min until the formation of
three products was observed (total amount of
Me3SiOTf: 10.5 mmol, 2.4 mg, 2.0 mL). The
reaction mixture was neutralized with Na2CO3
(200 mg), filtered over a pad of Celite, and the
filtrate was concentrated. The residue was
taken up in CH2Cl2 (100 mL), extracted with
water, 10% aq NaHCO3, water, dried over
MgSO4, and concentrated to an oil. Purifica-
tion by chromatography on silica gel (7:3“
2:3 toluene–EtOAc), followed by a second
purification on silica gel (100:0“97:3 CHCl3–
MeOH), afforded 25 (7 mg, 23%) as a syrup
(Rf 0.5 in 1:2 toluene–EtOAc), followed by 26
(9 mg, 30%) as a syrup (Rf 0.2) and 27 (10 mg,
33%) as a syrup (Rf 0.3).
1
(29%); [h]2D0 +11.6° (c 0.2, CHCl3); H NMR:
l 5.88 (m, 1 H, –CHꢀ), 5.49 (dd, 1 H, J3%,4%
=
J4%,5% 10.0 Hz, H-4%), 5.33 (dd, 1 H, J3,4 10.4,
J4,5 10.2 Hz, H-4), 5.33–5.25 (m, 2 H, ꢀCH2),
5.27 (ddd, 1 H, J5,6 1.8, J6,7a 5.9, J6,7b 7.2 Hz,
H-6), 5.26 (dd, 1 H, H-3%), 5.07 (dd, 1 H, J1%,2%
2.1, J2%,3% 5.2 Hz, H-2%), 5.06 (d, 1 H, H-1%),
4.94 (d, 1 H, J1,2 B1.0 Hz, H-1), 4.74 (ddd, 1
H, J5%,6% 1.3, J6%,7a% 4.9, J6%,7b% 8.4 Hz, H-6%), 4.51
(dd, 1 H, H-7a%), 4.46 (dd, 1 H, J7a%,7b% 8.5 Hz,
H-7b%), 4.30 (dd, 1 H, J6,7a 5.9 Hz, H-7a), 4.21
(dd, 1 H, J6,7b 7.2, J7a,7b 11.1 Hz, H-7b),
4.16–4.20 (m, 1 H, OCH2), 4.10 (dd, 1 H,
H-5%), 4.04 (dd, 1 H, J2,3 3.2 Hz, H-3), 4.03 (m,
1 H, H-2), 4.01 (dd, 1 H, H-5), 3.99–3.96 (m,
1 H, OCH2), 2.17 (s, 3 H), 2.14 (s, 6 H), 2.08,
2.07, and 2.01 (3 s, each 3 H, total 6 Ac).
Compound 27 was finally purified by chro-
matography on silica gel (98:2“93:7
CH2Cl2–MeOH). Yield: 8 mg (27%); [h]D20
Compound 25 was finally purified by HPLC
(1:1 toluene–EtOAc). Yield: 6.8 mg (22%);

50
A. Reiter et al. / Carbohydrate Research 317 (1999) 39–52
−7° (c 0.5, CHCl3); 1H NMR: l 5.91 (m, 1 H,
–CHꢀ), 5.53 (dd, 1 H, J3%,4% 9.8 Hz, H-4%), 5.40
(dd, 1 H, J3,4 9.8 Hz, H-4), 5.32 (dq, 1 H,
ꢀCH2 trans), 5.29 (ddd, 1 H, H-6), 5.28–5.26
(m, 1 H, ꢀCH2 cis), 5.22 (dd, 1 H, H-3%), 5.10
(dd, 1 H, J2%,3% 3.1 Hz, H-2%), 4.99 (d, 1 H, J1%,2%
2.1 Hz, H-1%), 4.74 (ddd, 1 H, H-6%), 4.58 (dd,
1 H, J6%,7a% 4.8, J7a%,7b% 8.4 Hz, H-7a%), 4.55 (d, 1
H, J1,2 0.8 Hz, H-1), 4.45 (dd, 1 H, J6%,7b% 8.8
Hz, H-7b%), 4.41 (dd, 1 H, J6,7a 5.4 Hz, H-7a),
4.35–4.37 (m, 1 H, OCH2), 4.24 (dd, 1 H, J5%,6%
1.1, J4%,5% 10.0 Hz, H-5%), 4.18–4.20 (m, 1 H,
OCH2), 4.14 (dd, 1 H, J6,7b 7.3, J7a,7b 11.1 Hz,
H-7b), 4.13 (dd, 1 H, H-2), 3.65 (dd, 1 H, J2,3
3.0 Hz, H-3), 3.58 (dd, 1 H, J5,6 2.4, J4,5 10.1
Hz, H-5), 2.16 (s, 3 H), 2.13 (s, 6 H), 2.08,
2.07, and 1.99 (3 s, each 3 H, total 6 Ac).
Method B. The trichloroacetimidate donor
23 was dried by coevaporation with toluene (3
mL). A solution of glycosyl donor 23 (14.2
mg, 0.016 mmol) and allyl alcohol (6.8 mL, 0.1
mmol) in dry CH2Cl2 (3 mL) was stirred at
vealed the formation of two main products 25,
26 and a minor amount of 27. The reaction
was stopped with triethylamine (0.05 mL), the
reaction mixture was diluted with CH2Cl2 (10
mL). The solids were removed by filtration
over a pad of Celite and the filtrate was
concentrated. Purification by chromatography
on silica gel (10:0“5:1 CH2Cl2–acetone) af-
forded a-allyl glycoside 25 (3.5 mg, 25%), 26
(3.4 mg, 28%) and 27 (0.6 mg, 5%). Com-
pound 26 (3.4 mg) was dissolved in pyridine (1
mL) and stirred for 24 h at ambient tempera-
ture with Ac2O (10 mL). The reaction mixture
was concentrated, coevaporated with toluene
(3 mL) and purified by chromatography on
silica gel (7:3“1:1 toluene–EtOAc), followed
by purification on the column of Sephadex
LH-20 (2:1, CH2Cl2–MeOH), to give com-
pound 25 (3.5 mg, 97%). Overall yield of 25:
53%.
Acetylation of 26.—A solution of 26 (7.0
mg, 9.6 mmol) and Ac2O (20 mL) in dry
pyridine (1.5 mL) was stirred for 24 h at
ambient temperature. The reaction mixture
was taken to dryness, coevaporated twice with
toluene (10 mL), and the residue was purified
by chromatography (100:0“97:3 CH2Cl2–
MeOH) to afford compound 25 as a solid.
Yield 6.5 mg (90%).
,
ambient temperature with powdered 4 A
molecular sieves for 1 h under N2. Boron
trifluoride etherate (0.02 mmol, 2.5 mL) was
added. After 2 h at 20 °C the reaction was
stopped with triethylamine (0.1 mL), the reac-
tion mixture was diluted with CH2Cl2 (10
mL). The solids were removed by filtration
over a pad of Celite and the filtrate was
concentrated. The residue was dried by co-
evaporation with dry toluene (3 mL) and
purified as described above to afford 25 (2.5
mg, 20%), 26 (4.6 mg, 38%) and 27 (1.8 mg,
15%). Compound 26 was dissolved in pyridine
(1 mL) and stirred for 24 h at ambient temper-
ature with Ac2O (20 mL). The reaction mixture
was concentrated and coevaporated with
toluene (3 mL). Purification by chromatogra-
phy on silica gel (10:0“5:1 CH2Cl2–acetone)
afforded the additional a-allyl glycoside 25
(4.7 mg). Overall yield for 25: 7.1 mg (58%).
Method C. The fluoride 24 was dried by
coevaporation with dry toluene (3 mL). A
solution of glycosyl donor 24 (12.5 mg, 0.017
mmol) and allyl alcohol (5.8 mg, 6.8 mL, 0.1
mmol) in dry CH2Cl2 (5 mL) was stirred at
Allyl (2,3,4-tri-O-acetyl-6,7-O-carbonyl-
glycero-h- -manno-heptopyranosyl)-(1“3)-
2,4,6,7-tetra-O-acetyl- -glycero-i- -manno-
L-
D
L
D
heptopyranoside (28).—A solution of 27 (8
mg, 11 mmol) in dry pyridine (1.5 mL) was
stirred with Ac2O (0.05 mL) for 12 h at rt. The
reaction mixture was evaporated, coevapor-
ated twice with toluene (10 mL), and purified
by silica gel chromatography (7:3“1:1
toluene–EtOAc) which afforded 28 as a
syrup. Yield 7.5 mg (90%); Rf 0.55 (1:2
toluene–EtOAc); [h]2D0 −1° (c 0.3, CHCl3); 1H
NMR: l 5.91 (m, 1 H, –CHꢀ), 5.49 (dd, 1
H, J3%,4% 9.8 Hz, H-4%), 5.41 (dd, 1 H, J1,2 B1.0
Hz, H-2), 5.33 (dd, 1 H, J3,4=J4,5 10.2 Hz,
H-4), 5.30 (dq, 1 H, ꢀCH2 trans), 5.25 (ddd, 1
H, H-6), 5.26–5.22 (m, 1 H, ꢀCH2 cis), 5.21
(dd, 1 H, J2%,3% 3.1 Hz, H-3%), 5.01 (d, 1 H, J1%,2%
2.0 Hz, H-1%), 4.92 (dd, 1 H, H-2%), 4.76 (ddd,
1 H, H-6%), 4.63 (d, 1 H, H-1), 4.56 (dd, 1 H,
J6%,7a% 8.4 Hz, H-7a%), 4.50 (dd, 1 H, J6%,7b% 5.6,
J7a%,7b% 8.8 Hz, H-7b%), 4.45 (dd, 1 H, J6,7a 5.7
,
ambient temperature with powdered 4 A
molecular sieves for 1 h under N2. Boron
trifluoride etherate (0.08 mmol, 10 mL) was
added. TLC analysis, after 3 h at 20 °C, re-

A. Reiter et al. / Carbohydrate Research 317 (1999) 39–52
51
amorphous solid. Yield 3.0 mg (67%), Rf 0.4
(5:5:1 THF–acetone–H2O); [h]2D0 +76° (c 0.5,
H2O); H NMR (D2O): l 5.98 (m, 1 H, –
Hz, H-7a), 4.34–4.40 (m, 1 H, OCH2), 4.17
(dd, 1 H, J6,7b 7.0, J7a,7b 11.4 Hz, H-7b),
4.10–4.15 (m, 1 H, OCH2), 3.97 (dd, 1 H, J5%,6%
1.5, J4%,5% 10.2 Hz, H-5%), 3.86 (dd, 1 H, J2,3 3.5,
J3,4 10.2 Hz, H-3), 3.58 (dd, 1 H, J5,6 2.4 Hz,
H-5), 2.16 (s, 6 H), 2.08 (s, 6 H), 2.06 (s, 6 H),
and 2.02 (s, 3 H, total 7 Ac).
1
CHꢀ), 5.33 (dq, 1 H, ꢀCH2 trans), 5.25 (dq, 1
H, ꢀCH2 cis), 5.18 (d, 1 H, J1%,2% 1.5 Hz, H-1%),
4.86 (d, 1 H, J1,2 1.7 Hz, H-1), 4.24-4.18 (m, 1
H, H-6%), 4.21–4.17 (m, 1 H, OCH2), 4.15 (dd,
1 H, J6%,7a% 6.5 Hz, H-7a%), 4.10 (dd, 1 H, J6%,7b%
9.2 Hz, H-7b%), 4.08–4.01 (m, 2 H, H-6,
OCH2), 4.07 (dd, 1 H, J2%,3% 3.2 Hz, H-2%), 4.00
(dd, 1 H, J2,3 3.2 Hz, H-2), 3.98 (t, 1 H,
J3,4=J4,5 9.6 Hz, H-4), 3.93–3.88 (m, 2 H,
H-3%, 4%), 3.92 (dd, 1 H, H-3), 3.76–3.65 (m, 3
H, H-7a, 7b, 5) and 3.63 (dd, 1 H, J4%,5% 9.5 Hz,
H-5%). Anal. Calcd for C18H31NO14: C, 44.54;
H, 6.44; N, 2.89. Found: C, 44.21; H, 6.17; N,
2.92.
Method B. To a stirred suspension of 26
(7.5 mg, 10 mmol) and ammonium hydrogen-
carbonate (500 mg) in 1:1 MeOH–THF (8
mL) 25% aq NH3 (2 mL) was added. Stirring
was continued for 60 h at ambient tempera-
ture, the reaction mixture was diluted with
MeOH, salts were removed by filtration and
the filtrate was concentrated to dryness. Purifi-
cation as described on Sephadex LH-20 using
MeOH as an eluent afforded 30. Yield 3.0 mg
(63%).
Allyl (7-O-carbamoyl-
heptopyranosyl)-(1“3)-
L
-glycero-h-
D
-manno-
-manno-
L
-glycero-i-
D
heptopyranoside (29).—To a suspension of 28
(7.5 mg, 10 mmol) and ammonium hydrogen-
carbonate (500 mg) in 1:1 MeOH–THF (8
mL) 25% aq NH3 (2 mL) was added. Stirring
was continued for 60 h at ambient tempera-
ture, the reaction mixture was diluted with
MeOH, the salts were removed by filtration
and the filtrate was concentrated to dryness.
The residue was purified by chromatography
on Sephadex LH-20 using MeOH as an eluent
giving 29 as amorphous solid. Yield 3.0 mg
1
(63%); [h]2D0 +8° (c 0.3, H2O); H NMR
(D2O): l 5.96 (m, 1 H, –CHꢀ), 5.33 (dq, 1 H,
ꢀCH2 trans), 5.26 (dq, 1 H, ꢀCH2 cis), 5.20 (d, 1
H, J1%,2% 1.6 Hz, H-1%), 4.75 (d, 1 H, J1,2 1.7 Hz,
H-1), 4.35–4.27 (m, 1 H, OCH2), 4.23–4.15
(m, 2 H, H-6%, OCH2), 4.15 (dd, 1 H, J6%,7a% 8.3
Hz, H-7a%), 4.12 (dd, 1 H, J6%,7b% 9.0 Hz, H-7b%),
4.08 (dd, 1 H, J2%,3% 2.9 Hz, H-2%), 4.03–3.97
(m, 2 H, H-2, 6), 3.94 (t, 1 H, J3,4 =J4,5 9.8
Hz, H-4), 3.91 (dd, 1 H, J4%,5% 8.4 Hz, H-4%),
3.90 (dd, 1 H, H-3%), 3.92 (dd, 1 H, J2,3 3.2, J3,4
9.8 Hz, H-3), 3.75–3.68 (m, 3 H, H-7a, 7b, 5%)
and 3.34 (dd, 1 H, J5,6 1.7 Hz, H-5).
3-(2-Ammoniumethylthio)propyl (7-O-car-
bamoyl-
(1“3)-
L
-glycero-h-
D
-manno-heptopyranosyl)-
-manno-heptopyranoside
L-glycero-i-
D
acetate (31).—A solution of 30 (2.5 mg, 0.005
mmol) and cysteamine hydrochloride (4.9 mg,
0.043 mmol) in water (0.1 mL) was treated as
described for 7 to give the spacer compound
31, which was further purified on Sephadex
LH-20 (MeOH) followed by chromatography
on Bio-Rad Econo-Pac CM ion-exchange car-
tridges using 0.01 M NH4Ac as eluent. Yield:
3.2 mg (99%), colorless syrup; [h]2D0 +41° (c
Allyl (7-O-carbamoyl-
L
-glycero-h-
D
-manno-
heptopyranosyl)-(1“3)-
L
-glycero-h-
D
-manno-
heptopyranoside (30)
Method A. To a stirred suspension of 25
(7.0 mg, 9 mmol) and ammonium hydrogen-
carbonate (800 mg) in 2:1 MeOH–THF (12
mL) 25% aq NH3 (3 mL) was added. Stirring
was continued for 72 h at ambient tempera-
ture, the reaction mixture was diluted with
MeOH, the salts were removed by filtration
and the filtrate was concentrated to dryness.
The residue was purified by silica gel chro-
matography (80:20:0“75:25:3 THF–ace-
tone–H2O), followed by a second chro-
matography (3:1“3:2 acetone–EtOH). Final
purification was performed on Sephadex LH-
20 using MeOH as an eluent giving 30 as
1
0.3, H2O); H NMR (D2O): l 5.19 (d, 1 H,
J1%,2% 1.4 Hz, H-1%), 4.81 (d, 1 H, H-1), 4.20–
4.16 (m, 2 H, H-6%, 7a%), 4.06 (dd, 1 H, H-7b%),
4.07 (dd, 1 H, J2%,3% 3.6 Hz, H-2%), 4.04–3.99
(m, 2 H, H-2, 6), 3.93 (t, 1 H, H-3), 3.90 (m,
2 H, H-3%, 4%), 3.77–3.67 (m, 4 H, H-7a, 7b, 5%,
OCH2), 3.63 (dd, 1 H, J4,5 9.9, J5,6 1.7 Hz,
H-5), 3.55 (t, 1 H, OCH2), 3.21 (t, 2 H,
NCH2), 2.85 (t, 2 H, SCH2), 2.67 (dt, 2 H,
SCH2), 1.92 (m, 2 H, CH2), and 1.91 (s, 3 H,

52
A. Reiter et al. / Carbohydrate Research 317 (1999) 39–52
Ac); 13C NMR (D2O): l 159.5 (CONH2),
102.59 (C-1%), 99.97 (C-1), 77.34 (C-3), 71.83
(C-5,5%), 70.89, 70.38 (C-2, 2%, 3%), 69.00 (C-6),
66.29 (C-4,4%,6%,7%, OCH2), 63.33 (C-7), 38.76
(NCH2), 28.69, 28.08 (2 SCH2, CH2) and
23.65 (Ac).
(OEAD). The authors thank Dr Puchberger
for recording the NMR spectra.
References
[1] O. Holst, H. Brade, Chemical structures of the core
region of lipopolysaccharides, in D.C. Morrison, J.L.
Ryan (Eds.), Bacterial Endotoxic Lipopolysaccharides,
Vol. I, CRC Press, Boca Raton, FL, 1992.
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Synthesis of BSA-conjugates 14 and 32.—A
solution of thiophosgene (2 mL, 26 mmol) in
CHCl3 (1 mL) was added to a solution of
compound 13 (3.0 mg, 19 mmol) in 0.1 M aq
NaHCO3 (1.5 mL) and the mixture was vigor-
ously stirred for 3 h at rt. The organic phase
was separated and the aqueous phase was
washed three times with CHCl3 (2 mL por-
tions) and finally purged with N2 until a clear
solution was obtained. The solution was trans-
ferred to a solution of BSA (Sigma®, 4.0 mg)
in 0.1 M aq NaHCO3–0.3 M NaCl (1 mL)
and stirred for 48 h at ambient temperature,
then passed through a Sephadex G-25 column
(1.6×50 cm, 0.01 M aq NaHCO3). Ninhy-
drin-positive fractions were combined and di-
alysed twice with water (2 L) for 24 h.
Lyophilization gave the BSA-conjugate 14 as
a fluffy white powder. Yield: 4.6 mg.
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BSA-conjugate 32 was prepared in a similar
manner using 3.2 mg of 31 and 3.0 mg of BSA
to give 3.2 mg of the neoglycoprotein 32.
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(1991) 1127–1145.
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Acknowledgements
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Financial support of this work by the Aus-
trian Science foundation (FWF-grant P11449
MOB) is gratefully acknowledged. Dr A. Za-
myatina has been a recipient of a scholarship
by the Austrian Academic Exchange Office
.