Synthesis of neoglycoproteins containing D-glycero-D-talo-oct-2-ulopyranosylonic acid (Ko) ligands corresponding to core units from Burkholderia and Acinetobacter lipopolysaccharide

Article abstract of DOI:10.1016/S0008-6215(00)00213-5

Glycal esters of Kdo derivatives were converted into 2,3-anhydro intermediates, which were transformed into D-glycero-D-talo-oct-2-ulopyranosylonic acid (Ko), as well as 3-O- and 4-O-p-nitrobenzoyl-Ko derivatives. The exo-allyl orthoester derivative, methyl {5,7,8-tri-O-acetyl-4-O-(4-nitrobenzoyl)-2,3-O-[(1-exo-allyloxy)-ethylide ne]-D-glycero-β-D-talo-oct-2-ulopyranos}onate, prepared from the 4-O-pNBz-protected Ko derivative, was elaborated into the α-Ko allyl ketoside, the reducing disaccharide α-Kdop-(2→4)-Ko and the disaccharide α-Kdop-(2→4)-Kop-(2→OAll). Conversely, methyl[4,5,7,8-tetra-O-acetyl-3-O-(4-nitrobenzoyl)-α-D-glycero-D-talo-2-o ctulopyranosyl bromide]onate [Carbohydr. Res., 244 (1993) 69-84], was coupled with a Kdo acceptor to give the disaccharide α-Kop-(2→4)-Kdop-(2→OAll) after orthoester rearrangement and deprotection. The allyl glycosides were treated with cysteamine and converted into neoglycoproteins. The ligands correspond to inner core units from Acinetobacter haemolyticus and Burkholderia cepacia lipopolysaccharides. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(00)00213-5

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Carbohydrate Research 329 (2000) 549–560
Synthesis of neoglycoproteins containing
D
-glycero- -talo-oct-2-ulopyranosylonic acid (Ko)
D
ligands corresponding to core units from Burkholderia
and Acinetobacter lipopolysaccharide
Norbert Wimmer a, Helmut Brade b, Paul Kosma a,*
aInstitute of Chemistry, Uni6ersity of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria
bResearch Center Borstel, D-23845 Borstel, Germany
Received 5 June 2000; accepted 10 July 2000
Abstract
Glycal esters of Kdo derivatives were converted into 2,3-anhydro intermediates, which were transformed into
-glycero- -talo-oct-2-ulopyranosylonic acid (Ko), as well as 3-O- and 4-O-p-nitrobenzoyl-Ko derivatives. The
exo-allyl orthoester derivative, methyl {5,7,8-tri-O-acetyl-4-O-(4-nitrobenzoyl)-2,3-O-[(1-exo-allyloxy)-ethylidene]-
glycero-b- -talo-oct-2-ulopyranos}onate, prepared from the 4-O-pNBz-protected Ko derivative, was elaborated into
the a-Ko allyl ketoside, the reducing disaccharide a-Kdop-(2“4)-Ko and the disaccharide a-Kdop-(2“4)-Kop-(2“
OAll). Conversely, methyl[4,5,7,8-tetra-O-acetyl-3-O-(4-nitrobenzoyl)-a- -glycero- -talo-2-octulopyranosyl bro-
D
D
D
-
D
D
D
mide]onate [Carbohydr. Res., 244 (1993) 69–84], was coupled with a Kdo acceptor to give the disaccharide
a-Kop-(2“4)-Kdop-(2“OAll) after orthoester rearrangement and deprotection. The allyl glycosides were treated
with cysteamine and converted into neoglycoproteins. The ligands correspond to inner core units from Acinetobacter
haemolyticus and Burkholderia cepacia lipopolysaccharides. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Lipopolysaccharides; Kdo; Ko; Neoglycoproteins
1. Introduction
glycero-
D
-talo-oct-2-ulopyranosylonic
acid
(Ko). Ko has first been detected as a con-
stituent of the main chain in the LPS core
from Acinetobacter calcoaceticus NCTC 10305
and Acinetobacter haemolyticus [4–6]. Mem-
bers of this genus are frequently involved in a
wide spectrum of nosocomial infections,
which are difficult to treat due to increasing
antibiotic resistance of the strains [7,8]. Ko
has also been found as a lateral substituent of
a Kdo residue in the inner core of Burkholde-
ria cepacia [9], a bacterium which is gaining
increasing biomedical interest, since it may
cause severe necrotizing pneumonias (cepacia-
syndrome) in patients with cystic fibrosis [10].
Lipopolysaccharides (LPS) are complex gly-
colipids located in the outer membrane of
Gram-negative bacteria. Within the core re-
gion of bacterial LPS, 3-deoxy- -manno-oct-
D
2-ulosonic acid (Kdo) provides the linkage of
the core to the lipid A domain [1]. In a few
bacterial strains, however, Kdo is replaced by
3-deoxy-
D
-threo-hex-2-ulosonic acid [2], 3-de-
oxy- -arabino-hept-2-ulosaric acid [3] or
D
D
-
* Corresponding author. Tel.: +43-1-360-066055; fax: +
43-1-360-066659.
E-mail address: pkosma@edv2.boku.ac.at (P. Kosma).
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00213-5

550
N. Wimmer et al. / Carbohydrate Research 329 (2000) 549–560
In continuation of previous work on the syn-
thesis of the disaccharide a-Kop-(2“6)-b-
ero- -talo-configured derivative 3 in 40%
D
D
-
overall yield. Hydrogenolysis of the benzyl
ester group followed by deacetylation and
Bio-Gel P-2 chromatography furnished
GlcpNAc [11], we report herein the synthesis
of disaccharide ligands containing a-(2“4)-
interlinked Ko and Kdo units. Neoglyco-
proteins derived from the allyl glycosides will
be used for the preparation and characteriza-
tion of Ko-specific monoclonal antibodies, as
well as for the further characterization of the
epitope specificities of known Kdo-reactive
monoclonal antibodies.
sodium
D
-glycero- -talo-oct-2-ulopyranosyl-
D
1 13
onate (4) in 95% yield. H and C NMR data
obtained for 4 (Table 1) indicated the presence
of a-pyranose (40%), b-furanose (40%), a-
1
furanose (15%) and b-pyranose (5%). The H
NMR signals for H-3, H-4 and H-5 of the
furanoses displayed a downfield shift (3.99–
4.28 ppm) out of the bulk region and could be
assigned to both anomeric forms considering
the chemical shift of the 13C NMR signal for
C-3 (74.36 for the a anomer and 76.87 for the
b anomer). Hydrolysis of the previously re-
ported a-2,3-anhydro-derivative 6, obtained
from the glycal methyl ester 5, gave a mixture
of compounds [11]. NMR analysis of the hy-
drolysis products indicated that acetyl migra-
tion from O-4 to O-3 had occurred. Hence,
this by-product was regarded as a suitable
precursor for the synthesis of a-(2“4)-linked
disaccharide derivatives 22 and 24. Upon
2. Results and discussion
The known glycal ester 1 [12] was used as
starting material for the synthesis of the
D
acid
-
glycero-
D
-talo-oct-2-ulopyranosylonic
(Ko, 4). An alternative synthetic approach to
obtain 4 has been published recently [13].
Epoxidation with m-chloroperbenzoic acid
gave the a-configured oxirane 2 [14], which
was hydrolyzed with wet silica gel. Subsequent
O-acetylation afforded the crystalline
D
-glyc-
Table 1
13C NMR data a for monosaccharide 4 and for the disaccharides 22, 24 and 31
Residue
Carbon
4 a-pyr
4 b-fur
4 a-fur
4 b-fur
22 a-pyr
24
31
a-K(d)o-(2“
1
2
3
4
5
6
7
8
n.d. b
98.23
72.50
67.12
69.14
72.17
70.22
63.87 c
176.36
n.d.
n.d.
n.d.
n.d
n.d.
176.16 e
100.77
35.46
n.d.
67.03
73.30
70.59
64.23
176.51
100.60
35.22
66.73
67.08
73.35
70.69
63.86
174.34
101.82
72.26
66.51
68.70
72.74
70.41
63.47
74.36
71.32
83.46
71.82 d
71.96 d
63.87 c
76.87
71.32
82.97
70.43
n.d.
72.92
68.19
68.52
74.48
69.80
64.64
63.80c
“4-a-K(d)op
1
2
3
4
5
6
7
8
176.64 e
98.51
72.22
67.37
69.65
n.d.
174.14
102.65
69.38
72.71
67.31
72.33
70.28
63.86
175.51
100.45
33.78
69.56
64.74
71.91
69.84
63.47
70.49
64.23
Allyl
1
2
3
65.15
134.45
118.27
64.57
134.42
117.69
a Spectra were recorded at 297 K and referenced to 1,4-dioxane (67.40 ppm).
b Not determined.
c Assignments may be reversed.
d Assignments may be reversed.
e Assignments may be reversed.

N. Wimmer et al. / Carbohydrate Research 329 (2000) 549–560
551
1
treatment of the material present in the hy-
drolysis mixture with 4-nitrobenzoyl chloride
and 4-N,N-dimethylaminopyridine in pyri-
dine, the 4-O-p-nitrobenzoyl derivative 10 was
isolated as the major product, together with
the previously described 3-O-p-nitrobenzoyl
compound 9 [11]. Conversion of 10 into the
bromide donor 11 (95%) was effected with
TiBr4. The reactivity of the glycosyl donor 11
was first investigated by its reaction with allyl
alcohol under Helferich conditions, which fur-
nished the exo-allyl orthoester derivative 13
(96%). The assignment of the exo-orientation
of the allyloxy group was based on the H
NMR chemical shift of the endo methyl group
observed at 1.82 ppm [15].
Orthoester rearrangement in the presence of
substoichiometric amounts of trimethylsilyl
trifluoromethanesulfonate [16] furnished ex-
clusively the a-allyl glycoside 14 (74%). De-
protection of 14 by Zemple´n de-O-acylation
and subsequent alkaline hydrolysis of the
methyl ester gave the previously known
sodium allyl
D
-glycero-a- -talo-oct-2-ulopyra-
D
nosidonate (15, 93%) [17]. Radical addition of
cysteamine hydrochloride to the allyl group
Scheme 1.

552
N. Wimmer et al. / Carbohydrate Research 329 (2000) 549–560
afforded the corresponding 3-(2-aminoethyl-
thio)propyl glycoside 16 (65%). It was acti-
vated with thiophosgene and coupled to
bovine serum albumin [18–20] (Scheme 1).
For the synthesis of the a-Kdop-(2“4)-
linked disaccharide derivatives, orthoester 13
was selectively de-O-acylated at O-4 using am-
monium hydrogencarbonate/aqueous NH3 at
0 °C, to afford the glycosyl acceptor 18 (78%).
Glycosylation of 18 with 2 equivalents of the
Kdo bromide donor 19 gave the (2“4)-linked
disaccharide (87%) in a 5:1 a/b ratio. The
disaccharide orthoester 20, obtained from the
foregoing anomeric mixture by column chro-
matography was hydrolyzed with dilute TFA
to afford the reducing disaccharide 21 (96%).
Similar to the reducing monosaccharide
derivative 3, the disaccharide remained stable
under alkaline conditions used for the removal
of the ester groups. Purification on Bio-Gel
P-2 finally gave the a-Kdop-(2“4)-Ko disac-
charide 22 (95%).
Trimethylsilyl
trifluoromethanesulfonate
promoted orthoester rearrangement of 20 fur-
nished the a-allyl disaccharide derivative 23.
The minor amount of the b-(2“4)-linked iso-
Scheme 2.

N. Wimmer et al. / Carbohydrate Research 329 (2000) 549–560
553
mer present could be separated at this stage.
Deprotection of 23, as described above, gave
the disaccharide a-Kdop-(2“4)-Kop-(2“
OAll) (24, 92%). The allyl glycoside was con-
verted into the spacer compound 25 and
coupled to BSA, to give the neoglycoconju-
gate 26 (Scheme 2).
trimethylsilyl trifluoromethanesulfonate in
CH2Cl2, affording the 5-O-Me3Si–ether
derivative 29 and the crystalline disaccharide
compound 30 in yields of 31 and 42%, respec-
tively (Scheme 3).
The NMR signal of H-3% of both com-
pounds 29 and 30 displayed a downfield shift
(5.79 and 5.74 ppm, respectively), confirming
the presence of an ester group at O-3. Re-
moval of the Me3Si group of 29 was accom-
plished by treatment with 2% HF in MeCN.
Zemple´n de-O-acylation and alkaline hydroly-
sis of the methyl ester groups of 30 afforded
the disaccharide a-Kop-(2“4)-Kdop-(2“
OAll) (31, 95%). The disaccharide is related to
the inner core of B. cepacia LPS and the 13C
NMR data (Table 1) are in good agreement
with the data of a similar methyl glycoside
obtained from native LPS [9]. Conversion of
31 into the spacered ligand 32 was performed
as described for similar conversions. The com-
pound was attached to BSA via thiophosgene
activation of the terminal amino group. Deter-
mination of the ligand/protein ratio 6.6, 2.4
and 1.9 mol/mol for 17, 26 and 33 was based
on MALDI TOF data, respectively. Immuno-
For the synthesis of the disaccharide a-
Kop-(2“4)-Kdop-(2“OAll), the previously
reported [11] 3-O-p-nitrobenzoyl bromide
donor 12 was used. Other disaccharide or-
thoester derivatives prepared from the Ko
donor 11 or methyl (3,4,5,7,8-penta-O-acetyl-
D
-glycero-a- -talo-oct-2-ulopyranosyl)onate
D
bromide led to the formation of hydrolysis
products, upon attempted rearrangement us-
ing trimethylsilyl trifluoromethanesulfonate or
boron trifluoride etherate. Helferich glycosyla-
tion of the 7,8-O-carbonyl acceptor derivative
27 [21] furnished the disaccharide nitroben-
zylidene orthoester 28 (85%). The presence of
the orthoester structure was inferred from the
1
upfield-shift of the H NMR signal of H-3%
(4.86 ppm). Rearrangement of the orthoester
into the a-configured glycoside was effected by
treatment with equimolar amount of
Scheme 3.

554
N. Wimmer et al. / Carbohydrate Research 329 (2000) 549–560
chemical results obtained with the neoglyco-
proteins will be reported elsewhere.
residue was taken up in CH2Cl2 (50 mL),
washed with satd aq NaHCO3 and dried
(Na2SO4). Concentration gave a syrup which
was chromatographed (1:1 toluene–EtOAc) to
give 3 as colourless crystals, mp 178–179 °C
(EtOAc–pentane). Yield: 300 mg (40%); [h]D20
3. Experimental
1
+74° (c 1.0, CHCl3). H NMR (CDCl3): l
General methods.—Melting points were de-
termined with a hot stage and are uncorrected.
Optical rotations were measured with a
7.38–7.33 (m, 5 H, arom H), 5.43 (dd, 1 H,
3
J3,4 3.7, J3,5 1.0 Hz, H-3), 5.39 (t, 1 H, J4,5 3.7
1
Hz, H-4), 5.35 (ddd, 1 H, H-5), 5.34 (ddd, 1
H, H-7), 5.21 and 5.10 (AB, 2 H, JA,B 11.9 Hz,
CH2), 4.53 (dd, 1 H, J8a,7 2.2, J8a,8b −12.5 Hz,
H-8a), 4.21 (dd, 1 H, J6,7 9.9, J6,5 1.6 Hz, H-6),
4.18 (dd, 1 H, J8b,7 3.3 Hz, H-8b), 2.15 (s, 3
H), 2.05 (s, 6 H), 1.99, 1.96 and 1.75 (3 s, each
3 H, total 6 Ac). Anal. Calcd for C27H32O15:
C, 54.40; H, 5.38. Found: C, 54.42; H, 5.28.
Perkin–Elmer 243 B polarimeter. H NMR
spectra were recorded at 297 K with Bruker
AC 300F and DPX instruments operating at
1
300 MHz for H using CDCl3 as solvent and
tetramethylsilane as internal standard, unless
stated otherwise. Coupling constants are given
in Hz (first order values). 13C NMR spectra
were measured at 75.47 MHz and referenced
to 1,4-dioxane (l 67.40). Homo- and het-
eronuclear 2D NMR spectroscopy was per-
formed with Bruker standard software. TLC
was performed on E. Merck precoated plates
(5×10 cm, layer thickness 0.25 mm, Silica
Gel 60F254); detection was effected by spray-
ing with anisaldehyde–H2SO4 [22]. For
column chromatography, silica gel (0.040–
0.063 mm) was used. Concentration of solns
was performed at reduced pressure and B
40 °C. UV-irradation was performed at 254
nm with a 176 W UV-lamp. Elemental analy-
ses were provided by Dr J. Theiner, Mikroan-
Sodium
(
D
-glycero- -talo-oct-2-ulos)onate
D
(4).—A soln of 3 (40 mg, 0.067 mmol) in
MeOH (10 mL) was stirred with 5% PdꢀC (40
mg) under H2 (atmospheric pressure) for 1 h
at rt. The catalyst was filtered off and the
filtrate was treated with 0.1 M methanolic
NaOMe (2 mL) for 2.5 h at rt. Dowex 50
cation-exchange resin (H+-form) was added
until the soln reached pH 7. The resin was
removed and the filtrate was concd. The
residue was purified on a Bio-Gel P-2 column
(2.5×100 cm, 95:1 water–EtOH), which af-
forded 4 as an amorphous powder. Yield: 17.6
mg (95%). [h]2D0 +11“ +9° after 16 h (c 0.5,
alytisches
Laboratorium,
Institut
fu¨r
Physikalische Chemie, University of Vienna.
MALDI-TOF mass spectra were obtained on
a Finnigan MAT instrument in the positive
ion mode using 2% 2,5-dihydroxybenzoic acid
as matrix, by Dr F. Altmann, Institut fu¨r
Chemie, University of Agricultural Sciences,
Vienna.
1
H2O); H NMR (D2O): l 4.31 (dd, 1 H, H-4
a-furanose), 4.28 (dd, 1 H, J4,5 4.6, J5,6 1.2 Hz,
H-5 b-furanose), 4.21 (t, 1 H, J4,3 5.9 Hz, H-4
b-furanose), 4.20 (dd, 1 H, H-5 a-furanose),
4.14 (d, 1 H, H-3 b-furanose), J3,5 1.0, J4,3 2.0
Hz, H-3 a-pyranose).
Methyl [4,5,7,8-tetra-O-acetyl-2,3-di-O-(4-
Benzyl
(2,3,4,5,7,8-hexa-O-acetyl- -glyc-
D
nitrobenzoyl) -
pyranos]onate (9) and methyl [3,5,7,8-tetra-O-
acetyl-2,4-di-O-(4-nitrobenzoyl)- -glycero-h-
-talo-oct-2-ulopyranos]onate (10).—A soln
D
-glycero-h-
D
-talo-oct-2-ulo-
ero-h- -talo-oct-2-ulopyranos)onate (3).—A
D
soln of 1 (600 mg, 1.25 mmol) and 3-chloro-
perbenzoic acid (800 mg) in CH2Cl2 (50 mL)
was stirred under reflux for 48 h. Ethyl acetate
(50 mL) and Silica Gel 60 (0.043-0.060 mm, 3
g) were added and the suspension was heated
at 40 °C for 48 h. After filtration, the solids
were washed with EtOAc and the filtrate was
concd. The residue was dissolved in dry pyri-
dine (10 mL), 4-N,N-dimethylamino pyridine
(10 mg) and Ac2O (2.5 mL) were added. The
soln was stirred for 15 h at rt and concd. The
D
D
of 5 (3.24 g, 8.07 mmol) and 3-chloroperben-
zoic acid (4.90 g, 28.4 mmol) in CH2Cl2 (22
mL) was stirred for 36 h under reflux. The
soln was diluted with EtOAc (5 mL) and
stirred with silica gel (5 g) under reflux for 12
h affording a heterogeneous mixture contain-
ing 7, 8 and furanose diols. The mixture was
dried with Na2SO4, filtered and concd.

N. Wimmer et al. / Carbohydrate Research 329 (2000) 549–560
555
The colorless syrup (1.30 g) was dissolved in
pyridine (30 mL) and stirred with 4-nitroben-
zoyl chloride (1.5 g, 8.1 mmol) and 4-N,N-
dimethylaminopyridine (15 mg) for 24 h. The
soln was diluted with toluene (200 mL) and
stirred with K2CO3 (150 mg, 1.1 mmol) for 30
min. The suspension was filtered, coevapo-
rated with toluene (150 mL) and concd. A
soln of the residue in CH2Cl2 (250 mL) was
washed with satd aq NaHCO3, the organic
phase was dried (Na2SO4) and evaporation of
the solvent afforded a yellow syrup, which
upon chromatography (2:1 n-hexane–EtOAc)
and crystallization (hexane–EtOAc) gave 9 as
slightly yellow crystals, mp 207 °C, lit. 206 °C
[11]. Yield: 710 mg (12%); [h]2D0 +49° (c 1.2,
CHCl3), lit. +51° (c 0.75, CHCl3) [11].
Methyl {5,7,8-tri-O-acetyl-4-O-(4-nitroben-
zoyl)-2,3-O-[(1-exo-allyloxy)-ethylidene]-
glycero-i- -talo-oct-2-ulopyranos}onate (13).
D
-
D
—A suspension of 11 (125 mg, 0.193 mmol),
allyl alcohol (200 mL, 2.9 mmol), Hg(CN)2
,
(220 mg, 0.87 mmol), and 4 A molecular
sieves (100 mg) in dry CHCl3 (3 mL) was
stirred for 2 h at 40 °C. The suspension was
diluted with CHCl3 (150 mL), washed with aq
KI (20%) and satd aq NaHCO3 and dried
(Na2SO4). Evaporation of the solvent and
chromatography (2:3 toluene–EtOAc) gave 13
as a colourless syrup. Yield: 117 mg (95%);
1
[h]2D0 +48° (c 2.1, CHCl3); H NMR (CDCl3):
l 8.32–8.16 (m, 4 H, arom H), 5.89 (m, 1 H,
ꢁCHꢀ), 5.47 (ddd, 1 H, J5,4 3.7 Hz, H-5), 5.44
(t, 1 H, H-4), 5.27 (dq, 1 H, ꢁCH2trans), 5.22
(m, 1 H, J7,6 9.8 Hz, H-7), 5.15 (dq, 1 H,
ꢁCH2cis), 4.82 (dd, 1 H, J3,4 4.0, J3,5 0.9 Hz,
H-3), 4.48 (dd, 1 H, J8a,7 2.3, J8a,8b −12.3 Hz,
H-8a), 4.34 (dd, 1 H, J8b,7 4.3 Hz, H-8b), 4.28
(dd, 1 H, H-6), 4.18 (m, 1 H, OCH2), 4.08 (m,
1 H, OCH2), 3.92 (s, 3 H, CO2Me), 2.08 (s, 6
H) and 2.04 (s, 3 H, 3 Ac) and 1.82 (s, 3 H,
endo-CH3). Anal. Calcd for C27H31NO16: C,
51.84; H, 5.00; N, 2.24. Found: C, 52.00; H,
4.86; N, 2.18.
Further elution gave 1.78 g (30%) of 10 as
yellow crystals; mp 192–195 °C (n-hexane–
1
EtOAc); [h]2D0 +95° (c 0.6, CHCl3); H NMR
(CDCl3): l 8.36–8.04 (m, 8 H, Ar H), 5.83
(dd, 1 H, H-3), 5.75 (t, 1 H, J3,4=J4,5 3.7 Hz,
H-4), 5.61 (ddd, 1 H, J5,6 1.2 Hz, H-5), 5.45
(d, 1 H, J7,6 9.8 Hz, H-7), 4.53 (dd, 1 H, J8a,7
2.2, J8a,8b −12.6 Hz, H-8a), 4.33 (dd, 1 H,
H-6), 4.23 (dd, 1 H, J8b,7 3.2 Hz, H-8b), 3.85
(s, 3 H, CO2Me), 2.12, 1.97, 1.68, and 1.55 (4
s, each 3 H, 4 Ac). Anal. Calcd for C31H29-
N2O19: C, 50.76; H, 3.99; N, 3.82. Found: C,
50.49; H, 3.88; N, 3.79.
Methyl [allyl 5,7,8-tri-O-acetyl-4-O-(4-ni-
trobenzoyl)-
D
-glycero-h- -talo-oct-2-ulopyran-
D
osid]onate (14).—Trimethylsilyl trifluoro-
methanesulfonate (0.120 mL, 0.579 mmol)
was added at 0 °C under Ar to a suspension of
Methyl
trobenzoyl)-
[3,5,7,8-tetra-O-acetyl-4-O-(4-ni-
-glycero-h- -talo-oct-2-ulopyran-
D
D
osyl bromide]onate (11).—A soln of 10 (405
mg, 0.552 mmol) and TiBr4 (800 mg, 2.18
mmol) in CH2Cl2 (50 mL) was stirred for 12 h
under reflux. The soln was diluted with CHCl3
(150 mL) and washed with ice-cold satd aq
NaHCO3, the organic phase was dried
(Na2SO4), and concd. Flash-chromatography
(2:1 toluene–EtOAc) afforded 11 as a slightly
yellow syrup. Yield: 377 mg (95%); [h]D20
,
13 (522 mg, 0.8345 mmol) and 4 A molecular
sieves (200 mg) in dry CH2Cl2 (5 mL). The
suspension was stirred for 2.5 h at rt. Then
triethylamine (0.2 mL) was added at 0 °C, the
suspension was stirred for 10 min, diluted with
CH2Cl2 (150 mL), filtered, and the filtrate was
washed with satd aq NaHCO3 (75 mL). The
organic layer was dried (Na2SO4) and concd
to dryness. The residue was chromatographed
(2:1 toluene–EtOAc), which afforded 14 as a
colourless syrup. Yield: 388 mg (74%); [h]D20
1
+125° (c 0.4, CHCl3); H NMR (CDCl3): l
8.32–8.02 (m, 4 H, Ar H), 5.99 (dd, 1 H, J3,4
3.6, J3,5 0.9 Hz, H-3), 5.97 (t, 1 H, J4,5 3.6 Hz,
H-4), 5.65 (ddd, 1 H, J5,6 1.8 Hz, H-5), 5.47
(d, 1 H, J7,6 9.8 Hz, H-7), 4.63 (dd, 1 H, H-6),
4.52 (dd, 1 H, J8a,7 2.2, J8a,8b −12.6 Hz,
H-8a), 4.30 (dd, 1 H, J8b,7 3.8 Hz, H-8b), 3.92
(s, 3 H, CO2Me), 2.13, 2.09, 2.08 and 2.04 (4
s, each 3 H, 4 Ac). Anal. Calcd for
C24H26NO15Br: C, 44.46; H, 4.04; N, 2.16.
Found: C, 44.51; H, 4.02; N, 2.22.
1
+53° (c 0.7, CHCl3); H NMR (CDCl3): l
8.30–8.01 (m, 4 H, arom H), 5.89 (m, 1 H,
ꢁCHꢀ), 5.72 (dd, 1 H, J3,4 3.6, J3,5 0.9 Hz,
H-3), 5.65 (t, 1 H, J4,5 3.7 Hz, H-4), 5.54 (ddd,
1 H, H-5), 5.43 (dt, 1 H, J7,6 10.0 Hz, H-7),
5.36 (dq, 1 H, ꢁCH2trans), 5.27 (dq, 1 H,
ꢁCH2cis), 4.73 (dd, 1 H, J8a,7 2.3, J8a,8b −12.4
Hz, H-8a), 4.32 (dd, 1 H, H-6), 4.28 (dd, 1 H,

556
N. Wimmer et al. / Carbohydrate Research 329 (2000) 549–560
0.0127 mmol) in 0.1 M NaHCO3 (1.5 mL) and
the mixture was vigorously stirred for 6 h at
rt. The organic phase was separated and the
aq phase was washed three times with CHCl3
(1 mL portions), and finally purged with N2
until a clear soln was obtained. The soln was
transferred to a soln of BSA (6.3 mg) in 0.1 M
aq NaHCO3/0.3 M aq NaCl buffer (1 mL)
and slowly stirred for 48 h at rt, then passed
through a Sephadex G-25 column (1.6×50
cm, 0.01 M aq NaHCO3). Ninhydrine-positive
fractions were combined and dialyzed twice
against water (4 L) for 48 h. Lyophilization
gave the BSA-conjugate 17. Yield: 6.6 mg.
MALDI-TOF-MS: M+ 69316; (determined
for BSA: 66431); 6.6 mol ligand/mol BSA.
Methyl {5,7,8-tri-O-acetyl-2,3-O-[(1-exo-al-
J8b,7 3.2 Hz, H-8b), 4.17 (m, 1 H, OCH2), 3.92
(m, 1 H, OCH2), 3.81 (s, 3 H, CO2Me), 2.10
(s, 3 H), 2.08 (s, 3 H), 2.06 (s, 3 H) and 2.00
(s, 3 H, 4 Ac). Anal. Calcd for C27H31NO16: C,
51.84; H, 5.00; N, 2.24. Found: C, 51.91; H,
5.03; N, 2.18.
Sodium (allyl
D
-glycero-h- -talo-oct-2-
D
ulopyranosid)onate (15).—A soln of 14 (35
mg, 0.056 mmol) in dry MeOH (8 mL) was
stirred with 0.1 M methanolic NaOMe (0.2
mL) for 1 h at rt. After deionization with
Dowex 50-AG WX8 (H+) cation-exchange
resin, a soln of the crude product in water (2
mL) was treated with 0.2 M NaOH (2 mL) for
2 h at rt. The pH of the soln was adjusted to
8.5 by addition of Dowex 50 resin. Filtration
and lyophilization of the filtrate gave a
residue, which was purified on Bio-Gel P-2
(2.6×100 cm, water) to afford 15 as an amor-
phous powder. Yield: 16.5 mg (93%). 1H
NMR data were identical to literature values
[17].
lyloxy)-ethylidene]-
D
-glycero-i- -talo-oct-2-
D
ulopyranos}onate (18).—A soln of 13 (50 mg,
0.082 mmol), NH4HCO3 (23 mg, 0.27 mmol)
and aq NH3 (25%, 0.1 mL) in MeOH (5 mL)
was stirred for 5 h at 0 °C. The soln was
passed through a column (10×3 cm) of silica
gel (EtOAc), dried (Na2SO4), concd and chro-
matographed (1:1 toluene–EtOAc) which fur-
nished 18 as a colourless syrup. Yield: 30 mg
Ammonium [3-(2-aminoethylthio)propyl
D
-
glycero-h- -talo-oct-2-ulopyranosid]onate hy-
D
drochloride (16).—A soln of 15 (12.5 mg,
0.0363 mmol) and cysteamine hydrochloride
(16 mg, 0.143 mmol) in water (0.36 mL) was
irradiated at 254 nm for 9 h at rt. The soln
was diluted with water (0.5 mL) and passed
through a column of Dowex AG-WX8 resin
(NH+4 form) using a gradient water“0.1 M
aq NH3 as eluant. Carbohydrate-containing
fractions were pooled, lyophilized and further
purified on Bio-Gel P-2 (2.6×100 cm, water)
to afford 16 as a colorless solid. Yield: 11 mg
1
(78%); [h]2D0 +34° (c 1.2, CHCl3); H NMR
(CDCl3): l 5.91 (m, 1 H, ꢁCHꢀ), 5.32 (ddd, 1
H, J5,4 4.0 Hz, H-5), 5.28 (dq, 1 H, ꢁCH2trans),
5.17 (dq, 1 H, ꢁCH2cis), 5.10 (dq, 1 H, J7,6 9.7
Hz, H-7), 4.66 (dd, 1 H, J3,4 4.0, J3,5 0.4 Hz,
H-3), 4.47 (dd, 1 H, J8a,7 2.4, J8a,8b −12.3 Hz,
H-8a), 4.30 (dd, 1 H, J8b,7 4.3 Hz, H-8b), 4.18
(m, 1 H, OCH2), 4.10 (m, 1 H, OCH2), 4.08
(dd, 1 H, J4,OH 9.2 Hz, H-4), 4.06 (dd, 1 H, J5,6
1.0 Hz, H-6), 3.87 (s, 3 H, CO2Me), 2.63 (d, 1
H, OH), 2.12, 2.07 and 2.06 (3 s, each 3 H, 3
Ac) and 1.79 (s, 3 H, endo-CH3). Anal. Calcd
for C20H28O13: C, 50.42; H, 5.92. Found: C,
50.24; H, 5.81.
1
(65%); [h]2D0 +48° (c 0.5, H2O); H NMR
(D2O): l 4.07 (m, 1 H, H-5), 4.02–3.93 (m, 4
H, H-3, H-4, H-7, H-8a), 3.72 (dd, 1 H, J8a,8b
−11.8, J8b,7 6.3 Hz, H-8b), 3.62 (d, 1 H, J6,7
8.7 Hz, H-6), 3.55 (m, 1 H, OCH2), 3.33 (m, 1
H, OCH2), 3.21 (t, 2 H, CH2N), 2.85 (t, 2 H,
SCH2), 2.66 (t, 2 H, CH2S) and 1.86 (m, 2 H,
Methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-h-
-manno - oct - 2 - ulopyranosyl)onate - (2“4)-
D
13
CH2). C NMR (D2O): l 174.64 (CO), 102.92
methyl {5,7,8-tri-O-acetyl-2,3-O-[(1-exo-allyl-
oxy) - ethylidene] - - glycero - i - - talo - oct-
(C-2), 72.74 (C-6), 72.48 (C-3), 70.42 (C-7),
69.22 (C-5), 67.51 (C-4), 64.12 (C-8), 62.89
(OCH2), 39.36 (CH2N), 29.51 (SCH2), 29.28
(CH2) and 28.54 (CH2S). MALDI-TOF MS:
m/z 809.0 2[M+Na+ −Cl−+NH4+].
D
D
2-ulopyranos}onate (20).—A suspension of 18
(95 mg, 0.2 mmol), 19 (167 mg, 0.346 mmol),
,
Hg(CN)2 (131 mg, 0.519 mmol) and 4 A
molecular sieves (80 mg) in dry CH3CN (4
mL) was stirred for 4 h at rt under Ar. A
second portion of 19 (40 mg, 0.083 mmol) and
Hg(CN)2 (35 mg, 0.138 mmol) was added, and
Synthesis of BSA-conjugate 17.—A soln of
thiophosgene (2.7 mL, 0.035 mmol) in CHCl3
(1 mL) was added to a soln of 16 (5.4 mg,

N. Wimmer et al. / Carbohydrate Research 329 (2000) 549–560
557
2.01 (s, 3 H), 2.00 (s, 3 H) and 1.98 (s, 3 H,
total 8 Ac). Anal. Calcd for C34H46O24: C,
48.69; H, 5.53. Found: C, 48.48; H, 5.80.
stirring was continued overnight at rt. The
mixture was diluted with CHCl3 (150 mL),
washed with aq KI (10%) and satd aq
NaHCO (75 mL), dried (Na2SO4) and concd
to drynes3s. The residue was chromatographed
(3:2“1:1 toluene–EtOAc), which afforded 20
as a colourless syrup. Yield: 153 mg (87%,
a:b=5:1). A second column chromatography
on silica gel (3:2“1:2 toluene–EtOAc) gave
pure a anomer 20 as a colourless syrup; [h]D20
Sodium (3-deoxy-h-
D
-manno-oct-2-ulopy-
-glycero-
ranosyl)onate-(2“4)-sodium
(
D
D
-
talo-oct-2-ulopyranos)onate (22).—A soln of
21 (24 mg, 0.029 mmol) in dry MeOH (5 mL)
was stirred with 0.1 M methanolic NaOMe
(0.2 mL) for 1 h at rt. The soln was neutral-
ized by addition of Dowex 50 AG-WX8 (H+)
cation-exchange resin, filtered, and concd. A
soln of the residue in water (1 mL) was treated
with 0.2 M NaOH (2 mL) for 3 h at rt. The
pH of the soln was adjusted to 8.0 by addition
of Dowex cation-exchange resin. Filtration
and lyophilization of the filtrate gave a
residue, which was purified on Bio-Gel P-2
(2.6×100 cm, water) to afford 22 as a white
amorphous powder yield. Yield: 14 mg (95%);
1
+77° (c 0.85, CHCl3); H NMR (CDCl3): l
5.98 (m, 1 H, ꢁCHꢀ), 5.37 (dd, J5%,4% 3.0 Hz,
H-5%), 5.33 (dq, 1 H, ꢁCH2trans), 5.30 (m, 1 H,
H-4%), 5.23 (dd, 1 H, H-5), 5.22 (dq, 1 H,
ꢁCH2cis), 5.10 (dt, 1 H, H-7%), 5.04 (dq, 1 H,
J7,6 9.5 Hz, H-7), 4.72 (dd, 1 H, J8a%,8b% −12.0,
J8a%,7% 3.2 Hz, H-8a%), 4.60 (dd, 1 H, J3,4 4.0 Hz,
H-3), 4.54 (t, 1 H, H-4), 4.44 (dd, 1 H, J8a,7
2.3, J8a,8b −12.5 Hz, H-8a), 4.26 (dd, 1 H,
J8b,7 4.0 Hz, H-8b), 4.18–4.12 (m, 2 H,
OCH2), 4.13 (dd, 1 H, H-6%), 4.00 (dd, 1 H,
H-8b%), 3.98 (dd, 1 H, H-6), 3.87 (s, 3 H,
CO2Me), 3.85 (s, 3 H, CO2Me), 2.08 (s, 3 H),
2.07 (s, 6 H), 2.02, 2.01, 1.98 and 1.97 (4 s,
each 3 H, total 7 Ac) and 1.78 (s, 3 H,
endo-CH3). Anal. Calcd for C37H50O24: C,
50.57; H, 5.73. Found: C, 50.38; 5.57.
1
[h]2D0+45“60° after 8 h (c 0.9, water); H
NMR (D2O): selected signals: l 4.16 (ddd, 1
H, J4%,3a% 12.3 Hz, H-4%), 3.55 (dd, 1 H, J6,5 0.8,
J6,7 8.5 Hz, H-6), 2.23 (dd, 1 H, J3e%,4% 5.0,
J3a%,3e% −13.2 Hz, H-3e%), 1.79 (dd, 1 H, J3a%,4%
12.3 Hz, H-3a%). MALDI-TOF MS: m/z 499.1
[M−H2O].
Methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-h-
D
- manno - oct - 2 - ulopyranosyl)onate - (2“4)-
Methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-h-
methyl (allyl 3,5,7,8-tetra-O-acetyl-
D
-glycero-
D
- manno - oct - 2 - ulopyranosyl)onate - (2“4)-
h- -talo-oct-2-ulopyranosid)onate
D
(23).—A
methyl (3,5,7,8-tetra-O-acetyl-
D
-glycero-h- -
D
suspension of 20 (80 mg, containing 0.076
mmol a anomer and 0.015 mmol b anomer),
Me3SiO–triflate (0.037 mL, 0.220 mmol) and
talo-oct-2-ulopyranos)onate (21).—A soln of
20 (10 mg, 0.011 mmol), trifluoroacetic acid (1
mL of a 2% soln in CH3CN) and water (0.06
mL) was stirred for 2.5 h at 40 °C. The soln
was neutralized with solid K2CO3 (5 mg, 0.036
mmol), filtered and concd. The residue was
chromatographed (2:3 toluene–EtOAc) and
crystallization (EtOAc-n-hexane) afforded 21,
mp 222–223 °C. Yield: 9.2 mg (96%) [h]D20
,
4 A molecular sieves (80 mg) was stirred in
dry CH2Cl2 (1 mL) for 1 h at rt under Ar.
K2CO3 (30 mg, 0.22 mmol) was added at 0 °C,
the suspension was filtered, diluted with
CH2Cl2 (150 mL), washed with satd aq
NaHCO3 (50 mL) and the organic phase was
dried (Na2SO4). The soln was concd and chro-
matographed (2:3 toluene–EtOAc), which af-
forded 23 as a colourless syrup. Yield: 51 mg
1
+60° (c 0.9, CHCl3); H NMR (CDCl3): l
5.45 (dd, 1 H, J3,4 3.7 Hz, H-3), 5.37 (dd, 1 H,
H-5), 5.22 (dt, 1 H, J7%,8a%=J7%,8b% 3.0 Hz, H-7%),
5.15 (m, 1 H, H-4%), 5.15 (dd, J5%,4% 4.0 Hz,
H-5%), 5.08 (dt, 1 H, J7,6 9.8 Hz, H-7), 4.91 (t,
1 H, H-4), 4.68 (dd, 1 H, J8a,7 2.9, J8a,8b −12.4
Hz, H-8a), 4.58 (dd, 1 H, J6,5 1.0 Hz, H-6),
4.32 (dd, 1 H, J6%,7% 10.0, J6%,5% 1.2 Hz, H-6%),
4.39 (dd, 1 H, H-8a%), 4.50 (s, 1 H, OH), 4.39
(dd, 1 H, H-8b%), 4.24 (m, 1 H, H-8b), 3.86 (s,
3 H, CO2Me), 3.80 (s, 3 H, CO2Me), 2.14 (s, 3
H), 2.13 (s, 3 H), 2.12 (s, 3 H), 2.06 (s, 6 H),
1
(80%); [h]2D0 +79° (c 0.8, CHCl3); H NMR
(CDCl3): 5.84 (m, 1 H, ꢁCHꢀ), 5.62 (dd, 1 H,
J3,4 4.0 Hz, J3,5 0.8 Hz, H-3), 5.39 (ddd, 1 H,
H-5%), 5.26 (dt, 1 H, J7%,8a% 2.8 Hz, H-7%), 5.25
(m, 1 H, ꢁCH2trans), 5.20 (dq, 1 H, ꢁCH2cis),
5.17 (m, 1 H, H-4%), 5.15 (ddd, 1 H, J5,4 4.0
Hz, H-5), 5.10 (dt, 1 H, J7,6 9.7 Hz, H-7), 4.89
(t, 1 H, H-4), 4.87 (dd, 1 H, J8a,8b −12.8, J8a,7
2.5 Hz, H-8a), 4.72 (dd, 1 H, J6,5 1.8 Hz, H-6),
4.67 (dd, 1 H, H-8b), 4.22 (dd, 1 H, J8a%,8b%

558
N. Wimmer et al. / Carbohydrate Research 329 (2000) 549–560
(68%); [h]2D0 +36° (c 0.2, water); H NMR
(D2O): l 4.18 (m, 1 H, H-4%), 4.17 (dd, 1 H,
J5,6 1.2 Hz, H-5), 4.04 (dd, 1 H, J5%,4% 3.2 Hz,
H-5%), 4.04–3.92 (m, 5 H, H-3, H-7, H-8a,
H-7%, H-8a%), 4.01 (t, 1 H, J4,5 3.1 Hz, H-4),
3.70 (m, 3 H, H-6, H-8b, H-8b%), 3.60 (dd, 1
H, J6%,7% 8.9, J6%.5% 1.0 Hz, H-6%), 3.52 (m, 1 H,
OCH2), 3.31 (m, 1 H, OCH2), 3.19 (t, 2 H,
CH2N), 2.86 (t, 2 H, SCH2), 2.68 (t, 2 H,
CH2S), 2.24 (dd, 1 H, J3e%,4% 5.0 Hz, H-3e%),
1.87 (m, 2 H, CH2), 1.80 (t, 1 H, J3a%,3e% −13.2,
J3a%,4% 13.2 Hz, H-3a%). MALDI-TOF MS: m/z
614.9 [M−HCl+Na+].
1
−12.5, H-8a%), 4.11 (dd, 1 H, J6%,5% 1.8 Hz,
H-6%), 4.03 (dd, 1 H, J8b%,7% 3.2 Hz, H-8b%), 3.86
(s, 3 H, CO2Me), 3.76 (s, 3 H, CO2Me), 2.17
(m, 1 H, J3a%,3e% −12.5 Hz, H-3e%), 2.13 (s, 3
H), 2.09 (m, 1 H, H-3a%), 2.11 (s, 3 H), 2.09 (s,
3 H), 2.07 (s, 3 H), 2.05 (s, 3 H), 2.02 (s, 3 H)
and 1.98 (s, 6 H, total 8 Ac). Anal. Calcd for
C37H50O24: C, 50.57; H, 5.73. Found: C, 50.48;
H, 5.54.
Sodium
pyranosyl)onate-(2“4)-sodium (allyl
ero-h- -talo-oct-2-ulopyranosidonate (24).—
(3-deoxy-h-
D
-manno-oct-2-ulo-
D
-glyc-
D
A soln of 23 (20 mg, 0.023 mmol) in dry
MeOH (4 mL) was stirred with 0.1 M
methanolic NaOMe (0.2 mL) for 1 h at rt.
The soln was neutralized by addition of
Dowex 50AG-WX8 (H+) cation-exchange
resin, filtered, and concd. The residue was
stirred with 0.2 M NaOH (1 mL) for 2 h at rt.
The pH of the soln was adjusted to 8.5 by
addition of Dowex cation-exchange resin. Af-
ter filtration, the filtrate was purified on Bio-
Gel P-2 (2.6×100 cm, water) to afford 24 as
a colorless solid. Yield: 12 mg (92%); [h]D20
Synthesis of BSA-conjugate 26.—A soln of
thiophosgene (1 mL, 0.013 mmol) in CHCl3 (1
mL) was added to a soln of 25 (2.8 mg, 0.0043
mmol) in 0.1 M aq NaHCO3 (1 mL) and the
mixture was vigorously stirred for 4 h at rt.
The soln was separated and processed as de-
scribed for 17, transferred to a soln of BSA
(3.2 mg) in 0.1 M aq NaHCO3/0.3 M aq NaCl
buffer (1 mL) and slowly stirred for 48 h at rt.
Work-up as described above gave the BSA-
conjugate 26, yield: 2.7 mg. MALDI-TOF
MS: m/z 68,020 (m/z BSA: 66431) (2.4 mol
ligand/mol BSA).
1
+47° (c 0.3, water); H NMR (D2O): l 5.94
(m, 1 H, ꢁCHꢀ), 5.32 (dq, 1 H, ꢁCH2trans),
5.21 (dq, 1 H, ꢁCH2cis), 4.17 (m, 1 H, H-4%),
4.14 (ddd, 1 H, H-5), 4.05 (dd, 1 H, J5%,4% 2.8
Hz, H-5%), 4.04–3.75 (m, 5 H, H-3, H-7, H-8a,
H-7%, H-8a%), 3.98 (m, 1 H, H-4), 3.90 (m, 1 H,
OCH2), 3.79 (m, 1 H, OCH2), 3.73-3.62 (m, 2
H, H-8b, H-8b%), 3.67 (dd, 1 H, J6%,7% 8.5 Hz,
H-6%), 3.58 (dd, 1 H, J6,7 8.6, J6,5 1.2 Hz, H-6),
2.23 (dd, 1 H, J3e%,4% 4.5 Hz, H-3e%), 1.79 (t, 1
H, J3a%,3e% 13.0 Hz, J3a%,4% 13.0 Hz, H-3a%).
MALDI-TOF MS: m/z 535.6 [M+Na+].
Methyl {{4,5,7,8-tetra-O-acetyl-2,3-O-[1-
exo-(allyl 7,8 - O - carbonyl - 3 - deoxy - 4 - yl - h-
-manno-oct-2-ulopyranosid)onate]-4-nitro-
D
benzylidene}- -glycero-i- -talo-oct-2-ulo-
D
D
pyranos}}onate (28).—A suspension of 27
(346 mg, 0.534 mmol), 12 (255 mg, 0.80
mmol), Hg(CN)2 (244 mg, 0.966 mmol),
,
HgBr2 (530 mg, 1.47 mmol) and 4 A molecu-
lar sieves (250 mg) in dry CH3CN (7 mL) was
stirred under Ar for 15 min at 0 °C and then
for 10 h at rt. The mixture was diluted with
CHCl3 (200 mL), filtered, washed with 10% aq
KI (250 mL) and satd aq NaHCO3 (50 mL)
and dried (Na2SO4). The soln was concd and
Ammonium (3-deoxy-h-
pyranosyl)onate]-(2“4)-ammonium [3-(2-ami-
noethylthio)propyl -glycero-h- -talo-oct-2-
D
-manno-oct-2-ulo-
D
D
ulopyranosid]onate hydrochloride (25).—A
soln of 24 (3.5 mg, 0.0063 mmol) and cys-
teamine hydrochloride (4.2 mg, 0.038 mmol)
in water (0.27 mL) was irradiated at 254 nm
for 4 h at rt. The soln was diluted with water
(0.5 mL) and passed through a column of
Dowex AG-WX8 resin (NH+4 form) using a
gradient water“0.1 M aq NH3. Carbohy-
drate-containing fractions were pooled,
lyophilized and further purified on Bio-Gel
P-2 (2.6×100 cm, water) to afford 25 as a
white amorphous powder. Yield: 2.8 mg
chromatography
(2:3
toluene–EtOAc“
EtOAc) afforded 28 as a colourless syrup.
Yield: 400 mg (85%); [h]2D0 +6° (c 1.0,
1
CHCl3); H NMR (CDCl3): l 8.32–7.88 (m, 4
H, arom H), 5.84 (m, 1 H, ꢁCHꢀ), 5.29 (dd, 1
H, J5%,4% 3.8 Hz, H-5%), 5.26 (t, 1 H, J4%,3% 3.9 Hz,
H-4%), 5.21 (dq, 1 H, ꢁCH2trans), 5.16 (dq, 1 H,
ꢁCH2cis), 4.92 (m, 2 H, H-7, 7%), 4.86 (dd, 1 H,
H-3%), 4.68 (dd, 1 H, J8a,8b −9.0 Hz, H-8a),
4.55 (t, 1 H, H-8b), 4.39 (dd, 1 H, J8a%,7% 2.5,

N. Wimmer et al. / Carbohydrate Research 329 (2000) 549–560
559
(c 0.7, CHCl3); 1H NMR (CDCl3): l 8.36–8.20
(m, 4 H, arom H), 5.84 (m, 1 H, ꢁCHꢀ), 5.74
(dd, 1 H, J3%,4% 3.6 Hz, H-3%), 5.44 (m, 1 H,
H-5%), 5.43 (m, 1 H, H-4%), 5.43 (m, 1 H, H-7%),
5.24 (dq, 1 H, ꢁCH2trans), 5.18 (dq, 1 H,
ꢁCH2cis), 4.92 (dd, 1 H, J8a%,8b% −12.8 Hz,
H-8a%), 4.90 (m, 1 H, H-7), 4.72 (dd, 1 H, J8a,8b
−9.0, J8a,7 6.8 Hz, H-8a), 4.54 (t, 1 H, H-8b),
4.35 (ddd, 1 H, H-4), 4.28 ( dd, 1 H, J6%,7% 9.8,
J6%,5% 1.7 Hz, H-6%), 4.09 (dd, 1 H, H-8b%), 4.00
(dd, 2 H, OCH2), 3.92 (dd, 1 H, H-6), 3.81 (s,
3 H, CO2Me), 3.66 (s, 3 H, CO2Me), 3.65 (dd,
1 H, H-5), 2.21 (d, 1 H, OH), 2.22 (m, 2 H,
J3a,3e 12.5 Hz, H-3a, H-3e), 2.14, 1.99, 1.93 and
1.88 (4 s, each 3 H, 4 Ac). Anal. Calcd for
C27H31NO16: C, 50.17; H, 4.89; N, 1.58.
Found: C, 50.38; H, 4.95; N, 1.48.
J8a%,8b% −12.5 Hz, H-8a%), 4.36 (ddd, 1 H, H-4),
4.19 (dd, 1 H, J6%,7% 9.8 Hz, H-6%), 4.19 (dd, 1 H,
J8b%,7% 4.5 Hz, H-8b%), 3.96 (dd, 1 H, H-5), 3.94
(s, 3 H, CO2Me), 3.88 (dd, 1 H, J6,7 5.0 Hz,
H-6), 3.80 (s, 3 H, CO2Me), 2.20 (m, 1 H, J3a,3e
−12.5 Hz, H-3e), 2.18 (m, 1 H, H-3a), 2.15,
2.08, 1.97 and 1.77 (4 s, each 3 H, 4 Ac). Anal.
Calcd for C37H43NO24: C, 50.17; H, 4.89; N,
1.58. Found: C, 50.38; H, 5.02; N, 1.48.
Methyl [4,5,7,8-tetra-O-acetyl-3-O-(4-nitro-
benzoyl)-
D
-glycero-h- -talo-oct-2-ulopyran-
D
osyl]onate-(2“4)-methyl (allyl 7,8-O-carb-
onyl-3-deoxy-5-O-trimethylsilyl-h- -manno-
D
oct-2-ulopyranosid)onate (29) and methyl
[4,5,7,8-tetra-O-acetyl-3-O-(4-nitrobenzoyl)-
D
-glycero-h-
(2“4)-methyl (allyl 7,8-O-carbonyl-3-deoxy-
h- -manno-oct-2-ulopyranosid)onate (30).—A
D
-talo-oct-2-ulopyranosyl]onate-
Deprotection of 29.—A soln of 29 (150 mg,
0.157 mmol) was desilylated by treatment with
2% HFꢀCH3CN (3 mL) and H2O (0.5 mL) for
2 h at rt. The pH of the soln was adjusted to
5 by addition of Dowex 50AG–1X8(OH−)
anion-exchange resin. The suspension was
filtered, the filtrate was dried (Na2SO4) and
concd to afford 30. Yield: 135 mg (97%).
D
suspension of 28 (400 mg, 0.451 mmol) and 4
,
A molecular sieves (200 mg) in dry CH2Cl2 (7
mL) was treated under Ar with Me3SiO–tri-
flate (0.081 mL, 0.452 mmol) for 5 min at 0 °C
and for 45 min at rt. The suspension was then
stirred with triethylamine (0.065 mL) at 0 °C
for 10 min, diluted with CH2Cl2 (150 mL),
filtered, the filtrate was washed with satd aq
NaHCO3 (50 mL) and the organic phase was
dried (Na2SO4). The soln was concd and chro-
matographed (2:1“1:2 toluene–EtOAc),
which afforded first the silylated product 29 as
a colourless syrup. Yield: 136 mg (31%); [h]D20
Sodium (
D
-glycero-h- -talo-oct-2-ulopyran-
D
osyl)onate-(2“4)-sodium (allyl 3-deoxy-h-
D
-
manno-oct-2-ulopyranosid)onate (31).—A soln
of 30 (105 mg, 0.119 mmol) in dry MeOH (7
mL) was stirred with 0.1 M methanolic
NaOMe (2 mL) for 2 h at rt. The soln was
deionized with Dowex 50AG-WX8 (H+)
cation-exchange resin, filtered, and concd. The
residue was treated with 0.2 M NaOH (5 mL)
for 2 h at rt, the pH was adjusted to 9 with
Dowex 50 resin. After filtration, the filtrate
was purified on Bio-Gel P-2 (2.6×100 cm,
water) to afford 31 as a white amorphous
powder. Yield: 62 mg (95%) [h]2D0 +68° (c 0.5,
H2O); 1H NMR (D2O): l 5.96 (m, 1 H, ꢁCHꢀ),
5.32 (dq, 1 H, ꢁCH2trans), 5.20 (dq, 1 H,
ꢁCH2cis), 4.16 (ddd, 1 H, J4,3a 12.6 Hz, H-4),
4.08 (dd, 1 H, H-5), 4.08 (m,1 H, H-5%), 4.03
(m, 1 H, H-7%), 4.02 (dd, 1 H, J3%,5% 1.0 Hz,
H-3%), 3.97 (t, 1 H, J4%,3% 3.2 Hz, H-4%), 3.91 (m,
1 H, H-7), 3.91 (dd, 1 H, J8a,8b −12.0, J8a,7 2.0
Hz, H-8a), 3.89 (dd, 1 H, J8a%,8b% −12.2 Hz,
H-8a%), 3.82 (m, 1 H, OCH2), 3.78 (m, 1 H,
OCH2), 3.75 (dd, 1 H, J8b%,7% 7.0 Hz, H-8b%),
3.63 (dd, 1 H, J6%,7% 7.8, J6%,5% 1.0 Hz, H-6%), 3.58
(dd, 1 H, J8b,7 5.0 Hz, H-8b), 3.54 (dd, 1 H, J6,5
1.0, J6,7 8.9 Hz, H-6), 2.03 (dd, 1 H, J3e,4
1
−2° (c 0.6, CHCl3). H NMR (CDCl3): l
8.34–8.21 (m, 4 H, arom H), 5.83 (m, 1 H,
ꢁCHꢀ), 5.79 (dd, 1 H, J3%,4% 3.6 Hz, H-3%), 5.47
(t, 1 H, H-4%), 5.45 (m, 1 H, H-7%), 5.44 (dd, 1
H, H-5%), 5.23 (dq, 1 H, ꢁCH2trans), 5.17 (dq, 1
H, ꢁCH2cis), 4.87 (dd, 1 H, J8a%,8b% –12.5 Hz,
H-8a%), 4.76 (m, 1 H, H-7), 4.54 (m, 2 H, H-8a,
H-8b), 4.37 (dd, 1 H, J6%,7% 9.5, J6%,5% 1.5 Hz,
H-6%), 4.19 (ddd, 1 H, H-4), 4.12 (dd, 1 H, J8b%,7%
4.4 Hz, H-8b%), 3.97 (dd, 2 H, OCH2), 3.97 (dd,
1 H, H-5), 3.79 (s, 3 H, CO2Me), 3.67 (m, 1 H,
H-6), 3.66 (s, 3 H, CO2Me), 2.34 (t, 1 H, J3a,3e
−12.0 Hz, H-3e), 2.23 (dd, 1 H, J3a,4 4.5 Hz,
H-3a), 2.09, 1.99, 1.94 and 1.87 (4 s, each 3 H,
4 Ac), 0.26 (s, 9 H, Me3Si). Anal. Calcd for
C40H51NO24 Si: C, 50.15; H, 5.37; N, 1.46.
Found: C, 50.18; H, 5.21; N, 1.40.
Further elution gave the main product 30 as
colourless crystals; mp 213–216 °C (n-hex-
ane–EtOAc). Yield: 166 mg (42%); [h]2D0 +53°

560
N. Wimmer et al. / Carbohydrate Research 329 (2000) 549–560
5.0 Hz, H-3e), 1.90 (t, 1 H, J3a,3e −12.6 Hz,
H-3a). MALDI-TOF-MS: m/z 537.9 [M+
Na+2H]+.
Acknowledgements
Technical assistance by Maria Hobel is
gratefully acknowledged. The authors thank
Dr Andreas Hofinger and Dr Michael
Puchberger for recording NMR spectra, and
Dr Fritz Altmann for providing the MALDI
data. Financial assistance by Fonds zur
Fo¨rderung der wissenschaftlichen Forschung
(FWF-grant P 12573 MOB) is gratefully
acknowledged.
Ammonium
pyranosyl)onate-(2“4)-ammonium
aminoethylthio)propyl 3-deoxy-h-
(
D
-glycero-h- -talo-oct-2-ulo-
D
[3-(2-
-manno-
D
oct-2-ulopyranosid]onate (32).—A soln of 31
(9.6 mg, 0.0172 mmol) and cysteamine hydro-
chloride (10.0 mg, 0.090 mmol) in water (0.86
mL) was irradiated at 254 nm for 3 h at rt.
The soln was diluted with water (0.75 mL)
and was passed through a column of Dowex
50AG-WX8 resin (NH+4 form) using a gradi-
ent water“0.1 M aq NH3. Carbohydrate-
containing fractions were pooled, lyophilized
and further purified on Bio-Gel P-2 (2.6×100
cm, water) to afford 32 as amorphous solid.
Yield: 10.4 mg (91%); [h]2D0 +57° (c 0.4, wa-
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