Synthesis of neoglycoproteins containing L-glycero-α-D-manno-heptopyranosyl-(1→4)- and -(1-→5)-linked 3-deoxy-α-D-manno-oct-2-ulopyranosylonic acid (Kdo) phosphate determinants

Article abstract of DOI:10.1039/a700497d

The disaccharide allyl glycosides 4,8,13 and the trisaccharide 33 have been prepared using heptopyranosyl trichloroacetimidate 1 or the disaccharide bromide 27 as glycosyl donors followed by efficient O-phosphorylarion via the amidite procedure. The allyl

Full text of DOI:10.1039/a700497d

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Synthesis of neoglycoproteins containing L-glycero-á-D-manno-
heptopyranosyl-(1→4)- and -(1→5)-linked 3-deoxy-á-D-manno-oct-2-
ulopyranosylonic acid (Kdo) phosphate determinants
Harald Sekljic,a Norbert Wimmer,a Andreas Hofinger,a Helmut Bradeb
and Paul Kosma *,a
a
Institute of Chemistry, University of Agricultural Sciences, Vienna, A-1190 Muthgasse,
Austria
b Research Center Borstel, Parkallee 22, D-23845 Borstel, Germany
The disaccharide allyl glycosides 4, 8, 13 and the trisaccharide 33 have been prepared using heptopyranosyl
trichloroacetimidate 1 or the disaccharide bromide 27 as glycosyl donors followed by efficient
O-phosphorylation via the amidite procedure. The allyl glycosides 4, 8 and 33 are converted into
3-(2-aminoethylthio)propyl glycosides and are coupled to bovine serum albumin. The resulting
neoglycoconjugates 6 and 35 containing spacer-linked H ep-(1→4)-K do and H ep-(1→5)-K do 4-phosphate-
(2→6)-â-G lcNAc residues correspond to part structures of the inner core region in bacterial
lipopolysaccharide, whereas compound 10 contains the artificial analogue H ep-(1→4)-K do 5-phosphate.
The compounds may be used in immunochemical characterisation of monoclonal antibodies.
the acid-labile glycosides of Kdo has found application both in
Introduction
the preparation of glycopolymers as well as neoglycoproteins
following introduction of a spacer moiety. Addition of cyste-
amine to the allyl group 11 and subsequent activation of the
terminal amino group with CSCl2, and in situ coupling to
ε-amino groups of lysine residues in bovine serum albumin
(BSA) affords neoglycoconjugates for immunochemical applic-
ations.12,13
Surface structures of bacterial cell walls play a prominent role
in the pathogenesis and virulence of bacterial infections.
Lipopolysaccharides (LPS), as essential constituents of the
outer membranes of Gram-negative bacteria, may be regarded
as target structures for the development of novel antibiotics by
inhibition of biosynthetic pathways or induction of protective
poly- and mono-clonal antibodies.1 LPS is composed of an
O-specific heteropolysaccharide chain, a core region and
lipid A, the latter part being responsible for endotoxic proper-
ties of LPS such as fever, inflammation, hypotension and
toxicity. In LPS of enteric and most other Gram-negative
bacteria, Kdo (3-deoxy--manno-oct-2-ulosonic acid) provides
the linkage of the core oligosaccharide to O-6 of the distal
glucosamine residue of lipid A. Furthermore, Kdo is frequently
substituted by either one or two additional Kdo unit(s) or by
phosphate residues, respectively.2 The presence of at least two
acidic functions in the inner core region seems to be of general
importance to maintain the functions and integrity of the outer
membrane.
Results and discussion
For the synthesis of disaccharide 4 corresponding to a part
structure found in the core region of Hafnia alvei strains 32 and
1192, known 14 diol glycosyl acceptor derivative 2 was treated
with a small excess of the trichloroacetimidate donor 115 and
0.35 mol. equiv. of trimethylsilyl trifluoromethanesulfonate
(triflate) (TMSOTf) which afforded regioselectively the α-
(1→4)-linked crystalline disaccharide derivative 3 (J1Ј,2Ј 1.5 Hz
at δ 5.06, JC-1Ј,H-1Ј 172 Hz) in 61% isolated yield. The structure of
1
compound 3 was assigned on the basis of the H NMR data,
Kdo phosphates have been reported to occur in LPS from
Haemophilus,3 Bordetella pertussis,4 Vibrionaceae,5 and Bacter-
oides6 species. Furthermore, whereas the inner core structure of
the deep rough mutant of Haemophilus influenzae I-69 RdϪ/
bϩ—being composed of Kdo 4- and Kdo 5-phosphate linked to
lipid A—constitutes the smallest functional LPS core found
thus far, substitution by an -glycero--manno-heptopyranosyl
residue in position 5 of Kdo 4-phosphate is quite common. By
contrast, substitution at O-4 with a heptosyl moiety has been
found in the inner core region of Hafnia alvei strains.7 The
potential role of enzymes responsible for the phosphorylation
within the biosynthetic assembly of the core remains to be stud-
ied. Given the frequent presence of heptose-Kdo phosphate
entities present in different bacterial genera, the development
and characterisation of antibodies recognising phosphorylated
core structures8,9 is desirable. For immunisation procedures
with structurally well defined immunogens, the oligosaccharide
derivatives have to be linked to protein carriers, since polyacryl-
amide derivatives,10 which are multivalent haptens useful for
enzyme linked immunoassay (EIA)-inhibition studies, are only
weakly immunogenic. Previously, the use of allyl glycosides for
which showed the presence of a hydroxy group coupled to H-5
of the Kdo-residue, whereas H-4 displayed only spin-couplings
to H-5 and the geminal protons at C-3. Removal of the ester
groups by reaction with sodium methoxide and aq. NaOH
furnished the deprotected allyl glycoside derivative 4 in 93%
yield. 13C NMR data for compound 4 (Table 1) were in full
agreement with the structure assigned. C-4 of the Kdo unit
experienced a downfield shift to δC 71.90, whereas C-5 and C-3
were shifted slightly upfield (δC 63.85 and 33.89, respectively)
compared with unsubstituted Kdo. Addition of cysteamine
hydrochloride under radical conditions (UV irradiation at 254
nm) proceeded in an Anti-Markownikow fashion to give the
3-(2-aminoethylthio)propyl disaccharide compound 5 in 77%
1
yield. In the H and 13C NMR spectra of compound 5, com-
plete absence of the allyl signals was observed, whereas the
respective signals of the spacer group were readily seen at δ
39.16 (C᎐N), 29.31, 29.30 and 28.40.
For comparison purposes the unnatural phosphorylated
disaccharide derivative 8 was synthesized. Reaction of com-
pound 3 with bis(2-cyanoethyl) N,N-diisopropylphosphor-
amidite16 and 1H-tetrazole followed by in situ oxidation with
J. Chem. Soc., Perkin Trans. 1, 1997
1973

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Table 1 13C NMR chemical shifts (δC) and carbon–phosphorus
coupling constants (J/Hz) for compounds 4, 8, 13 and 33, measured at
pD 7.5–8.5
degradation due to an intramolecular reaction of the terminal
amino group with the 4-O-phosphomonoester group was pre-
viously13 noted for the spacer derivative of α-Kdo 4-phosphate.
In order to avoid hydrolysis of the 4-phosphate, the synthesis of
a trisaccharide derivative with the spacer moiety attached to a
β-configured 2-acetamido-2-deoxyglucopyranosyl residue was
performed.
Chemical shifts (δC)
4
8
13
33
Hep-(1→
Within the first approach, the coupling reaction of the
trichloroacetimidate donor 1 with the Kdo-(2→6)-GlcNAc
derivative 16—readily accessible from the previously
described 13 disaccharide 15—in the presence of TMSOTf, Sn
triflate or Ag triflate, respectively, did not furnish the expected
O-glycoside 17 but gave rise to the formation of the unstable
imidate compound 18, which was isolated in low yield (Scheme
3). The structural assignment of compound 18 was based on
the presence of an OH-group coupled to H-5 of the Kdo unit
C-1
C-2
C-3
C-4
C-5
C-6
C-7
98.31
70.17
71.45
66.97
72.73a
69.80
64.13
97.70
70.59
71.44
67.08
72.71
69.89
64.25
101.25b
71.07
71.32
67.51
73.10
70.05
64.18
101.16b
70.95
71.35
67.69
72.91
70.91
63.82c
→Kdo-(2→
1
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
176.03
100.79
33.89
71.90
63.85
72.31a
71.06
63.85
175.96
100.77
34.01
69.89
67.11 (5.3)
72.57
175.82
101.02b
35.17
70.63 (4.3)
73.32 (4.7)
72.72
175.71
101.12b
35.27
69.48 (3.8)
73.54 (4.5)
72.98
and the absence of an NH-signal in the H NMR spectrum.
Owing to the propensity of the acetamido group to compete in
the O-glycosidation reaction, the sequence of glycosylation
steps was therefore reversed by first preparing the disaccharide
donor Hep-(1→5)-Kdo 27.
The 4-O-(p-methoxybenzyl) ether derivative 20 was prepared
in 64% yield—together with a small amount (9%) of the methyl
ester 21—by treatment of compound 1923 with dibutyltin oxide
(DBTO)–p-methoxybenzyl chloride in toluene (Scheme 4).
After separation of the products by silica gel chromatography,
compound 21 was subjected to glycosylation with acetimidate 1
in the presence of TMSOTf. Since separation of an orthoester
derivative formed as a by-product was difficult, the p-meth-
oxybenzyl ester derivative 20 was used. TMSOTf-catalysed
coupling of compound 20 with the trichloroacetimidate donor
1 afforded the α-(1→5)-linked disaccharide derivative 22 in 64%
yield. The presence of the α-anomeric configuration was evi-
dent from the value of the heteronuclear coupling constant
JC-1Ј,H-1Ј (173.1 Hz).
69.89
63.48
70.77
64.18
70.17
64.44c
→GlcNAc
C-1
C-2
C-3
C-4
C-5
C-6
101.12b
56.33
74.88
71.35
75.02
63.17
Allyl group
C-1
C-2
65.05
134.78
118.10
64.98
134.82
117.95
65.34
134.67
118.81
71.35
134.31
118.80
22.98
C-3
CH3
CO
In order to provide a suitable disaccharide donor allowing for
the introduction of the 4-O-phosphate group at a later stage of
the synthesis, the cyclic silyl ether blocking group was cleaved
by treatment with TBAF. Subsequent deacetylation under
Zemplén conditions and O-acetylation with Ac2O–pyridine
afforded the methyl ester derivative 23 in 73% overall yield.
Removal of the benzyl ether groups via hydrogenolysis with
Pd–carbon (5%) in MeOH proceeded quantitatively to give
the reduced oligosaccharide derivative 24 as an α,β-mixture.
Chloroacetylation of diol 24 was attempted under various con-
ditions. Whereas reactions with 2,6-dimethylpyridine (lutidine)–
triethylamine–chloroacetic anhydride or 2,6-dimethylpyridine–
chloroacetyl chloride in dichloromethane did not proceed in
good yield, treatment with chloroacetic anhydride in pyridine at
0 ЊC for 35 min afforded a 71% yield of 25 and 26. The ano-
meric mixture could be resolved by silica gel chromatography.
The anomeric chloroacetates were then converted—by treat-
ment with TiBr4—into the bromide derivative 27, which was
immediately used in the glycosidation reaction with the previ-
ously reported 24 glucosamine acceptor derivative 28. Promo-
tion of the reaction by a 1:3 mixture of HgBr2–Hg(CN)2 in
nitromethane gave the α-(2→6)-linked trisaccharide derivative
30 (28%), traces of the β-linked isomer and a substantial
amount (50% based on substrate 26) of the glycal ester deriv-
ative 29, which were separated by silica gel chromatography
(Scheme 5). Assignment of the α-anomeric configuration of the
Kdo unit in compound 30 was based on the 1H NMR chemical
shift value of H-4Ј and the small chemical shift difference
between the equatorial and axial protons at C-3Ј. The 4Ј-O-
chloroacetyl group was then removed with hydrazine dithiocar-
bonate25 to afford the alcohol 31 in 94% yield. Subsequent
phosphorylation similar to the preparation of compound 7
proceeded smoothly, and furnished the protected phosphotri-
ester trisaccharide derivative 32 in high yield (99%). Removal of
the blocking groups was accomplished by successive treatment
175.38
a,b,c Assignments may be reversed within a column.
m-chloroperbenzoic acid (MCPBA) furnished the phospho-
triester derivative 7 in 67% yield. Removal of the blocking
groups under alkaline conditions went smoothly to give the
5-O-phosphorylated disaccharide derivative 8 in near quan-
titative yield (96%). The presence of the 5-O-phos-
phomonoester in compound 8 was evident from the downfield
shift of C-5 (δC 67.11) of the Kdo residue compared with that
of compound 4 (δC 63.85), and the carbon–phosphorus coup-
ling (J 5.3 Hz).
The disaccharide derivative 13, which corresponds to the
inner core structure of bacterial strains such as Haemophilus
influenzae,17 H. ducreyi,18 Vibrio cholerae,7 V. salmonicida 19
and Bordetella pertussis20 was prepared from the previously
described 21 -glycero-α--manno-heptopyranosyl-(1→5)-Kdo
derivative 11. O-Phosphorylation of the equatorial 4-OH group
in compound 11 afforded the phosphotriester derivative 12 in
70% yield. Deprotection of compound 12 was accomplished
by sequential removal of the tetraisopropyldisiloxane-1,3-diyl
group using tetrabutylammonium fluoride (TBAF)22 followed
by treatment with sodium methoxide and aq. NaOH which fur-
nished the target allyl glycoside Hep-(1→5)-Kdo 4-phosphate
13 in 83% yield. The purity and structure of compound 13
1
were confirmed by H, 13C and 31P NMR spectroscopy. The
C-5 and C-4 signals of Kdo were shifted upfield and displayed
a heteronuclear coupling constant JC,P of 4.7 and 4.3 Hz,
respectively (Table 1), whereas the phosphate residue was
observed at δP 1.32. Conversion of compound 13 into the
spacer derivative 14, however, met with difficulties since the 3-
(2-aminoethylthio)propyl glycoside 14 was dephosphorylated
upon chromatography on Bio-Gel P2 (Scheme 2). A similar
1974
J. Chem. Soc., Perkin Trans. 1, 1997

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Table 2 1H NMR chemical shifts (δ) and coupling constants (J/Hz) for compounds 4, 8, 13 and 33
Chemical shifts (δ)
4
8
13
33
Hep-(1→
H-1
H-2
H-3
H-4
H-5
H-6
H-7a
H-7b
5.11 (1.7)
3.97
3.86
5.14 (1.6)
3.99 (3.0)
3.85
5.18 (1.7)
4.11 (3.3)
3.97 (9.6)
3.82 (9.8)
4.05 (1.9)
3.85
5.20 (1.7)
4.11 (3.4)
3.96 (9.6)
3.77 (9.8)
4.10 (2.7)
3.94
3.86
3.85
3.61 (1.5)
4.05 (6.6)
3.73
3.59 (1.6)
4.03 (6.7)
3.72
n.d.
3.65 (5.6, 11.6)
3.88 (4.8, 11.1)
n.d.
3.75
3.74
→Kdo-(2→
H-3a
H-3e
H-4
H-5
H-6
1.90 (12.2)
2.11 (13.1)
4.16 (5.0)
4.25 (2.9)
3.61 (1.0)
1.97 (12.7)
2.06 (13.0)
4.25 (5.1)
4.68 (2.7)
3.58 (10.0)
n.d.
2.00 (12.8)
2.24 (13.1)
4.53 (4.8)
4.28 (2.1)
3.71 (10.1)
3.99
1.95 (12.8)
2.23
4.45 (4.3)
4.27
3.72
3.83
H-7
3.96 (9.0)
H-8a
H-8b
3.93 (2.8)
3.65 (6.3, 11.6)
n.d.
3.64 (6.4, 11.7)
n.d.
3.73 (7.6, 11.6)
3.92 (2.9)
3.63 (6.5, 11.5)
→GlcNAc
H-1
H-2
H-3
H-4
H-5
H-6a
H-6b
4.55
3.73
~3.50
n.d.
n.d.
n.d.
n.d.
n.d., Not determined.
O
O
AcO
OAc
C
C
O
AcO
OAc
AcO
O
+
AcO
O
O
O
HO
O
AcO
AcO
i
O
HO
AcO
AcO
O
NH
HO
CCl3
O
C
CO2Me
CO2Me
O
OAll
OAll
1
2
3
ii
v
O
C
HO
OH
HO
OH
O
O
O
OAc
HO
HO
O
(NC[CH2]2O)2P
O
AcO
HO
HO
O
AcO
O
O
CO2R2
OR1
O
CO2Me
OAll
O
AcO
AcO
7
4
5
6
R1 = All, R2 = Na+
R1 = [CH2]3S[CH2]2NH2•HCl, R2 = NH4
R1 = [CH2]3S[CH2]2NHCSNH–BSA
iii
iv
+
ii
OH
OH
O
HO
R2O
8
9
R1 = All, R2 = Na+, H+
R1 = [CH2]3S[CH2]2NH2•HCl, R2 = NH4
O
P
iii
iv
+
HO O
HO
O
10 R1 = [CH2]3S[CH2]2NHCSNH–BSA
O
CO2R2
OR1
HO
HO
HO
All = CH2CH CH2
Scheme 1 Reagents and conditions: i, TMSOTf, CH2Cl2; ii, (a) MeONa–MeOH; (b) aq. NaOH; iii, aq. HSCH2CH2NH2ؒHCl, hν; iv, (a) CSCl2, aq.
NaHCO3, (b) BSA, aq. NaHCO3–NaCl; v, bis(2-cyanoethyl) N,N-diisopropylphosphoramidite, 1H-tetrazole, CH2Cl2; then MCPBA
J. Chem. Soc., Perkin Trans. 1, 1997
1975

View Article Online
1
300F instrument operating at 300 MHz for H using CDCl3 as
solvent and tetramethysilane as the internal standard, if not
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 (δC 67.40). 31P NMR spectra were
recorded at 121.49 MHz using orthophosphoric acid as
external standard (δP 0.00). Homo- and hetero-nuclear 2D
NMR spectroscopy were performed with Bruker standard soft-
ware. TLC was performed on Merck precoated plates (5 × 10
cm; layer thickness 0.25 mm, Silica Gel 60F254); spots were
detected by spraying with anisaldehyde–H2SO4. For column
chromatography, silica gel (0.040–0.063 mm) was used. Con-
centration of solutions was performed at reduced pressure at
temperatures <40 ЊC. Elemental analyses were provided by
Dr J. Theiner, Mikroanalytisches Laboratorium, Institut für
Physikalische Chemie, Universität Wien. Centrifugation was
performed on a Heraeus Labofuge model 200.
AcO
OAc
AcO
O
Si
Si
O
O
AcO
AcO
O
O
O
CO2Me
OAll
HO
11
i
OAc
AcO
O
AcO
AcO
AcO
Si
Si
O
O
O
O
Methyl [allyl O-(2Ј,3Ј,4Ј,6Ј,7Ј-penta-O-acetyl-L-glycero-á-D-
manno-heptopyranosyl)-(1Ј→4)-methyl-7,8-O-carbonyl-3-deoxy-
á-D-manno-oct-2-ulopyranosid]onate 3
O
O
NCCH2CH2O
NCCH2CH2O
CO2Me
OAll
P
O
A suspension of compound 1 (150 mg, 0.27 mmol), compound
2 (75 mg, 0.24 mmol) and molecular sieves 4 Å (300 mg) in dry
CH2Cl2 (3 cm3) was stirred at ambient temperature for 2.5 h
under N2. After cooling of the mixture to 0 ЊC, a solution of
TMSOTf in CH2Cl2 (0.4 ; 205 mm3, 0.083 mmol) was added.
Stirring was continued for 2 h and triethylamine (0.2 cm3) was
added. The suspension was diluted with EtOAc (30 cm3) and
filtered over Celite. The filtrate was washed with saturated aq.
NaHCO3 and dried (Na2SO4). Removal of solvents followed by
column chromatography on silica gel [toluene–EtOAc (1:1)]
afforded compound 3 (104 mg, 61%) as crystals, mp 206–208 ЊC
(from hexane–EtOAc); [α]D20 ϩ56 (c 0.9, CHCl3); δH 5.84 (1 H, m,
12
ii
OH
HO
HO
HO
O
HO
OH
HO
O
O
P
᎐CH), 5.32 (1 H, t, J 9.9, 4Ј-H), 5.26 (1 H, dd, J 3.0, 3Ј-H), 5.25
᎐
O
HO
–O
CO2H
O
(1 H, m, ᎐CH2trans), 5.22–5.20 (2 H, m, 2Ј- and 6Ј-H), 5.18 (1
᎐
Na+
H, m, ᎐CH2cis), 5.06 (1 H, d, J 1.5, 1Ј-H), 4.97 (1 H, ddd, J7,6 3.8,
᎐
OR
J7,8a 6.8, J7,8b 8.6, 7-H), 4.76 (1 H, dd, J 8.9, 8-Ha), 4.56 (1 H, t,
8-Hb), 4.27 (1 H, dd, J7Ј,6Ј 6.7, J7Јa,7Јb 11.0, 7Ј-Ha), 4.22 (1 H, ddd,
J4,5 3.0, J4,3e 5.6, J4,3a 11.1, 4-H), 4.17 (1 H, dd, J7Јb,6Ј 4.5, 7Ј-Hb),
4.10 (1 H, dd, J5Ј,6Ј 2.3, 5Ј-H), 4.03 (1 H, br s, 5-H), 3.99–3.96 (3
H, m, 6-H, OCH2), 3.80 (3 H, s, CO2Me), 2.49 (1 H, br s, OH),
2.21–2.10 (2 H, m, 3-H2) and 2.19, 2.14, 2.09, 2.03 and 2.00
(each 3 H together s, 5 × Ac) (Found: C, 49.9; H, 5.6. C30H40O20
requires C, 50.0; H, 5.6%).
13 R = All
14 R = [CH2]3S[CH2]2NH2•HCl
iii
Scheme
2
Reagents and conditions: i, bis(2-cyanoethyl) N,N-
diisopropylphosphoramidite, 1H-tetrazole, CH2Cl2; then MCPBA; ii,
(a) TBAF, THF; (b) MeONa–MeOH; (c) aq. NaOH; iii, aq. HSCH2-
CH2NH2ؒHCl, hν
of compound 32 with TBAF in tetrahydrofuran (THF) fol-
lowed by reaction with NaOMe and aq. NaOH to give the
target trisaccharide derivative 33 in 68% yield. The 31P NMR
spectrum of compound 33 displayed a singlet at δP 2.16.
Introduction of the spacer function by addition of cysteam-
ine hydrochloride provided the stable 3-(2-aminoethylthio)-
propyl derivative 34 (68%).
The neoglycoproteins 6, 10 and 35 were prepared via in situ
activation of the spacer compounds 5, 9 and 34 with thiophos-
gene and coupling of the resulting isothiocyanate to bovine
serum albumin (BSA). The glycoconjugates were isolated by gel
chromatography on Sephadex G-25 and subsequent dialysis
against water. The ratio of ligand to protein was determined by
measuring the amount of phosphate26 or of Kdo by the thio-
barbiturate assay after hydrolysis in 0.1  HCl as described 27
and a protein assay, respectively. Immobilised ligands were in
the range 115–154 nmol/mg BSA which corresponds to 8–11
mol/mol (Table 3).
Sodium [allyl O-(L-glycero-á-D-manno-heptopyranosyl)-(1Ј→4)-
(3-deoxy-á-D-manno-oct-2-ulopyranosid]onate 4
A solution of disaccharide 3 (24 mg, 0.033 mmol) in 0.1 
methanolic NaOMe (2 cm3) was stirred for 1.5 h at room tem-
perature. Dowex AG50 W-X8 resin (Hϩ-form) was added
until pH 6 was attained. The resin was removed and the filtrate
was taken to dryness and then treated with 0.1  aq. NaOH (2
cm3) for 2 h. The pH of the solution was adjusted to 8.5 by
adding Dowex resin, the suspension was filtered, and the resi-
due obtained after lyophilisation of the filtrate was purified on
a Bio-Gel P2 column [2.5 × 100 cm; EtOH–water (95:5)] to
afford compound 4 (15.3 mg, 93%) as a syrup, [α]D20 ϩ80 (c 0.8,
water); δ (D O) (inter alia) 5.99 (1 H, m, ᎐CH), 5.35 (1 H, dq,
᎐
H
2
J 17.2, 1.6, ᎐CH
), 5.23 (1 H, dq, J 10.3, 1.3, ᎐CH2cis) and
᎐
᎐
2trans
3.98–3.92 and 3.86–3.86 (2 H, m, OCH2). Additional 1H NMR
and 13C NMR data are presented in Tables 1 and 2 (Found: C,
41.0; H, 6.4. C18H29NaO14ؒ2H2O requires C, 40.9; H, 6.3%).
Ammonium [3-(2-aminoethylthio)propyl O-(L-glycero-á-D-
manno-heptopyranosyl)-(1Ј→4)-3-deoxy-á-D-manno-oct-2-
ulopyranosid]onate hydrochloride 5
Experimental
General methods
Mps were determined with a Kofler hot stage and are uncor-
rected. Optical rotations were measured with a Perkin-Elmer
243 B polarimeter; [α]D -values are given in units of 10Ϫ1 deg cm2
gϪ1. 1H NMR spectra were recorded at 297 K with a Bruker AC
A solution of compound 4 (5.6 mg, 0.011 mmol) and cysteam-
ine hydrochloride (10.3 mg, 0.081 mmol) in water (180 mm3) was
placed in a quartz vial and irradiated at 254 nm for 15 h at room
temperature. The reaction mixture was applied onto a column
1976
J. Chem. Soc., Perkin Trans. 1, 1997

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O
O
HO
O
CO2R
O
pMBnO
AcO
OAc
O
15 R = pMBn
16 R = Me
i
OAc
O
Si
O
OAll
O
AcO
AcO
O
AcNH
Si
O
NH
C
+
CCi3
1
16
X
ii
O
AcO
OAc
OAc
O
HO
O
AcO
AcO
O
O
CO2Me
pMBnO
O
O
O
O
O
Si
O
OAll
O
CO2Me
pMBnO
O
N
C
Si
O
OAc
O
O
Si
O
OAll
O
O
OAc
OAc
O
AcNH
Si
OAc
OAc
17
18
pMBn = p-MeOC6H4CH2
Scheme 3 Reagents: i, MeONa–MeOH; ii, TMSOTf, CH2Cl2
Table 3 Chemical analysis of the neoglycoconjugates 6, 10 and 35
Phosphate
(nmol/ml)
BSA
(µg/ml)
Ligand/protein quotient
(nmol/mg)
Compound
Structure
6
10
35
Hep-(1→4)-Kdo
860
781
806
115
131
154
Hep-(1→4)-Kdo 5P
102
124
Hep-(1→5)-Kdo 4P (2→6)-βGlcNAc
(0.5 × 10 cm) of Dowex AG50 W-X8 resin (NH4ϩ-form) and
eluted with water → 0.1  aq. NH3. Fractions (2.5 cm3) were
collected and immediately neutralised by addition of 0.1  aq.
NH3. Ninhydrin-positive fractions (10–15) were pooled, lyophil-
ised and purified on Bio-Gel P2 as described for compound 4
which furnished compound 5 (5.3 mg, 77%) as an amorphous
solid, [α]D20 ϩ71 (c 0.3, water); δH (D2O) 5.10 (1 H, d, J 1.5, 1Ј-H),
4.25 (1 H, d, 5-H), 4.14 (1 H, ddd, J4,5 3.0, J4,3e 5.2, J4,3a 12.0, 4-
H), 4.06 (1 H, dt, J 6.2, 6Ј-H), 3.99–3.90 (3 H, m, 2Ј- and 7-H, 8-
Ha), 3.87–3.84 (2 H, m, 3Ј- and 4Ј-H), 3.75–3.73 (2 H, m, 7Ј-H2),
molecular sieves 4 Å (200 mg) in dry CH2Cl2 (5 cm3) was stirred
for 30 min at room temperature under N2. 1H-Tetrazole (15 mg,
0.21 mmol) and bis(2-cyanoethyl) N,N-diisopropylphos-
phoramidite (48 mg, 0.18 mmol) were added in four portions
over a period of 64 h. The suspension was then cooled to
Ϫ20 ЊC and a solution of MCPBA (13 mg, 0.075 mmol) in
CH2Cl2 (1 cm3) was added. The reaction was quenched after
one hour by addition of 10% aq. Na2S2O5 (0.2 cm3). The sus-
pension was centrifuged at 5000 rpm and solids were washed
thoroughly with EtOAc–MeOH (1:1). The supernatant and
washings were combined, concentrated, and purified by silica
gel chromatography (EtOAc) which gave phosphotriester 7 as a
syrup (17 mg, 67%), [α]D20 ϩ42 (c 0.3, CHCl3); δH 5.83 (1 H, m,
3.67 (1 H, dd, J8b,7 7.3, J
12.5, 8-Hb), 3.62–3.58 (2 H, m, 6-
8b,8a
and 5Ј-H), 3.49 and 3.55 (2 H, dt, OCH2), 3.22 (2 H, t, NCH2),
2.88 and 2.72 (4 H, t, SCH2), 2.09 (1 H, dd, J3e,3a 12.5, 3-He), 1.89
(1 H, t, 3-Ha) and 1.89 (2 H, m, CH2); δC 176.14 (C-1), 100.66
(C-2), 98.00 (C-1Ј), 72.75 (C-5Ј), 72.20 (C-6), 71.50 and 71.44
(C-4, -3Ј), 70.96 (C-2Ј), 70.10 (C-7), 69.85 (C-6Ј), 66.91 (C-4Ј),
64.29, 63.86, and 63.63 (C-5, -8, -7Ј), 62.29 (OCH2), 33.81
(C-3), 39.16 (NCH2) and 29.31, 29.30 and 28.40 (2 × SCH2,
CH2) (Found: C, 38.6; H, 7.0; N, 4.6; S, 5.1. C20H40ClN2O14Sؒ
H2O requires C, 38.9; H, 6.85; N, 4.5; S, 5.2%).
᎐CH), 5.50 (1 H, dd, J
᎐
3.3, J1Ј,2Ј 1.6, 2Ј-H), 5.35 (1 H, t,
2Ј,3Ј
J4Ј,3Ј = J4Ј,5Ј = 10.3, 4Ј-H), 5.22 (1 H, dq, J 17.2, 1.6, ᎐CH2trans),
᎐
5.19 (2 H, m, ᎐CH2cis, 6Ј-H), 5.14 (1 H, d, 1Ј-H), 5.11 (1 H, dd,
᎐
3Ј-H), 5.07 (1 H, m, 7-H), 4.89 (1 H, dd, J5,6 2.3, J5,4 8.8, 5-H),
4.69 (1 H, t, J8a,8b = J8a,7 = 8.9, 8-Ha), 4.50 (1 H, dd, J8b,7 5.3,
8-Hb), 4.42–4.26 (6 H, m, 7Ј-Ha, 4-H, 2 × OCH2CH2CN),
4.14 (1 H, dd, J7bЈ,7aЈ 9.7, J7bЈ,6Ј 7.0, 7Ј-Hb), 4.08 (1 H, dd, J5Ј,6Ј
2.0, 5Ј-H), 4.00–3.95 (2 H, m, OCH2), 3.89 (1 H, ddd, J6,7 7.0,
6-H), 3.81 (3 H, s, CO2Me), 2.91 (2 H, t, CH2CN), 2.81 (2 H,
m, CH2CN), 2.23–2.13 (2 H, m, 3-H2) and 2.17, 2.15, 2.09,
2.03 and 1.94 (each 3 H, s, total 5 × Ac) (Found: C, 47.3;
H, 5.2; N, 3.15. C36H47N2O23P requires C, 47.6; H, 5.2; N,
3.1%).
Methyl {allyl O-(2Ј,3Ј,4Ј,6Ј,7Ј-penta-O-acetyl-L-glycero-á-D-
manno-heptopyranosyl)-(1Ј→4)-7,8-O-carbonyl-5-O-[bis(2-
cyanoethyl)phosphoryl]-3-deoxy-á-D-manno-oct-2-ulopyranosid}-
onate 7
A suspension of compound 3 (20.0 mg, 0.028 mmol) and
J. Chem. Soc., Perkin Trans. 1, 1997
1977

View Article Online
AcO
OAc
AcO
O
Si
OH
Si
19
R
1 = H, R2 = Me
O
O
O
AcO
AcO
20 R1 = R2 = pMBn
Si
OAll
O
i
O
21 R1 = pMBn, R2 = Me
+
O
OAc
O
O
HO
AcNH
Si
AcO
O
O
OBn
R1O
CO2Me
CO2R2
CAO
AcO
OAc
AcO
Br
27
28
AcO
O
OAc
AcO
AcO
AcO
i
OR1
O
AcO
OAc
AcO
R1O
+
20
O
ii
AcO
AcO
O
O
O
NH
AcO
AcO
OBn
C
pMBnO
OAc
O
CCl3
CO2R2
AcO
O
1
22 R1 = SiPri2OSiPri2-,R2 = pMBn
23 R1 = Ac, R2 = Me
iii
CAO
CO2Me
29
AcO
iv
OAc
AcO
+
AcO
OAc
O
AcO
AcO
OAc
O
OAc
O
AcO
R2O
AcO
AcO
24 R1 = OH, R2 = H
OAc
O
25 R1 = β-CA, R2 = CA
26 R1 = α-CA, R2 = CA
v
AcO
O
CO2Me
vi
27
R
1 = Br, R2 = CA
O
CO2Me
O
RO
R1
CA = ClCH2CO
O
Scheme
4
Reagents: i, (a) DBTO, toluene; (b) TBAB,
Si
OAll
O
p-MeOC6H4CH2Cl; ii, TMSOTf, CH2Cl2; iii, (a) TBAF, THF; (b)
MeONa–MeOH; (c) Ac2O, pyridine; iv, H2, Pd–carbon, MeOH; v,
CA2O, pyridine; vi, TiBr4, CH2Cl2
O
O
AcNH
Si
Allyl O-(L-glycero-á-D-manno-heptopyranosyl)-(1Ј→4)-3-deoxy-
á-D-manno-oct-2-ulopyranosidonic acid 5-(sodium hydrogen
phosphate) 8
30 R = CA
31 R = H
32 R = PO(OCH2CH2CN)2
ii
iii
A solution of compound 7 (13 mg, 0.014 mmol) and 0.1 
methanolic NaOMe (0.3 cm3) in dry MeOH (5 cm3) was stirred
for 20 h at ambient temperature. The pH of the solution was
adjusted to 6.0 by adding Dowex AG50W-X8 resin (Hϩ-
form). The resin was filtered off, and the filtrate was concen-
trated, and treated with 0.2  aq. NaOH (0.6 cm3) for 4 h at
room temperature. The pH of the solution was adjusted to 7.5
by addition of Dowex resin (Hϩ-form), the suspension was
filtered, and the filtrate was concentrated. The residue was
purified on Bio-Gel P2 as described for compound 4, which
afforded compound 8 as a syrup (8.5 mg, 96%); [α]D20 ϩ83 (c 0.5,
iv
HO
OH
OH
O
HO
OH
HO
HO
O
O
O
–
+
HO
CO2 NH4
P
O
+
NH4 –O
water); δ (D O) (inter alia) 5.96 (1 H, m, CH᎐), 5.34 (1 H, dq,
O
᎐
H
2
O
J 17.3, 1.6, ᎐CH
), 5.21 (1 H, dq, J 10.3, 1.2, ᎐CH2cis), 3.97–
᎐
᎐
2trans
OR
HO
HO
3.89 and 3.84–3.79 (2 H, m, OCH2). Additional 1H NMR and
13C NMR data are given in Tables 1 and 2 (Found: C, 35.9;
H, 5.6. C18H30NaO17Pؒ 2H2O requires C, 36.1; H, 5.55%).
AcNH
33 R =All
v
34 R = [CH2]3S[CH2]2NH2•HCl
35 R = [CH2]3S[CH2]2NHCSNH–BSA
vi
3-(2-Aminoethylthio)propyl O-(L-glycero-á-D-manno-
heptopyranosyl)-(1Ј→4)-3-deoxy-á-D-manno-oct-2-
ulopyranosidonate 5-(ammonium hydrogen phosphate)
hydrochloride 9
A solution of compound 8 (5 mg, 8.1 µmol) and cysteamine
hydrochloride (8.8 mg, 78 µmol) in water (0.2 cm3) was irradiated
at 254 nm for 20 h. The solution was processed as described for
compound 5 to afford spacer compound 9 (2.7 mg, 65%) as a
syrup, [α]D20 ϩ53 (c 0.27, water); δH (D2O) 5.20 (1 H, d, J2Ј,1Ј 1.6,
1Ј-H), 4.59 (1 H, dd, J5,4 9.8, J5,6 2.0, 5-H), 4.17 (1 H, m, 4-H),
4.07 (1 H, dd, J2Ј,3Ј 3.0, 2Ј-H), 4.04 (1 H, m, J6Ј,7Ј 6.0, 6Ј-H),
Scheme 5 Reagents and conditions: i, HgBr2, Hg(CN)2, MeNO2; ii,
HDTC, 2,6-lutidine, HOAc; iii, bis(2-cyanoethyl) N,N-diisopropyl-
phosphoramidite, 1H-tetrazole, CH2Cl2; then MCPBA; iv, (a) TBAF,
THF; (b) MeONa–MeOH; (c) aq. NaOH; then NH4OH; v, aq.
HSCH2CH2NH2ؒHCl, hν; vi (a) CSCl2, aq. NaHCO3; (b) BSA, aq.
NaHCO3–NaCl
3.99–3.93 (2 H, m, 7-H, 8-Ha), 3.85–3.82 (2 H, m, 3Ј- and 5Ј-
H), 3.75 (2 H, d, 7Ј-H2), 3.68–3.30 (5 H, m, 4Ј-, 6- and 8-Hb,
OCH2), 3.21 (2 H, t, NCH2), 2.86 (2 H, t, SCH2), 2.70 (2 H, t,
SCH2) and 2.01–1.85 (4 H, m, 3-H2, CH2); δC 176.18 (C-1),
1978
J. Chem. Soc., Perkin Trans. 1, 1997

View Article Online
100.67 (C-2), 97.44 (C-1Ј), 72.80 (C-5Ј), 72.80 (C-6), 71.47
(C-3Ј), 70.47 (C-2Ј), 70.01 and 69.87 (C-7, -6Ј), 69.69 (C-4),
67.13 (C-4Ј), 66.15 (C-5), 64.50 (C-8), 63.70 (C-7Ј), 62.20
(OCH2), 39.18 (NCH2), 34.03 (C-3) and 29.39, 29.13 and 28.62
(2 × SCH2,CH2) (Found: C, 33.2; H, 6.5; N, 6.1; S, 4.5.
C20H45ClN3O17PSؒH2O requires C, 33.55; H, 6.6; N, 5.9; S,
4.5%).
CH2Ar), 4.44 (1 H, m, J7Ј,8aЈ 6.3, J7Ј,8bЈ 5.6, 7Ј-H), 4.19 (1 H, m,
OCHH allyl), 4.19 (1 H, dd, J8Јa,8bЈ 8.5, 8Ј-Ha), 4.17 (1 H, m,
3-H), 4.12 (1 H, br s, 5Ј-H), 4.00–3.86 (3 H, m, 4Ј-H, 8Ј-Hb,
OCHH allyl), 3.81 (1 H, m, 6-Ha), 3.79 (3 H, s, ArOMe), 3.74 (1
H, dd, J6,7Ј 7.5, 6Ј-H), 3.74 (3 H, s, CO2Me), 3.53–3.46 (2 H, m,
5- and 6-Hb), 3.40 (1 H, m, 4-H), 3.20 (1 H, m, J2,3 9.1, 2-H),
2.26 (1 H, br s, OH), 2.24 (1 H, dd, J3eЈ,3aЈ 12.8, J3eЈ,4Ј 4.6, 3Ј-He),
2.01 (1 H, t, 3Ј-Ha), 1.95 (3 H, s, NHAc), 1.40 and 1.39 (each
3 H, together s, 2 × Me) and 1.05–0.97 (28 H, m, 4 × PriSi)
(Found: C, 58.1; H, 8.0; N, 1.5. C43H71NO15Si2 requires C, 57.5;
H, 8.0; N, 1.6%).
Methyl {allyl O-(2Ј,3Ј,4Ј,6Ј,7Ј-penta-O-acetyl-L-glycero-á-D-
manno-heptopyranosyl)-(1Ј→5)-4-O-[bis(2-cyanoethyl)-
phosphoryl]-3-deoxy-7,8-O-(1,1,3,3-tetraisopropyldisiloxane-
1,3-diyl)-á-D-manno-oct-2-ulopyranosid}onate 12
A suspension of compound 11 (40 mg, 0.042 mmol) and
molecular sieves 4 Å (100 mg) in CH2Cl2 (5 cm3) was treated
with five portions of bis-(2-cyanoethyl) N,N-diisopropyl-
phosphoramidite (total amount 63 mg, 0.23 mmol) and 1H-
tetrazole (17 mg, 0.24 mmol) for 96 h at room temperature. The
solution was cooled to Ϫ20 ЊC and a solution of MCPBA (31
mg) in CH2Cl2 (1 cm3) was added. After 90 min, 10% aq.
Na2S2O5 (0.25 cm3) was added and the reaction mixture was
processed as described for compound 7 to give compound 12 (32
mg, 70%) as a syrup, [α]D20 ϩ28 (c 0.5, CHCl3); δH 5.86 (1 H, m,
Methyl {1-O-allyl-2,6-dideoxy-2-[2-(2Љ,3Љ,4Љ,6Љ,7Љ-penta-O-
acetyl-L-glycero-á-D-manno-heptopyranosyl)ethylimino]-3,4-O-
(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-â-D-glucopyranos-6-yl
3Ј-deoxy-7Ј,8Ј-O-isopropylidene-4Ј-O-(p-methoxybenzyl)-á-D-
manno-oct-2Ј-ulopyranosid}onate 18
A suspension of compounds 16 (20 mg, 0.022 mmol) and 1 (25
mg, 0.044 mmol) and molecular sieves 4 Å (0.2 g) in CH2Cl2 (4
cm3) was stirred under N2 for 4 h. A solution of 0.02 
TMSOTf in CH2Cl2 (0.15 cm3) was added in two portions dur-
ing 2 h at room temperature. CH2Cl2 (2 cm3) and triethylamine
(10 mm3) were added, and the suspension was filtered, and
washed with EtOAc. The filtrate was concentrated, and chroma-
tographed on silica gel [toluene–EtOAc (3:2)] which gave com-
pound 18 (3.7 mg, 13%) as a syrup, [α]D20 ϩ20 (c 0.15, CHCl3); δH
7.22 (2 H, m) and 6.86 (2 H, m) (together ArH), 6.37 (1 H, d,
CH᎐), 5.37 (1 H, dd, J3Ј,2Ј 3.1, J3Ј,4Ј 10.3, 3Ј-H), 5.37–5.32 (1 H,
᎐
m, 6Ј-H), 5.30 (1 H, dd, J2Ј,1Ј 1.8, 2Ј-H), 5.29 (1 H, t, J5Ј,4Ј 10.3,
4Ј-H), 5.28 (1 H, dq, J 1.6, ᎐CH2trans), 5.21 (1 H, d, 1Ј-H), 5.13 (1
᎐
H, dq, J 10.4, 1.3, ᎐CH2cis), 4.90 (1 H, m, 4-H), 4.44–4.29 (8 H,
᎐
m, 5- and 7-H, 5Ј-H, 7Ј-Ha, 2 × OCH2CH2CN), 4.22 (1 H, dd,
J7aЈ,7bЈ 11.7, J7bЈ,6Ј 9.0, 7Ј-Hb), 4.18 (1 H, dd, J6,5 1.0, J6,7 5.0, 6-H),
4.16 (1 H, ddt, OCH2), 4.08 (1 H, t, J8a,8b 9.5, J8a,7 9.5, 8-Ha),
3.92 (1 H, ddt, OCH2), 3.83 (1 H, d, 8-Hb), 3.80 (3 H, s,
CO2Me), 2.83 (4 H, t, 2 × CH2CN), 2.46 (1 H, dd, J3e,3a 12.3,
J3e,4 4.5, 3-He), 2.22 (1 H, t, J3a,4 12.3, 3-Ha), 2.13 (3 H, s), 2.11
(3 H, s), 2.01 (6 H, s) and 1.97 (3 H, s) (total 5 × Ac) and 0.97–
1.05 (28 H, m, 4 × PriSi) (Found: C, 50.8; H, 6.6; N, 2.5.
C47H75N2O23PSi2 requires C, 50.3; H, 6.7; N, 2.5%).
J2Љ,1Љ 1.7, 1Љ-H), 5.76 (1 H, m, CH᎐), 5.38 (1 H, dd, J3Љ,4Љ 10.1, J3Љ,2Љ
᎐
3.3, 3Љ-H), 5.30 (1 H, t, J4Љ,5Љ 10.1, 4Љ-H), 5.30–5.26 (1 H, m, 6Љ-
H), 5.22 (1 H, dd, 2Љ-H), 5.14 (1 H, dq, J 17.3, 1.6, ᎐CH2trans),
᎐
5.07 (1 H, dq, J 10.5, 1.6, ᎐CH2cis), 4.53 and 4.49 (2 H, AB
᎐
system, JA,B 11.2, CH2Ar), 4.46 (1 H, m, 7Ј-H), 4.37 (1 H, d, J1,2
7.7, 1-H), 4.23–4.11 (6 H, m, 5Ј-H, 8Ј-Ha, 5Љ-H, 7Ј-H2 and
OCHH allyl), 3.97–3.87 (3 H, m, 4Ј-H, 8Ј-Hb, OCHH allyl),
3.82–3.79 (4 H, m, 6-Ha, ArOMe), 3.74–3.70 (4 H, m, 6Ј-H,
CO2Me), 3.60 (1 H, t, J3,2 7.9, 3-H), 3.57–3.45 (3 H, m, 4- and
5-H, 6-Hb), 3.21 (1 H, t, 2-H), 2.27 (1 H, s, OH), 2.24 (1 H,
dd, J3Јa,3Јe 13.0, J3Јe,4Ј 4.7, 3Ј-He), 2.18 (3 H, s) and 2.13 (3 H, s)
(together 2 × Ac), 2.01 (4 H, m, 3Ј-Ha, Ac), 1.99 (3 H, s) and
1.98 (6 H, s) (together CMe and 2 × Ac), 1.40 (6 H, s, CMe2)
and 1.06–0.98 (28 H, m, 4 × PriSi) (Found: C, 55.2; H, 7.2; N,
1.0. C60H93NO26Si2 requires C, 55.4; H, 7.2; N, 1.1%).
Allyl O-(L-glycero-á-D-manno-heptopyranosyl)-(1Ј→5)-3-deoxy-
á-D-manno-oct-2-ulopyranosidonic acid 4-(sodium hydrogen
phosphate) 13
A solution of compound 12 (27 mg, 0.024 mmol) in THF (5
cm3) was stirrred with a 1  solution of TBAF in THF (36
mm3) for 30 min at room temperature. Methanol (0.3 cm3) was
added and the solution was concentrated and treated with 0.1
 methanolic NaOMe (0.5 cm3) in MeOH (5 cm3) for 48 h at
4 ЊC. The pH of the solution was adjusted to 7.0 by addition
of Dowex 50 resin (Hϩ-form) and the filtrate obtained upon
removal of the resin was evaporated to dryness. The residue was
dissolved in water (2 cm3) and the solution was stirred with
0.2  aq. NaOH (1 cm3) for 22 h. Work-up of the solution as
described for compound 8 afforded compound 13 as a syrup
(12.3 mg, 83%), [α]D20 ϩ67 (c 0.3, water); δH (D2O) 5.69 (1 H,
p-Methoxybenzyl [benzyl 3-deoxy-4-O-(p-methoxybenzyl)-7,8-
O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-â-D-manno-oct-2-
ulopyranosid]onate 20 and methyl [benzyl 3-deoxy-4-O-(p-
methoxybenzyl)-7,8-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)-â-D-manno-oct-2-ulopyranosid]onate 21
A solution of compound 19 (2 g, 4.24 mmol) and DBTO (2.6 g,
10.4 mmol) in toluene (120 cm3) was heated under reflux with
continuous separation of water for 24 h. After cooling of the
mixture to 90 ЊC, molecular sieves 4 Å (1 g) were added. Tetra-
butylammonium bromide (TBAB) (2.72 g, 8.4 mmol) and
p-methoxybenzyl chloride (2.76 cm3, 20.4 mmol) were added
and the reaction was kept at 90 ЊC for 4.5 h. MeOH (1.2 cm3)
was added dropwise and stirring was continued for 30 min. The
suspension was filtered over Celite, and washed successively
with EtOAc and water. The organic layer was dried (Na2SO4)
and evaporated. Purification of the residue on silica gel
[toluene–EtOAc (20:1)] afforded a mixture of products 20 and
21 which were separated by further chromatography [hexane–
EtOAc (15:1 → 5:1)]. Yield for compound 20: 2.23 g (65%),
syrup, [α]D20 ϩ0.6 (c 1.6, CHCl3); δH 7.28–7.18 (9 H, m, ArH),
6.87–6.83 (4 H, m, ArH), 5.09 and 4.99 (2 H, AB system, JA,B
11.5, CH2Ar), 4.78 and 4.40 (2 H, AB system, JA,B 11.5,
CH2Ar), 4.51 and 4.40 (2 H, AB system, JA,B 11.5, CH2Ar), 4.30
(1 H, m, J7,8a 1.5, J7,8b 8.6, J7,6 8.6, 7-H), 4.28 (1 H, dd, J8a,8b
12.0, 8-Ha), 4.00 (1 H, br s, 5-H), 3.79 (3 H, s, OMe), 3.78 (3 H,
s, OMe), 3.62 (1 H, dd, 8-Hb), 3.38 (1 H, ddd, J4,5 2.6, J4,3a 12.2,
J4,3e 4.4, 4-H), 3.28 (1 H, d, 6-H), 2.59 (1 H, dd, J3a,3e 12.5, 3-He),
m, ᎐CH), 5.37 (1 H, dq, ᎐CH
), 5.24 (1 H, dq, ᎐CH
)
᎐
᎐
᎐
2trans
2cis
and 4.01–3.90 (m, OCH2). Additional H and 13C NMR data
are presented in Tables 1 and 2 (Found: C, 36.4; H, 5.6.
C18H30NaO17Pؒ H2O requires C, 36.6; H, 5.5%).
1
Methyl [2-acetamido-1-O-allyl-2,6-dideoxy-3,4-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)-â-D-glucopyranos-6-yl 3Ј-
deoxy-7Ј,8Ј-O-isopropylidene-4Ј-O-(p-methoxybenzyl)-á-D-
manno-oct-2Ј-ulopyranosid]onate 16
A solution of compound 15 (122 mg, 0.12 mmol) in MeOH (30
cm3) was stirred with 0.1  methanolic NaOMe (1.5 cm3) for 5
h at room temperature. The pH of the solution was adjusted to
7.0 by addition of Dowex (Hϩ)-resin. The resin was removed
and the filtrate was concentrated, and purified on silica gel
[toluene–EtOAc (1:1)] to give compound 16 as a syrup (100 mg,
93%), [α]D20 ϩ33 (c 1.4, CHCl3); δH 7.23 (2 H, m) and 6.86 (2 H,
m) (together ArH), 5.84 (1 H, m, CH᎐), 5.50 (1 H, d, JN H ,2 7.8,
᎐
NH), 5.21 (1 H, dq, J 17.1, 1.6, ᎐CH2trans), 5.16 (1 H, dq, J 10.4,
᎐
1.2, ᎐CH2cis), 4.96 (1 H, d, J1,2 8.4, 1-H), 4.52 (2 H, d, J 2.6,
᎐
J. Chem. Soc., Perkin Trans. 1, 1997
1979

View Article Online
2.24 (1 H, d, OH), 2.09 (1 H, t, 3-Ha) and 1.09–0.99 (28 H, m,
4 × PriSi) (Found: C, 63.1; H, 7.8. C43H62O11Si2 requires C, 63.7;
H, 7.7%).
ArOMe), 3.61 (3 H, s, CO2Me), 3.38 (1 H, ddd, J4,3e 3.5, J4,3a
12.8, J4,5 2.5, 4-H), 2.61 (1 H, dd, J3a,3e 12.5, 3-He), 2.16 (3 H, s,
Ac), 2.12–2.08 (10 H, m, 3-Ha and 3 × Ac) and 2.02, 2.00 and
1.97 (each 3 H, s, total 3 × Ac) (Found: C, 56.5; H, 5.7.
C45H56O22 requires C, 56.9; H, 5.9%).
Yield for compound 21: 0.26 g (9%), syrup [α]D20 Ϫ0.6 (c 0.9,
CHCl3); δH 7.31–7.22 (7 H, m, ArH), 6.89–6.86 (2 H, m, ArH),
4.82 and 4.48 (2 H, AB system, JA,B 11.6, CH2Ar), 4.57 and 4.49
(2 H, AB system, JA,B 11.6, CH2Ar), 4.34 (1 H, m, J7,8a 1.6, J7,8b
9.0, J7,6 8.4, 7-H), 4.33 (1 H, dd, J8a,8b 12.3, 8-Ha), 4.06 (1 H, br
s, 5-H), 3.80 (3 H, s, ArOMe), 3.75 (1 H, dd, 8-Hb), 3.66 (3 H, s,
CO2Me), 3.43 (1 H, ddd, J4,3a 12.3, J4,3e 4.6, J4,5 2.7, 4-H), 3.34
(1 H, d, J6,5 1.1, 6-H), 2.55 (1 H, dd, J3e,3a 12.5, 3-He), 2.30 (1 H,
dd, OH), 2.13 (1 H, t, 3-Ha) and 1.10–1.0 (28 H, m, 4 × PriSi)
(Found: C, 61.3; H, 8.0. C36H56O10Si2 requires C, 61.1; H,
7.95%).
Methyl O-(2Ј,3Ј,4Ј,6Ј,7Ј-penta-O-acetyl-L-glycero-á-D-manno-
heptopyranosyl)-(1Ј→5)-7,8-di-O-acetyl-3-deoxy-D-manno-oct-
2-ulopyranosylonate 24
A solution of compound 23 (580 mg) in dry MeOH (30 cm3)
was stirred with 5% Pd/C (150 mg) under H2 at atmospheric
pressure for 90 min. The catalyst was removed by filtration over
Celite and the filtrate was taken to dryness to afford com-
pound 24 as a syrup (450 mg, ~100%), [α]D20 ϩ14 (c 1.5, CHCl3);
δH 5.43 (1 H, dd, J3Ј,4Ј 9.8, J3Ј,2Ј 3.3 , 3Ј-H), 5.29 (1 H, t, J4Ј,5 9.7,
4Ј-H), 5.28 (1 H, m, J6Ј,7Јa 4.7, J6Ј,7bЈ 7.1, J6Ј,5Ј 3.3, 6Ј-H), 5.16 (1
H, dd, J2,1Ј 2.3, 2Ј-H), 5.10 (1 H, m, J7,6 9.5, J7,8a 2.6, J7,8b 3.6, 7-
H), 4.94 (1 H, d, 1Ј-H), 4.47 (1 H, dd, J8a,8b 12.3, 8-Ha), 4.40 (1
H, dd, J7Јa,7Јb 11.8, 7Ј-Ha), 4.38 (1 H, dd, 5Ј-H), 4.21 (1 H, dd,
8-Hb), 4.18 (1 H, dd, 7Ј-Hb), 4.15 (1 H, dd, J6,5 0.8, 6-H), 4.12 (1
H, m, 4-H), 3.94 (1 H, br s, 5-H), 3.89 (3 H, s, CO2Me), 3.87 (1
H, d, J3e,OH 2.1, 2-OH), 2.74 (1 H, d, J4,OH 8.7, 4-OH), 2.30 (1 H,
m, 3-He), 2.16, 2.13 and 2.10 (each 3 H, s), 2.06 (6 H, s), 2.03
and 2.02 (each 3 H, s) (together 7 × Ac), 1.97 (1 H, m, 3-Ha)
(Found: C, 48.1; H, 5.7. C30H42O21ؒ1/2 H2O requires C, 48.2; H,
5.8%).
p-Methoxybenzyl [benzyl O-(2Ј,3Ј,4Ј,6Ј,7Ј-penta-O-acetyl-L-
glycero-á-D-manno-heptopyranosyl-(1Ј→5)-3-deoxy-4-O-(p-
methoxybenzyl)-7,8-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)-â-D-manno-oct-2-ulopyranosid]onate 22
A suspension of compounds 20 (1.45 g, 1.79 mmol) and 1 (2.02
g, 3.58 mmol) in CH2Cl2 (20 cm3) was stirred with molecular
sieves 4 Å (1 g) for 90 min at room temperature. A solution of
TMSOTf (0.16 cm3, 0.895 mmol) in CH2Cl2 (1 cm3) was added
under N2. After 20 min, the reaction was quenched by addition
of triethylamine (0.1 cm3). The suspension was filtered over
Celite, and washed successively with EtOAc and water. The
organic phase was dried (Na2SO4), concentrated and purified
by silica gel chromatography [hexane–EtOAc (5:1)] which fur-
nished compound 22 (1.39 g, 64%) as a syrup, [α]D20 ϩ11 (c 2.7,
CHCl3); δH 7.31–7.18 (9 H, m, ArH), 6.87–6.82 (4 H, m, ArH),
5.37 (1 H, dd, J2Ј,1Ј 1.6, J2Ј,3Ј 3.4, 2Ј-H), 5.32 (1 H, dd, J3Ј,4Ј 10.0,
3Ј-H), 5.18 and 5.01 (2 H, AB system, JA,B 11.7, OCH2Ar), 5.16
(1 H, t, J4Ј,5Ј 10.0, 4Ј-H), 5.15 (1 H, d, 1Ј-H), 4.91 (1 H, dt, J6Ј,5Ј
2.6, J6Ј,7bЈ 2.1, 6Ј-H), 4.81 and 4.51 (2 H, AB system, JA,B 11.8,
CH2Ar), 4.60 (1 H, dd, 5Ј-H), 4.55 and 4.16 (2 H, AB system,
JA,B 10.9, OCH2Ar), 4.16–4.05 (2 H, m, 8-Ha, 7Ј-Ha), 4.07 (1 H,
d, J4,5 2.3, 5-H), 3.96 (1 H, dd, J8a,8b 13.2, J8b,7 3.9, 8-Hb), 3.91 (1
H, m, 7-H), 3.88 (1 H, dd, J7aЈ,7bЈ 11.9, 7Ј-Hb), 3.81 (3 H, s,
OMe), 3.80 (3 H, s, OMe), 3.57 (1 H, d, J6,7 9.3, 6-H), 3.43 (1 H,
dd, J4,3a 12.4, J4,3e 4.1, 4-H), 2.68 (1 H, dd, J3e,3a 12.4, 3-He), 2.14
(1 H, t, 3-Ha), 2.11, 2.05 and 2.01 (each 3 H, s, total 3 × Ac),
1.95 (6 H, s, 2 × Ac) and 1.06–0.97 (28 H, m, 4 × PriSi) (Found:
C, 59.6; H, 7.0. C60H84O22Si2 requires C, 59.4; H, 7.0%).
Methyl [chloroacetyl O-(2Ј,3Ј,4Ј,6Ј,7Ј-penta-O-acetyl-L-glycero-
á-D-manno-heptopyranosyl)-(1Ј→5)-7,8-di-O-acetyl-4-O-
chloroacetyl-3-deoxy-â-D-manno-oct-2-ulopyranosid]onate 25
and methyl [chloroacetyl O-(2Ј,3Ј,4Ј,6Ј,7Ј-penta-O-acetyl-L-
glycero-á-D-manno-heptopyranosyl)-(1Ј→5)-7,8-di-O-acetyl-4-
O-chloroacetyl-3-deoxy-á-D-manno-oct-2-ulopyranosid]onate 26
A solution of diol 24 (420 mg, 0.57 mmol) in pyridine (15
cm3) was cooled to 0 ЊC and stirred with chloroacetic anhydride
(1.8 g, 10.5 mmol) for 35 min. MeOH (4 cm3) was added, and
the solution was concentrated, and co-evaporated three times
with toluene (50 cm3). Purification of the residue on silica gel
[toluene–EtOAc (1:1)] furnished compound 25 as the faster
migrating isomer (124 mg, 25%), syrup, [α]D20 ϩ24 (c 0.6, CHCl3);
δH 5.45 (1 H, dd, J3Ј,2Ј 3.4, J3Ј,4Ј 10.0, 3Ј-H), 5.35–5.30 (1 H, m,
6Ј-H), 5.29 (1 H, t, J4Ј,5Ј 10.0, 4Ј-H), 5.17 (1 H, ddd, J4,3e 4.5, J4,3a
12.6, J4,5 2.5, 4-H), 5.08 (1 H, m, J7,8a 2.2, J7,8b 3.3, J7,6 9.8, 7-H),
5.06 (1 H, dd, J2Ј,1Ј 2.0, 2Ј-H), 4.82 (1 H, d, 1Ј-H), 4.61 (1 H, d,
6-H), 4.54 (1 H, dd, J8a,8b 12.5, 8-Ha), 4.30–4.23 (3 H, m, 8-Hb,
7Ј-Ha, 5Ј-H), 4.22 (2 H, s, ClCH2CO), 4.19 (2 H, s, ClCH2CO),
4.15 (1 H, m, 7Ј-Hb), 4.09 (1 H, br s, 5-H), 3.78 (3 H, s, CO2Me),
2.47 (1 H, dd, J3e,3a 12.6, 3-He), 2.26 (1 H, t, 3-Ha) and 2.17,
2.13, 2.10, 2.08, 2.05, 2.04 and 2.03 (each 3 H, s, total 7 × Ac)
(Found: C, 45.5; H, 4.6; Cl, 7.9. C34H44Cl2O23 requires C, 45.8;
H, 5.0; Cl, 7.95%).
Further elution of the column afforded stereoisomeric com-
pound 26 as a syrup (224 mg, 46%), [α]D20 ϩ46 (c 0.3, CHCl3); δH
5.39 (1 H, dd, J3Ј,2Ј 3.4, J3Ј4Ј 9.9, 3Ј-H), 5.33–5.29 (2 H, m, 4- and
6Ј-H), 5.28 (1 H, t, J4Ј,5Ј 9.8, 4Ј-H), 5.14 (1 H, m, J7,6 9.7, J7,8a 2.4,
7-H), 5.09 (1 H, dd, J2Ј,1Ј 2.2, 2Ј-H), 4.84 (1 H, d, 1Ј-H), 4.67 (1
H, dd, J8a,8b 12.3, 8-Ha), 4.28–4.09 (6 H, m, 5-, 5Ј- and 6-H, 8-
Hb and 7Ј-H2), 4.23 (2 H, s, ClCH2CO), 4.20 (2H, s, ClCH2-
CO), 3.83 (3 H, s, CO2Me), 2.35 (1 H, dd, J3a,3e 12.6, J3e,4 5.0, 3-
He), 2.27 (1 H, t, J3a,4 12.2, 3-Ha), 2.16, 2.14, 2.09 and 2.05 (each
3 H, s), 2.04 (6 H, s) and 2.03 (3 H, s) (together 7 × Ac) (Found:
C, 45.8; H, 4.7; Cl, 7.5%).
Methyl [benzyl O-(2Ј,3Ј,4Ј,6Ј,7Ј-penta-O-acetyl-L-glycero-á-D-
manno-heptopyranosyl)-(1Ј→5)-7,8-di-O-acetyl-3-deoxy-4-O-
(p-methoxybenzyl)-â-D-manno-oct-2-ulopyranosid]onate 23
A solution of compound 22 (800 mg, 0.66 mmol) in THF (60
cm3) was treated with a 1.1  solution of TBAF in THF (1.2
cm3) for 1 h at room temperature. MeOH (85 cm3) was added
and the solution was evaporated to dryness. A solution of the
residue in dry MeOH (70 cm3) was stirred with 0.1  meth-
anolic NaOMe (28 cm3) for 4.5 h. The pH of the solution was
adjusted to 7.5 by addition of Dowex resin (Hϩ-form) and the
suspension was filtered. The filtrate was evaporated and dried.
The residue was dissolved in pyridine (30 cm3), and the solution
was cooled to 0 ЊC and stirred with acetic anhydride (1 cm3) for
4 h. MeOH (5 cm3) was added and the solution was stirred for
15 min and concentrated. Co-evaporation with toluene (3 × 20
cm3) afforded a syrup, which was subjected to silica gel chroma-
tography [toluene–EtOAc (1:1)] to give compound 23 as a syrup
(760 mg, 73%), [α]D20 ϩ11 (c 0.8, CHCl3); δH 7.34–7.21 (7 H, m,
ArH), 6.88–6.85 (2 H, m, ArH), 5.39 (1 H, dd, J3Ј,2Ј 3.4, J4Ј,3Ј
10.1, 3Ј-H), 5.24 (1 H, t, J4Ј,5Ј 10.1, 4Ј-H), 5.21 (1 H, dd, J2Ј,1Ј 1.7,
2Ј-H), 5.16 (1 H, m, J6Ј,5Ј 2.2, J6Ј,7bЈ 4.7, J6Ј,7Јa 7.2, 6Ј-H), 5.08 (1
H, m, J7,8a 3.4, J7,8b 2.4, J7,6 9.4, 7-H), 4.85 (1 H, d, 1Ј-H), 4.80
and 4.46 (2 H, AB system, JA,B 11.8, CH2Ar), 4.63 and 4.40 (2
H, AB system, JA,B 11.9, CH2Ar), 4.47 (1 H, dd, J8a,8b 12.2, 8-
Ha), 4.45 (1 H, br d, 5Ј-H), 4.38 (1 H, dd, 8-Hb), 4.19–4.16 (2 H,
m, 7Ј-H2), 3.91 (1 H, d, 6-H), 3.82 (1 H, br s, 5-H), 3.81 (3 H, s,
Methyl O-(2Ј,3Ј,4Ј,6Ј,7Ј-penta-O-acetyl-L-glycero-á-D-manno-
heptopyranosyl)-(1Ј→5)-7,8-di-O-acetyl-4-O-chloroacetyl-3-
deoxy-á-D-manno-oct-2-ulopyranosyl)onate bromide 27
A solution of stereoisomers 25 and 26 (285 mg, 0.32 mmol) and
titanium tetrabromide (733 mg, 2 mmol) was kept at 4 ЊC for
42 h. The solution was diluted with ice-cold CHCl3 (100 cm3)
and extracted with saturated aq. NaHCO3. The organic layer
1980
J. Chem. Soc., Perkin Trans. 1, 1997

View Article Online
was dried (Na2SO4), evaporated to dryness and immediately
used for the glycosylation step. Yield for compound 27: 278 mg
(99%), syrup, [α]D20 ϩ66 (c 0.6, CHCl3); δH 5.51 (1 H, ddd, J4,5 2.8,
J4,3e 4.4, J4,3a 11.8, 4-H), 5.37 (1 H, dd, J2Ј,3Ј 3.2, J4Ј,3Ј 9.9, 3Ј-H),
5.28 (1 H, t, J4Ј,5Ј 9.5, 4Ј-H), 5.30–5.25 (1 H, m, 6Ј-H), 5.17 (1 H,
dt, J7,6 9.8, J7,8a 2.0, 7-H), 5.02 (1 H, dd, J2Ј,1Ј 2.0, 2Ј-H), 4.82 (1
H, d, 1Ј-H), 4.53 (1 H, dd, J8a,8b 12.6, 8-Ha), 4.41 (1 H, br d, 6-
H), 4.32–4.10 (7 H, m, 5-, 8-Hb, 5Ј-H, 7Ј-H2 and ClCH2CO),
3.94 (3 H, s, CO2Me), 2.72 (1 H, dd, J3a,3e 13.6, 3-He), 2.40 (1 H,
t, 3-Ha) and 2.17, 2.14, 2.10 and 2.09 (3 H each, s) and 2.04
(9 H, s) (together 7 × Ac).
with toluene (50 cm3), concentrated and chromatographed on
silica gel [EtOAc–toluene (2.5:1)] which afforded compound 31
as a syrup (53 mg, 94%), [α]D20 ϩ12 (c 0.4, CHCl3); δH 5.88 (1 H,
m, CH᎐), 5.47 (1 H, d, J
8.1, NH), 5.40 (1 H, dd, J3Љ,2Љ 3.3,
᎐
N H ,2
J3Љ,4Љ 9.6, 3Љ-H), 5.28 (1 H, dq, J 17.2, 1.6, ᎐CH2trans), 5.27 (1 H, t,
᎐
J4Љ,5Љ 9.6, 4Љ-H), 5.24–5.16 (3 H, m, 7Ј- and 6Љ-H, ᎐CH2cis), 5.13
᎐
(1 H, t, 2Љ-H), 4.92 (1 H, d, J1Љ,2Љ 2.2, 1Љ-H), 4.90 (1 H, d, J1,2 8.3,
1-H), 4.80 (1 H, dd, J8Јa,8Јb 12.3, J8aЈ,7Ј 2.3, 8Ј-Ha), 4.32 (1 H, dd,
J5Љ,6Љ 3.1, 5Љ-H), 4.32 (1 H, dd, J7Љa,7bЉ 11.7, J7aЉ,6Љ 5.3, 7Љ-Ha), 4.28
(1 H, dt, OCHH), 4.18 (1 H, dd, J7bЉ,6Љ 7.0, 7Љ-Hb), 4.17–4.03 (5
H, m, 3-, 4Ј- and 6Ј-H, 8Ј-Hb, OCHH), 3.90 (1 H, br s, 5Ј-H),
3.81 (1 H, d, J6a,6b 9.0, J6a,5 <1.5, 6-Ha), 3.78 (3 H, s, CO2Me),
3.60 (1 H, t, J6b,5 8.5, 6-Hb), 3.53 (1 H, t, J5,4 8.3, 5-H), 3.37 (1 H,
t, 4-H), 3.26 (1 H, m, 2-H), 2.80 (1 H, d, JOH ,4Ј 9.1, OH), 2.24 (1
H, dd, J3Јa,3Јe 12.5, J3Јe,4Ј 4.5, 3Ј-He), 2.15, 2.12, 2.08, 2.07, 2.05,
2.03 and 2.02 (3 H each, s, together 7 × Ac), 1.96 (3 H, s,
NHAc), 1.91 (1 H, t, 3Ј-Ha) and 1.07–0.99 (28 H, m, 4 × PriSi)
(Found: C, 51.9; H, 6.7; N, 1.2. C53H85NO27Si2 requires C, 52.0;
H, 7.0; N, 1.1%).
Methyl O-(2Ј,3Ј,4Ј,6Ј,7Ј-penta-O-acetyl-L-glycero-á-D-manno-
heptopyranosyl)-(1Ј→5)-7,8-di-O-acetyl-2,6-anhydro-4-O-
chloroacetyl-3-deoxy-D-manno-oct-2-enoate 29 and methyl [2-
acetamido-1-O-allyl-2,6-dideoxy-3,4-O-(1,1,3,3-tetraisopropyl-
disiloxane-1,3-diyl)-â-D-glucopyranos-6-yl O-(2Љ,3Љ,4Љ,6Љ,7Љ-
penta-O-acetyl-L-glycero-á-D-manno-heptopyranosyl)-(1Љ→5Ј)-
7Ј,8Ј-di-O-acetyl-4Ј-O-chloroacetyl-3Ј-deoxy-á-D-manno-oct-2Ј-
ulopyranosid]onate 30
A solution of compound 27 (267 mg, 0.3 mmol) in MeNO2 (2
cm3) was added to a suspension of compound 28 (290 mg, 0.57
mmol), mercury() cyanide (68 mg, 0.27 mmol), mercury()
bromide (32 mg, 0.09 mmol) and molecular sieves 4 Å (0.5 g) in
dry MeNO2 (2 cm3) at room temperature under N2. After 60 h,
the suspension was diluted with EtOAc (50 cm3), filtered over
Celite and washed with MeOH–EtOAc (1:1). Filtrate and
washings were combined, concentrated, then diluted with
CH2Cl2 (100 cm3). The organic layer was extracted in turn with
saturated aq. NaHCO3, then with 10% aq. KI, and dried
(Na2SO4). Purification of the residue obtained upon concentra-
tion on silica gel [toluene–EtOAc (1:1)] gave, first the glycal
ester 29 as a syrup (120 mg, 50%), [α]D20 Ϫ135 (c 0.3, CHCl3); δH
5.95 (1 H, dd, J3,4 3.2, J3,5 1.0, 3-H), 5.71 (1 H, m, J4,5 4.3, J4,6
1.3, 4-H), 5.46 (1 H, m, J7,6 9.6, 7-H), 5.33–5.26 (3 H, m, 3Ј-, 4Ј-
and 6Ј-H), 5.15 (1 H, t, J2Ј,1Ј 2.2, J2Ј,3Ј 2.2, 2Ј-H), 4.94 (1 H, d, 1Ј-
H), 4.68 (1 H, dd, J8a,8b 12.5, J8a,7 2.6, 8-Ha), 4.40 (1 H, br d,
J7aЈ,7bЈ 9.6, 7Ј-Ha), 4.30 (1 H, br s, 5-H), 4.25–4.12 (5 H, m, 6-H,
8-Hb, 7Ј-Hb, ClCH2CO), 3.82 (3 H, s, CO2Me) and 2.17, 2.13,
2.12, 2.08, 2.05, 2.04 and 2.00 (each 3 H, s, together 7 × Ac)
(Found: C, 42.5; H, 5.8. C25H41ClO21 requires C, 42.1; H, 5.8%).
Pooling and evaporation of the following fractions afforded
starting material 28 (140 mg, 48% recovery); finally trisacchar-
ide 30 was obtained as a syrup (110 mg, 28%), [α]D20 ϩ33 (c 0.3,
Methyl {2-acetamido-1-O-allyl-2,6-dideoxy-3,4-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)-â-D-glucopyranos-6-yl O-
(2Љ,3Љ,4Љ,6Љ,7Љ-penta-O-acetyl-L-glycero-á-D-manno-
heptopyranosyl)-(1Љ→5Ј)-7Ј,8Ј-di-O-acetyl-4Ј-O-[bis(2-
cyanoethyl)phosphoryl]-3Ј-deoxy-á-D-manno-oct-2Ј-
ulopyranosid}onate 32
A suspension of compound 31 (48 mg, 0.039 mmol), bis(2-
cyanoethyl) N,N-diisopropylphosphoramidite (63 mg, 0.234
mmol), 1H-tetrazole (27 mg, 0.39 mmol) and molecular sieves
4 Å (0.3 g) in CH2Cl2 (6 cm3) was stirred for 23 h at room
temperature under N2. The reaction vessel was cooled to
Ϫ20 ЊC and a solution of MCPBA (67 mg) in CH2Cl2 (2 cm3)
was added. After 30 min, 10% aq. Na2S2O5 (0.5 cm3) was added
and the suspension was centrifuged. Solids were repeatedly
washed with EtOAc–MeOH (1:1). Washings and supernatant
were combined, concentrated and purified by two successive
column chromatographic separations on silica gel [EtOAc–
MeOH (40:1); then EtOAc–MeOH–hexane (10:1:1)] to give
phosphotriester 32 as a syrup (55 mg, 99%); δH 5.92 (1 H, m,
CH᎐), 5.46 (1 H, d, JN H ,2 8.4, NH), 5.40 (1 H, dd, J3Љ,2Љ 3.2, J3Љ,4Љ
᎐
10.0, 3Љ-H), 5.31 (1 H, t, J4Љ,5Љ 10.0, 4Љ-H), 5.31–5.17 (3 H, m, 6Љ-
H and ᎐CH ), 5.12 (1 H, m, J7Ј,6Ј 9.3, 7Ј-H), 5.03 (1 H, dd, J2Љ,1Љ
᎐
2
1.9, 2Љ-H), 4.98 (1 H, d, 1Љ-H), 4.89–4.82 (3 H, m, J1,2 8.6, J8aЈ,8bЈ
12.8, 1- and 4Ј-H, 8Ј-Ha), 4.37–4.23 (7 H, m, 5Љ-H, 7Ј-Ha,
OCHH, 2 × OCH2CH2CN), 4.19–4.07 (5 H, m, 5Ј- and 6Ј-H,
8Ј- and 7Љ-Hb, OCHH2), 4.02 (1 H, t, J3,4 9.0, 3-H), 3.85 (1 H, d,
J6a,6b 8.7, 6-Ha), 3.80 (3 H, s, CO2Me), 3.65–3.54 (2 H, m, 5-H
and 6-Hb), 3.40–3.30 (2 H, m, 2- and 4-H), 2.83–2.79 (4 H, m,
2 × CH2CN), 2.38 (1 H, dd, J3eЈ3Јa 12.3, J3eЈ,4Ј 4.1, 3Ј-He), 2.29 (1
H, t, J3Јa,4Ј 12.2, 3Ј-Ha), 2.16, 2.10, 2.09, 2.08, 2.02, 2.01 and 2.00
(each 3 H, s, together 7 × Ac), 1.96 (3 H, s, NHAc) and 1.06–
0.99 (28 H, m, 4 × PriSi) (Found: C, 50.1; H, 6.3; N, 2.9.
C59H92N3O30PSi2 requires C, 50.4; H, 6.6; N, 2.9%).
CHCl3); δH 5.90 (1 H, m, CH᎐), 5.45 (1 H, d, J
7.6, NH),
᎐
N H ,2
5.41 (1 H, dd, J3Љ,2Љ 3.4, J3Љ,4Љ 10.0, 3Љ-H), 5.37–5.28 (3 H, m, 4Ј-
and 6Љ-H, ᎐CH2trans), 5.28 (1 H, t, J4Љ,5Љ 10.1, 4Љ-H), 5.18 (1 H,
᎐
dq, J 10.4, 1.6, ᎐CH2cis), 5.11 (1 H, m, J7Ј,6Ј 9.5 , 7Ј-H), 5.01
᎐
(1 H, dd, J2Љ,1Љ 2.0, 2Љ-H), 4.88 (1 H, d, J1,2 8.4, 1-H), 4.86 (1 H,
dd, J8Јa,8Јb 12.1, J8aЈ,7Ј 2.2, 8Ј-Ha), 4.80 (1 H, d, 1Љ-H), 4.32 (1 H,
ddt, OCH2), 4.27–4.07 (8 H, m, 5Ј- and 6Ј-H, 8Ј-Hb, 5Љ-H,
7Љ-H2 and ClCH2CO), 4.06 (1 H, t, J3,4 7.7, 3-H), 3.84 (1 H, dd,
J6a,6b 8.0, J6a,5 1.8, 6-Ha), 3.79 (3 H, s, CO2Me), 3.66 (1 H, t, J6b,5
8.0, 6-Hb), 3.54 (1 H, m, J5,4 9.0, 5-H), 3.39 (1 H, dd, 4-H), 3.31
(1 H, ddd, J2,3 10.2, 2-H), 2.27 (1 H, dd, J3Јe,3Јa 12.5, J3Јe,4Ј 4.7,
3Ј-He), 2.17–2.12 (7 H, m, 3Ј-Ha and 2 × Ac), 2.10, 2.08, 2.04,
2.03 and 2.02 (each 3 H, s, total 5 × Ac), 1.96 (3 H, s, NHAc)
and 1.10–0.99 (28 H, m, 4 × PriSi) (Found: C, 50.8; H, 6.5; N,
1.0. C55H86ClNO28Si2 requires C, 50.8; H, 6.7; N, 1.1%).
Ammonium [2-acetamido-1-O-allyl-2,6-dideoxy-â-D-
glucopyranos-6-yl O-(L-glycero-á-D-manno-heptopyranosyl)-
(1Љ→5Ј)-3-deoxy-á-D-manno-oct-2Ј-ulopyranosid]onate 4Ј-
(ammonium hydrogen phosphate) 33
A solution of compound 32 (22 mg, 0.015 mmol) in THF (5
cm3) was stirred with 1  TBAF in THF (23 mm3, 0.023 mmol)
for 2 h at ambient temperature. MeOH (1 cm3) was added and
the solution was concentrated, and treated with 0.1  meth-
anolic NaOMe (0.5 cm3) and MeOH (3 cm3) for 2 h. The pH of
the solution was adjusted to 6.5 by addition of Dowex 50 resin
(Hϩ-form), the resin was filtered off, and the filtrate was taken
to dryness which afforded the de-O-acetylated compound (18.9
mg) as a syrup. The residue was dissolved in water (2 cm3) and
the solution was stirred with 0.2  aq. NaOH (1 cm3) for 15 h.
The solution was cooled to 0 ЊC and quickly passed through
a column (5 × 0.5 cm) of Dowex resin (Hϩ-form); the
Methyl [2-acetamido-1-O-allyl-2,6-dideoxy-3,4-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)-â-D-glucopyranos-6-yl O-
(2Љ,3Љ,4Љ,6Љ,7Љ-penta-O-acetyl-L-glycero-á-D-manno-hepto-
pyranosyl)-(1Љ→5Ј)-7Ј,8Ј-di-O-acetyl-3Ј-deoxy-á-D-manno-
oct-2Ј-ulopyranosid]onate 31
A solution of chloroacetate 30 (60 mg, 0.046 mmol) in 2:1
lutidine–HOAc (5 cm3) was cooled to 0 ЊC and treated with
hydrazine dithiocarbonate (HDTC) (0.37 cm3, 0.138 mmol) for
30 min at 0 ЊC, then was stirred at room temperature for an
additional 60 min. The solution was co-evaporated six times
J. Chem. Soc., Perkin Trans. 1, 1997
1981

View Article Online
carbohydrate-containing effluents were immediately neutralised
by addition of 0.1  aq. NH3 and lyophilised. Final purification
of the material on Bio-Gel P2 afforded the target trisaccharide
33 as a hygroscopic solid (8.9 mg, 68%), [α]D20 ϩ33 (c 0.6, water);
Acknowledgements
The authors gratefully acknowledge help and support by
Professor Hans Paulsen and financial support of this work by
the Austrian Science Foundation (FWF-grant P 11449-MOB).
δ (D O) 5.90 (1 H, m, ᎐CH), 5.31 (1 H, dq, J 17.3, 1.6,
᎐
H
2
᎐CH
), 5.25 (1 H, dq, J 10.6, 1.3, ᎐CH2cis), 4.34 and 4.15
᎐
1
᎐
2trans
References
(2 H, ddt, OCH2) and 2.03 (3 H, s, NHAc). Additional H
and 13C NMR data are presented in Tables 1 and 2 (Found:
C, 37.9; H, 6.7; N, 4.9. C26H50N3O22Pؒ2H2O requires C, 37.9;
H, 6.6; N, 5.1%).
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Ammonium {2-acetamido-1-O-[3-(2-aminoethylthio)propyl]-2,6-
dideoxy-â-D-glucopyranos-6-yl O-(L-glycero-á-D-manno-
heptopyranosyl)-(1Љ→5Ј)-3Ј-deoxy-á-D-mannno-oct-2Ј-
ulopyranosid}onate 4Ј-(ammonium hydrogen phosphate)
hydrochloride 34
An aq. solution of compound 33 (6 mg, 0.007 mmol) and
cysteamine hydrochloride (2.5 mg, 0.022 mmol) (0.2 cm3) was
irradiated at 254 nm for 15 h at room temperature. The solution
was processed as described for compound 9, which afforded
compound 34 as a syrup (4.3 mg, 68%), [α]D20 ϩ32 (c 0.3, water);
δH (D2O) (inter alia) (1 H, br s, 1Љ-H), 4.50 (1 H, d, J1,2 8.5, 1-H),
4.48 (1 H, m, 4Ј-H), 4.26 (1 H, br s, 5Ј-H), 4.10 (1 H, m, 2Љ-H),
4.08 (1 H, dd, J6Ј,7Ј 8.0, 6Ј-H), 4.01–3.82 (6 H, m, 7Ј-H, 8Ј-Ha, 3Љ-
and 6Љ-H, 7Љ-Ha and OCHH), 3.82–3.65 (5 H, m, 2-, 4Љ- and 5Љ-
H, 7Љ-Hb, OCHH), 3.64 (1 H, dd, J8bЈ,7 5.6, J8bЈ,8aЈ 10.7, 8Ј-Hb),
3.60–3.48 (5 H, m, 3-, 4-, 5-H and 6-H2), 3.22 (2 H, t, NCH2),
2.86 (2 H, t, SCH2), 2.64 (2 H, t, SCH2), 2.24 (1 H, dd, J3eЈ,3aЈ
13.0, J3eЈ,4Ј 4.4, 3Ј-He), 2.03 (3 H, s, NHAc), 1.97 (1 H, t, J3aЈ,4Ј
12.7, 3Ј-Ha) and 1.84 (2 H, m, CH2); δC 175.60 and 175.32
(NHCO, C-1Ј), 102.24 (C-1), 101.16 (C-1Љ), 100.93 (C-2Ј), 75.13
(C-5), 74.91 (C-3), 73.46 (C-5Ј), 72.96 and 72.91 (C-6Ј, -5Љ),
71.34 (C-3Љ), 70.99 (C-4), 70.91 (C-7Ј, -2Љ), 70.08 (C-6Љ), 69.85
(C-4Ј), 69.37 (OCH2), 67.62 (C-4Љ), 64.40 and 63.94 (C-8Ј, -7Љ),
62.87 (C-6), 56.39 (C-2), 38.59 (CN), 35.21 (C-3Ј), 29.25, 29.02
and 27.70 (CSC, CH2) and 23.00 (CH3) (Found: C, 36.3; H, 6.8;
N, 5.9; S, 3.3. C28H58ClN4O22PSؒH2O requires C, 36.6; H, 6.6;
N, 6.1; S, 3.5%).
14 P. Kosma, J. Gass, G. Schulz, R. Christian and F. M. Unger,
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Synthesis of BSA-conjugates 6, 10 and 35
A solution of thiophosgene (2 mm3, 0.026 mmol) in CHCl3 (2
cm3) was added to a solution of compound 5 (5.3 mg, 0.009
mmol) in 0.1  aq. NaHCO3 (1.5 cm3) and the mixture was
vigorously stirred for 4 h at room temperature. The organic
phase was separated, and the aqueous layer was washed three
times with CHCl3 (2 cm3 portions) and purged with N2 until a
clear solution was obtained. The solution was transferred to a
solution of BSA (SIGMA; 5 mg) in 0.1  aq. NaHCO3–0.3 
aq. NaCl (1 cm3). The solutions were stirred slowly at room
temperature for 72 h, then passed through a Sephadex G-25
column (1.6 × 50 cm; 0.01  aq. NaHCO3). Ninhydrin-positive
fractions were pooled, and dialysed twice against distilled
water (2000 cm3) for 24 h. Lyophilisation gave the BSA-
conjugate 6 (6.0 mg) as a fluffy powder. Yield for conjugate 10
(4.5 mg of 9, 2 mm3 of CSCl2, 5.0 mg of BSA): 4.0 mg. Yield
for conjugate 35 (3.3 mg of 34, 2 mm3 of CSCl2, 4.0 mg of
BSA): 5.0 mg.
27 H. Brade, C. Galanos and O. Lüderitz, Eur. J. Biochem., 1983, 131,
195.
Paper 7/00497D
Received 21st January 1997
Accepted 17th March 1997
1982
J. Chem. Soc., Perkin Trans. 1, 1997