Application of β-1,4-galactosyltransferase in the synthesis of complex branched-chain oligosaccharide mimics of fragments of the capsular polysaccharide of Streptococcus pneumoniae type 14

Article abstract of DOI:10.1039/a804272a

The chemoenzymic synthesis is described of β-D-Galp-(1→4)-β-D-Glcp-(1→6)-[β-D-Galp-(1→4)]- β-D-GlcpNAc-(1→O[CH₂] ₃O→4)-β-D-Glcp-(1→OCH₂CH=CH₂) 32 and β-D-Galp-(1→4)-β-D-GlcpNAc-(1→ O[CH₂] ₃O→4)-β-D-Glcp-(1→6)-[β-D-Galp-(1→4)]- β-D-GlcpNAc-(1→O[CH₂] ₃O→4)-β-D-Glcp-(1→OCH₂-CH=CH₂) 33, representing hexa- and octasaccharide mimics of fragments of the Streptococcus pneumoniae type 14 polysaccharide. In a chemical approach the intermediate linear oligosaccharide mimics 30 and 31 were synthesized, wherein both terminal and non-terminal N-acetyl-β-D-glucosamine residues were not yet galactosylated. The alkyl-bridged derivatives were found to be good acceptor substrates for bovine milk β-1,4-galactosyltransferase. Reaction of the anomeric allyl functions with cysteamine under UV-irradiation gave the corresponding 3-(2-aminoethylthio)propyI glycosides 34 and 35, suitable for further coupling of the oligosaccharide mimics to protein carriers.

Full text of DOI:10.1039/a804272a

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Application of â-1,4-galactosyltransferase in the synthesis of complex
branched-chain oligosaccharide mimics of fragments of the capsular
polysaccharide of Streptococcus pneumoniae type 14
Jutta Niggemann,* Johannis P. Kamerling and Johannes F. G. Vliegenthart
Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, PO Box 80.075,
NL-3508 TB Utrecht, The Netherlands
The chemoenzymic synthesis is described of β-D-Galp-(1→4)-â-D-Glcp-(1→6)-[â-D-Galp-(1→4)]-â-D-
GlcpNAc-(1→O[CH ] O→4)-â-D-Glcp-(1→OCH CH᎐CH ) 32 and â-D-Galp-(1→4)-â-D-GlcpNAc-(1→
᎐
2
3
2
2
O[CH2]3O→4)-â-D-Glcp-(1→6)-[â-D-Galp-(1→4)]-â-D-GlcpNAc-(1→O[CH2]3O→4)-â-D-Glcp-(1→OCH2-
CH᎐CH ) 33, representing hexa- and octasaccharide mimics of fragments of the Streptococcus pneumoniae
᎐
2
type 14 polysaccharide. In a chemical approach the intermediate linear oligosaccharide mimics 30 and
31 were synthesized, wherein both terminal and non-terminal N-acetyl-â-D-glucosamine residues were not
yet galactosylated. The alkyl-bridged derivatives were found to be good acceptor substrates for bovine milk
â-1,4-galactosyltransferase. Reaction of the anomeric allyl functions with cysteamine under UV-
irradiation gave the corresponding 3-(2-aminoethylthio)propyl glycosides 34 and 35, suitable for
further coupling of the oligosaccharide mimics to protein carriers.
(1→3)-β--Galp-(1→4)-β--Glcp-(1→ trisaccharide backbone
Introduction
structure is substituted by a flexible acyclic spacer. For an easy
access to branched-chain oligosaccharide mimics, also the
β-1,4-galactosyltransferase-catalyzed galactosylation of N-
acetyl-β--glucosamine residues of linear oligosaccharide mim-
ics, using uridine-5Ј-diphosphogalactose (UDP-Gal) as donor,
was investigated.
Pneumococcal infections are major causes of bacterial
pneumonia, otitis media and meningitis and despite the avail-
ability of antibiotic therapy is still a significant cause of mor-
tality throughout the world.1,2 Owing to the rapid course of the
disease and the emergence of antibiotic-resistant strains,3–5 dis-
ease prevention by vaccination is highly desirable. The current
polyvalent pneumococcal vaccines Pneumovax 23 (Merck,
Sharp and Dohme) and Pnu-Immune 23 (Lederle-Praxis),
which contain the capsular polysaccharides of 23 of the most
common serotypes,6 offer 90% protection in immunocompetent
adults, but are inadequate in the population at greatest risk
for serious pneumococcal infections.7,8 The immune response
to T-cell-independent (TI) polysaccharide antigens is poor
in infants and young children up to the age of 2 years. The
development of more immunogenic conjugate vaccines for
serotypes responsible for most pediatric diseases (e.g. 6B, 14,
18C, 19F, and 23F) is thus of great importance.9,10
Owing to its availability and flexibility both in donor and
acceptor substrate specificity, β-1,4-galactosyltransferase is
one of the most extensively studied mammalian glycosyl-
transferases.15,16 Two findings stimulated our approach to
branched-chain oligosaccharide mimics by enzymic galactosyl-
ation. β-1,4-Galactosyltransferase (UDP-Gal: -glucose β-1,4-
galactosyltransferase [EC 2.4.1.22]), which uses -glucose as
the preferred acceptor in the presence of α-lactalbumin, is
regarded as transferring in vivo galactose to only terminal
N-acetyl-β--glucosamine residues.17,18 However, 6-O-glyco-
sylated N-acetyl-β--glucosamine derivatives like α--Fucp-
(1→6)-β--GlcpNAc,19
α-Neup5Ac(OMe)-(2→6)-β--Glcp-
The Streptococcus pneumoniae type 14 polysaccharide (Pn
14-PS) consists of a branched tetrasaccharide repeating unit11
which is structurally identical with the asialo core antigen of
the type III group B Streptococcus (GBS III) capsular poly-
saccharide:12
NAc,19 and β--Galp-(1→4)-β--Glcp-(1→6)-β--GlcpNAc-
(1→OAll)20 were found to be substrates of the enzyme.
Furthermore, a large variety of aglycones are readily tolerated
by the enzyme, and even increasing galactosylation has been
observed for N-acetyl-β--glucosamine glycosides of hydro-
phobic aglycones.21,22 This so called hydrophobic effect was
confirmed by studies on recognition- and binding-subsites of
the enzyme using diantennary alkyl-bridged oligosaccharides
with terminal N-acetyl-β--glucosamine residues.23,24
→6)-β--GlcpNAc-(1→3)-β--Galp-(1→4)-β--Glcp(1→
4
↑
1
Here, we report on the chemoenzymic synthesis of alkyl-
bridged oligosaccharide mimics of fragments of the Strepto-
coccus pneumoniae type 14 polysaccharide containing an agly-
cone spacer for the subsequent attachment to carrier proteins.
β--Galp
Structural similarities between antigenic determinants of the
Pn 14 polysaccharide and human oligosaccharide structures,
which may give rise to the induction of autoantibodies and
suppression of the immune response, may be responsible for the
poor immunogenicity of the Pn 14 polysaccharide among the
pneumococcal capsular polysaccharides.13 Evidence for cross-
reactivity with human tissue was found by immunization of
both rabbits and mice with Pn 14-PS.14
Results and discussion
The convergent synthetic strategy for the preparation of the key
linear oligosaccharide mimic derivatives 18 and 19 involves the
glycosylation of the common trisaccharide mimic acceptor 9
with either disaccharide 11 or trisaccharide mimic 17 thioethyl
glycoside donors. In a series of protecting-group manipu-
lations key compounds 18 and 19 can be converted into the
As autoreactive antibodies were preferentially reactive with
lactose, we designed mimics of the Pn 14 polysaccharide in
which the galactose moiety of the repeating →6)-β--GlcpNAc-
J. Chem. Soc., Perkin Trans. 1, 1998
3011

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OBn
O
HO
BnO
+
Br
TBDMSO
OTMSE
OBn
1
2
i
OAc
O
OBn
O
AcO
AcO
O
BnO
TBDMSO
SEt
OTMSE
NPhth
OBn
3
5
ii
iii
OTBDPS
O
OAc
OBn
O
O
RO
RO
O
BnO
AcO
AcO
HO
+
+
SEt
OTMSE
Br
NPhth
OBn
NPhth
12
4
6 R = H
7 R = MBz
iv
v
viii
OAc
OR
OAc
O
OAc
O
OBn
O
OR
O
O
O
O
AcO
MBzO
MBzO
AcO
AcO
AcO
BnO
O
RO
O
OTMSE
OTMSE
O
O
NPhth
AcO
NPhth
O
AcO
CCl3
OBn
OAc
OR
NH
8 R = TBDPS
9 R = H
10
13 R = Bn
14 R = Ac
vi
ix
vii
x
OAc
O
OAc
O
OAc
OAc
O
O
AcO
AcO
O
R
SEt
AcO
AcO
AcO
O
O
OAc
AcO
OAc
NPhth
OAc
11
15 R = OH
16 R = OCNHCCl3
17 R = β-SEt
xi
TMSE = (CH2)2SiMe3, TBDMS = SiMe2CMe3, TBDPS = SiPh2CMe3, MBz = p-MeC6H4CO
xii
Scheme 1 Reagents and yields: i, NaH (96%); ii, p-TsOH (97%); iii, (a) NaOMe, MeOH; (b) TBDPSCl (72%); iv, MBzCl (90%); v, NIS, AgOTf
(85%); vi, AcCl, MeOH (89%); vii, EtSH, BF3ؒEt2O (94%); viii, AgOTf (76%); ix, (a) H2, Pd–C; (b) Ac2O, pyridine (84%); x, TFA (96%); xi, CCl3CN,
DBU (89%); xii, EtSH, BF3ؒEt2O (87%).
unprotected penta- and hexasaccharide mimics β--Galp-
(1→4)-β--Glcp-(1→6)-β--GlcpNAc-(1→O[CH2]3O→4)-β-
The 4-O-alkylated glycoside 3 was prepared in 96% yield by
reaction of 2-(trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-β--gluco-
pyranoside25 2 with 3-(tert-butyldimethylsilyloxy)propyl brom-
ide26 1 in a 1:1 mixture of DMF and THF. Subsequent removal
of the acid-sensitive tert-butyldimethylsilyl (TBDMS) group
with p-TsOH in aq. acetonitrile gave glycosyl acceptor 4 in
97% yield.
For the synthesis of the trisaccharide mimic acceptor 9 a
combination of tert-butyldiphenylsilyl (TBDPS) and p-methyl-
benzoyl (MBz) protecting groups was chosen, which had
already successfully been used in the preparation of the β--
Galp-(1→4)-β--Glcp-(1→6)-β--GlcpNAc-(1→OAll) trisac-
charide fragment of the S. pneumoniae type 14 polysacchar-
ide.20 The suitably protected 6-O-silylated β-thioglycoside
-Glcp-(1→OCH CH᎐CH ) 30 and β--GlcpNAc-(1→O-
᎐
2
2
[CH2]3O→4)-β--Glcp-(1→6)-β--GlcpNAc-(1→O[CH2]3O→
4)-β--Glcp-(1→OCH CH᎐CH ) 31, respectively, being the
᎐
2
2
acceptor structures for β-1,4-galactosyltransferase.
Both the synthesis of the trisaccharide mimic acceptor 9 and
donor 17 required the 2-(trimethylsilyl)ethyl (TMSE) 4-O-(3-
hydroxypropyl) glycoside 4 as intermediate (Scheme 1). For
anomeric protection the 2-(trimethylsilyl)ethyl group was
chosen, as TMSE glycosides are compatible with a wide range
of different reaction conditions as well as being easy removable
by reaction with TFA in dichloromethane without affecting the
glycosidic bonds.25
3012
J. Chem. Soc., Perkin Trans. 1, 1998

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OAc
OAc
OAc
O
O
OAc
O
O
AcO
AcO
O
O
AcO
SEt
AcO
O
OAc
AcO
OAc
MBzO
OAc
OBn
AcO
OAc
O
i
11
BnO
O
OTMSE
O
MBzO
O
NPhth
OBn
OH
18
OBn
O
O
MBzO
MBzO
BnO
O
OTMSE
O
OAc
NPhth
NPhth
O
OBn
O
9
AcO
AcO
O
O
AcO
O
i
OAc
MBzO
OBn
OAc
O
BnO
O
OTMSE
O
MBzO
OAc
O
NPhth
OAc
O
O
OBn
AcO
AcO
SEt
AcO
O
O
NPhth
19
OAc
17
Scheme 2 Reagents and yields: i, NIS, AgOTf (18: 42%; 19: 66%).
donor 7 was prepared in 65% overall yield by deacetylation
of ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-1-thio-β--
glucopyranoside27 5, followed by selective silylation of the
primary HO-6 group with tert-butyldiphenylsilyl chloride
(TBDPSCl) (→ 6) and subsequent p-methylbenzoylation of
the HO-3 and HO-4 groups with MBzCl. N-Iodosuccinimide
(NIS) promoted glycosylation of the primary hydroxy group of
the 4-O-(3-hydroxypropyl) glycoside 4 with β-thioethyl glyco-
side 7 in the presence of a catalytic amount of silver trifluoro-
methanesulfonate (AgOTf)28,29 afforded the desired trisacchar-
ide mimic 8 in 85% yield. Selective desilylation with acetyl
chloride in 1:1 methanol–toluene30 gave the desired trisacchar-
ide mimic acceptor 9 in 89% yield.
lished earlier as a consequence of MBz group migration during
de-N-phthaloylation.20 Thus, compounds 18 and 19 were first
de-O-p-methylbenzoylated and de-O-acetylated with sodium
methoxide in methanol, and subsequently de-N-phthaloyl-
ated with ethylenediamine in butan-l-ol35 for 24 h at 80 ЊC.
Re-N,O-acetylation gave compounds 20 (95%) and 21
(97%), respectively (Scheme 3). Removal of the benzyl
groups by hydrogenolysis with 10% Pd–C as catalyst in acetic
acid, followed by O-acetylation, gave the per-O-acetylated
2-(trimethylsilyl)ethyl glycosides 22 and 23 in 72% and 84%
yield, respectively.
In subsequent reactions compounds 22 and 23 were con-
verted into allyl glycosides 26 and 29, respectively, by treatment
with TFA in dichloromethane [→ hemiacetal sugars 24 (90%)
and 27 (84%)], followed by imidation with trichloroacetonitrile
in the presence of DBU [→ 25 (85%) and 28 (82%)], and
subsequent allylation with allyl alcohol and BF3ؒEt2O [→ 26
(61%) and 29 (59%)]. Deacetylation with sodium methoxide in
methanol furnished the linear glycosyl mimics 30 (86%) and 31
(92%), respectively.
The bovine β-1,4-galactosyltransferase-catalyzed syntheses
of the branched-chain oligosaccharide mimics 32 and 33 was
achieved by transfer of galactosyl groups from UDP-galactose
to the N-acetyl-β--glucosamine residues of intermediates 30
and 31, respectively (Scheme 4). Initial reaction rates were
determined under standard conditions with a coupled enzyme
assay for UDP.36 With N-acetyl--glucosamine taken as a refer-
ence (with an assigned relative rate of 100) the pentasaccharide
mimic 30, containing a non-terminal 6-O-substituted N-acetyl-
β--glucosamine residue, showed good acceptor activity (35%).
The hexasaccharide mimic 31, containing both terminal and
non-terminal N-acetyl-β--glucosamine residues, was found to
be a weaker acceptor (15%).
Oligosaccharide mimics 32 and 33 were then prepared on a
preparative scale. Alkaline phosphatase was added to the
incubation mixtures to prevent feedback inhibition by
released UDP37,38 and to promote a high conversion of
the acceptor [→ 32 (85%) and 33 (80%)]. Fast-atom bom-
bardment (FAB) mass spectrometry confirmed the intro-
duction of one galactosyl residue in compound 32 and the
presence of two galactosyl residues in compound 33. The 13C
NMR spectra of compounds 32 and 33 showed the expected
As the anomeric TMSE group of glycosyl acceptor 9 is not
compatible with Lewis acid-induced activation of glycosyl
imidates,31 the β-thioethyl group was chosen for anomeric acti-
vation of both disaccharide donor 11 and trisaccharide mimic
donor 17.
As per-acetylated α-lactose, which is formed by acetylation
of lactose with acetic anhydride in pyridine, does not form the
corresponding thioglycoside upon reaction with ethanethiol in
the presence of Lewis acid,32 the thiolactoside 11 was prepared
in 94% yield by reaction of lactosylimidate 1033 with ethane-
thiol in the presence of boron trifluoride–diethyl ether.
For the preparation of the trisaccharide mimic donor 17 first
the 4-O-(3-hydroxypropyl) glucoside 4 was coupled with glyco-
syl bromide 1234 in the presence of AgOTf to give compound
13 in 76% yield. Hydrogenolytic cleavage of the benzyl groups
of compound 13 with 10% Pd–C as catalyst in acetic acid
and subsequent O-acetylation with acetic anhydride in pyridine
gave hexaacetate 14 in 84% overall yield. Then, TMSE glyco-
side 14 was converted into β-thioglycoside 17 by selective
removal of the anomeric 2-(trimethylsilyl)ethyl group with TFA
in dichloromethane (→ 15, 96%), followed by imidation with
trichloroacetonitrile in the presence of DBU (→ 16, 89%)
and subsequent glycosylation with ethanethiol in the presence
of BF3ؒEt2O (→ 17, 87%).
NIS/AgOTf-promoted reactions of glycosyl acceptor 9 with
thioglycosides 11 and 17 gave the key 6Ј-O-glycosylated com-
pounds 18 (42%) and 19 (66%), respectively (Scheme 2).
De-N-phthaloylation of intermediates 18 and 19 was
achieved in high yield by applying a reaction sequence estab-
J. Chem. Soc., Perkin Trans. 1, 1998
3013

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Table 1 1H NMR data (COSY, TOCSY, ROESY) of compound 32
18, 19
Glca
GlcNAc
Glcb
Gala
Galb
i
Proton (δH)
OR
OR'
O
H-1c
H-2
H-3
H-4
H-5
Ha-6
Hb-6
4.47
3.29
3.43
3.73
3.56
3.91
3.89
4.54
3.73
3.69
3.83
3.71
4.28
3.96
4.56
3.39
3.68
3.63
3.60
3.98
3.82
4.45
3.55
3.67
3.92
3.74
d
4.54
3.54
3.66
3.92
3.72
d
O
AcO
AcO
R'O
O
OTMSE
O
NHAc
OR'
20, 21 R' = Bn
22, 23 R' = Ac
ii
d
d
iii
OCH2CH᎐CH2
4.37, 4.21 (2 m, each 1 H)
5.98 (m, 1 H)
5.40–5.27 (m, 2 H)
2.05 (s, 3 H)
᎐
OR
O
OCH2CH᎐CH2
᎐
OR'
O
OCH2CH᎐CH2
᎐
R'O
R'O
R'O
O
R''
NHCOCH3
O
OCH2CH2CH2O
1.87–1.82 (m, 2 H)
NHAc
OR'
a,b Glca: Glcβ(1-OAll). Glcb: Glc(β1→6)GlcNAc. Gala: Gal(β1→4)-
24, 27 R' = Ac, R'' = OH
25, 28 R' = Ac, R'' = α-OCNHCCl3
26, 29 R' = Ac, R'' = β-OAll
c
Glc. Galb: Gal(β1→)GlcNAc. J1,2 Coupling constants were >7 Hz,
iv
indicating β-configuration for all monosaccharide residues. d Not
v
determined.
30, 31 R' = H, R'' = β-OAll
vi
Table 2 1H NMR data (COSY, TOCSY, ROESY) of compound 33
All = CH2CH=CH2
Proton (δH
in ppm)
Glca
GlcNAca Glcb
GlcNAcb
Gala
Galb
OAc
OAc
H-1c
H-2
H-3
H-4
H-5
Ha-6
Hb-6
4.47
3.29
3.43
d
3.57
d
4.53
3.73
3.69
3.83
3.72
4.28
3.94
4.49
3.31
3.46
3.68
3.58
3.98
3.82
4.53
3.73
d
3.74
d
d
d
4.47
3.54
3.68
3.93
3.74
d
4.53
3.54
d
3.93
d
d
d
O
AcO
AcO
O
= R
20, 22, 24, 25, 26
21, 23, 27, 28, 29
AcO
O
OAc
OAc
OAc
O
NHAc
O
AcO
AcO
O
O
AcO
= R
= R
d
d
OAc
OAc
OCH2CH᎐CH2
OCH2CH᎐CH2
OCH2CH᎐CH2
NHCOCH3
4.39, 4.22 (2 m, each 1 H)
5.98 (m, 1 H)
5.40–5.27 (m, 2 H)
2.05 (s, 6 H)
᎐
᎐
᎐
OH
OH
O
O
HO
O
HO
30
31
OH
OCH2CH2CH2O
1.90–1.80 (m, 4 H)
HO
OH
a,b Glca: Glcβ(1-OAll). Glcb: Glc(β1→6)GlcNAca. Gala: Gal(β1→4)-
GlcNAcb. Galb: Gal(β1→)GlcNAca. c,d As in Table 1.
OH
NHAc
O
O
HO
HO
O
O
HO
= R
OH
either 10% H2SO4 in EtOH, 0.2% orcinol in 20% methanolic
H2SO4, or 1% KMnO4 in 0.2  aq. Na2CO3. Solutions were
concentrated under reduced pressure at <40 ЊC. Column chrom-
atography was performed on Silica Gel 60 (0.063–0.200 mm,
Merck). Gel-permeation chromatography was performed on
Toyopearl HW-40S (Supelco) (2.0 × 60 cm). UV-irradiations
were performed in quartz vials at 254 nm using a Cole-Parmer
50 W high-intensity UV lamp. Optical rotations were measured
with a Perkin-Elmer 241 polarimeter. [α]D-Values are given in
OH
Scheme 3 Reagents and yields: i, (a) NaOMe, MeOH; (b) H2NCH2-
CH2NH2, butan-l-ol, 80 ЊC; (c) Ac2O, pyridine (20: 95%; 21: 97%); ii,
(a) H2, Pd–C; (b) Ac2O, pyridine (22: 72%; 23: 84%); iii, TFA (24: 90%;
27: 84%); iv, CCl3CN, DBU (25: 85%; 28: 82%); v, AllOH, BF3ؒEt2O
(26: 61%; 29: 59%); vi, NaOMe, MeOH (30: 86%; 31: 92%).
additional anomeric signals, corresponding to mono- (32)
and di-galactosylation (33), respectively. Furthermore, 2D
COSY,† TOCSY† and ROESY† spectra confirmed the struc-
tures of compounds 32 (Table 1) and 33 (Table 2), with inter-
residual nuclear Overhauser effects (NOEs) between 1-H of
the transferred galactose and 4-H of terminal and non-terminal
N-acetylglucosamine residues, respectively.
The allyl glycosides 32 and 33 were then converted by reac-
tion with cysteamine39 under UV-irradiation into the 3-(2-
aminoethylthio)propyl glycosides 34 and 35, respectively, suit-
able for conjugation to carrier proteins.
1
10Ϫ1 deg cm2 gϪ1. H NMR spectra (300 MHz) were recorded
with a Bruker AC 300 spectrometer; only selected NMR data
are reported. Two-dimensional double-quantum filtered 1H–1H
1
correlated spectra (2D DQF H–1H COSY), two-dimensional
TOCSY spectra with 100 ms and 150 ms mixing sequences, and
2D 1H ROESY spectra (300 ms mixing sequence) were recorded
at 300 K using a Bruker AMX 500 spectrometer. Chemical
shifts (δ) are given in ppm relative to the signal for internal
Me4Si (δ 0, CDCl3) or acetone (δ 2.225, D2O). J-Values are
given in Hz. 13C NMR spectra (75.5 MHz) were recorded with a
Bruker AC 300 spectrometer; δC(ppm)-values are given relative
to the signal for CDCl3 (δC 76.9) or internal acetone (δC 31.08).
13C signals of SiMe3 groups (<0 ppm) are not listed. Owing to
overlap not all 13C signals are present in appropriate numbers.
Fast-atom bombardment mass spectrometry (FABMS) was
performed on a JEOL JMS SX/SX 102A four-sector mass
spectrometer, equipped with a JEOL MS-FAB 10 D FAB gun.
Elemental analyses were carried out by H. Kolbe Mikro-
analytisches Laboratorium (Mülheim an der Ruhr, Germany).
All compounds for which elemental analytical data are not
available were chromatographically homogeneous and NMR
and mass spectral data were in full agreement with the assigned
structures.
Conjugation of the type 14 oligosaccharide mimics to
CRM197 (cross-reactive material) and immunological studies
are in progress.
Experimental
General procedures
Reactions were monitored by TLC on Silica Gel 60 F254
(Merck) with detection either by UV light or charring with
† 2D COSY: 2-dimensional chemical-shift–correlation spectroscopy;
TOCSY: phase-sensitive 2-dimensional total correlation spectroscopy;
ROESY: rotating-frame nuclear Overhauser enhancement spectroscopy.
3014
J. Chem. Soc., Perkin Trans. 1, 1998

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OH
NHAc
O
O
HO
HO
O
O
HO
O
OH
OH
OH
O
OH
HO
HO
HO
O
OAll
O
OH
O
O
O
O
HO
HO
NHAc
O
OH
OH
HO
31
HO
OH
OH
O
O
HO
O
OAll
O
HO
NHAc
OH
i
30
i
OH
OH
HO
NHAc
O
O
O
HO
O
O
O
HO
HO
O
OH
OH
OH
OH
O
OH
O
O
NHAc
O
HO
HO
HO
O
R
OH
O
OH
O
O
O
HO
HO
HO
OH
OH
O
OH
HO
OH
O
OH
OH
O
O
NHAc
O
HO
HO
HO
O
R
33 R = OAll
35 R = O(CH2)3S(CH2)2NH2
ii
O
HO
OH
OH
32 R = OAll
34 R = O(CH2)3S(CH2)2NH2
ii
Scheme 4 Reagents and yields: i, UDP-Gal, β-1,4-galactosyltransferase (32: 85%; 33: 80%); ii, cysteamine hydrochloride, hν (34: 78%; 35: 85%).
Materials
pyranoside25 2 (1.28 g, 2.3 mmol) in dry THF (10 ml). The
solution was stirred for 3 h at 0 ЊC. MeOH (3 ml) was added
and the solution was poured onto ice–water, then extracted with
CH2Cl2, and the organic layer was washed with water, dried
(MgSO4), and concentrated. Column chromatography (toluene–
EtOAc, 40:1) of the residue gave title compound 3 (1.60 g, 96%);
TLC (toluene–EtOAc, 10:1) Rf 0.35 (2), 0.57 (3); [α]D ϩ4 (c 1,
in CHCl3) (Found: C, 68.2; H, 8.6. C41H62O7Si2 requires C, 68.1;
H, 8.6%); δH(300 MHz; CDCl3) 7.34–7.23 (15 H, m, 3 × Ph),
4.94, 4.87, 4.75 and 4.71 (4 H, 2 AB systems, JA,B 11.0,
2 × PhCH2O), 4.64 and 4.56 (2 H, AB system, JA,B 12.2,
PhCH2O), 4.38 (1 H, d, J1,2 7.7, 1-H), 1.70–1.69 (2 H, m,
OCH2CH2CH2O), 1.08–1.02 (2 H, m, CH2Si), 0.86 [9 H, s,
C(CH3)3], 0.03 [9 H, s, Si(CH3)3] and 0.01 [6 H, s, Si(CH3)2]; δC-
(75.5 MHz; CDCl3) 138.6–138.2 and 128.2–127.4 (Ar-C), 103.0
(C-1), 84.5, 82.2, 78.3 and 75.0 (C-2, -3, -4, -5), 75.4, 74.7, 73.4,
69.8, 69.2, 67.3 and 60.0 (C-6, 3 × PhCH2O, OCH2CH2CH2O
and OCH2CH2Si), 33.6 (OCH2CH2CH2O), 25.8 [C(CH3)3], 18.4
[C(CH3)3 and CH2Si]; FABMS of C41H62O7Si2 (M, 722.4) m/z
721.5 (M Ϫ H)Ϫ.
Bovine milk β-1,4-galactosyltransferase (EC 2.4.1.22),‡ UDP-
galactose, β-nicotinamide adenine dinucleotide (reduced form;
β-NADH), phospho(enol)pyruvate, pyruvate kinase (EC
2.7.1.40, type III from rabbit muscle),§ -lactic dehydrogenase
(EC 1.1.1.27, type XI from rabbit muscle),¶ and alkaline phos-
phatase (EC 3.1.3.1, type I from bovine intestine)|| were obtained
from Sigma.
Measurement of galactosyltransferase activity
Initial reaction rates were determined under standard condi-
tions at 20 ЊC in 500 µl sodium cacodylate buffer (100 m, pH
7.5) containing 10 m MnCl2, 50 m KCl, 0.2 m UDP-galac-
tose, 1 m phospho(enol)pyruvate, 0.3 m β-NADH, 25 U
pyruvate kinase, 25 U -lactic dehydrogenase, 10 m acceptor,
and 20 U β-1,4-galactosyltransferase. Formation of UDP was
followed by monitoring the decrease in absorbance at 340 nm.
2-(Trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-4-O-[3-(tert-butyl-
dimethylsilyloxy)propyl]-â-D-glucopyranoside 3
To a suspension of sodium hydride (60% dispersion in oil; 0.4 g)
in dry DMF (10 ml) was added at 0 ЊC under argon a solution
of 3-(tert-butyldimethylsilyloxy)propyl bromide26 1 (1.00 g, 3.9
mmol) and 2-(trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-β--gluco-
2-(Trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-4-O-(3-hydroxy-
propyl)-â-D-glucopyranoside 4
To a solution of compound 3 (0.90 g, 1.2 mmol) in CH3CN (80
ml) were added water (9 ml) and p-TsOH monohydrate (50 mg).
The solution was stirred for 1.5 h at rt, neutralized with tri-
ethylamine, and concentrated. To the residue were added
CH2Cl2 and water, and the organic layer was separated, washed
with water, dried (MgSO4), and concentrated. Column chrom-
atography (toluene–EtOAc, 4:1) of the residue gave title com-
pound 4 (0.71 mg, 97%) isolated as a syrup; TLC (toluene–
EtOAc, 1:1) Rf 0.92 (3), 0.69 (4); [α]D ϩ9 (c 1, CHCl3) (Found:
‡ (1 unit will transfer 1.0 µmol of galactose from UDP-galactose
to glucose per min at pH 8.4 at 30 ЊC in the presence of 0.2 mg
α-lactalbumin).
§ (1 unit will convert 1.0 µmol of phospho(enol)pyruvate to pyruvate
per min at pH 7.6 at 37 ЊC).
¶ (1 unit will reduce 1.0 µmol of pyruvate to -lactate per min at pH 7.5
at 37 ЊC).
|| (1 unit will hydrolyse 1.0 µmol of p-nitrophenyl phosphate per min at
pH 10.4 at 37 ЊC).
J. Chem. Soc., Perkin Trans. 1, 1998
3015

View Article Online
C, 69.2; H, 7.9. C35H48O7Si requires C, 69.05; H, 7.95%); δH(300
MHz; CDCl3) 7.36–7.17 (15 H, m, 3 × Ph), 4.96 and 4.92 (2 H,
AB system, JA,B 10.9, PhCH2O), 4.75 and 4.71 (2 H, AB system,
JA,B 11.0, PhCH2O), 4.64 and 4.58 (2 H, AB system, JA,B 12.1,
PhCH2O), 4.39 (1 H, d, J1,2 7.7, 1-H), 2.37 (s, OH), 1.70–1.69 (2
H, m, OCH2CH2CH2O), 1.08–1.02 (2 H, m, CH2Si), 0.03 [9 H,
s, Si(CH3)3]; δC(75.5 MHz; CDCl3) 138.5–138.0 and 128.2–
127.4 (Ar-C), 103.0 (C-1), 84.4, 82.2, 78.4 and 74.8 (C-2, -3, -4,
-5), 75.3, 74.6, 73.4, 71.4, 68.9, 67.3 and 61.0 (C-6, 3 × PhCH2O,
OCH2CH2CH2O and OCH2CH2Si), 32.5 (OCH2CH2CH2O)
and 18.4 (CH2Si); FABMS of C35H48O7Si (M, 608.3) m/z 631.4
(M ϩ Na)ϩ; 607.4 (M Ϫ H)Ϫ.
J4Ј,5Ј 9.5, 4Ј-H), 5.47 (1 H, d, J1Ј,2Ј 8.4, 1Ј-H), 4.91 and 4.68 (2 H,
AB system, JA,B 11.1, PhCH2O), 4.78 and 4.63 (2 H, AB system,
JA,B 11.0, PhCH2O), 4.56 and 4.53 (2 H, AB system, JA,B 12.2,
PhCH2O), 4.50 (1 H, dd, 2Ј-H), 4.29 (1 H, d, J1,2 7.6, 1-H), 2.37
and 2.28 (each 3 H, 2 s, 2 × COC6H4CH3), 1.71–1.64 (2 H, m,
OCH2CH2CH2O), [1.06 (9 H, s, C(CH3)3], 1.06–1.02 (2 H, m,
CH2Si) and 0.03 [9 H, s, Si(CH3)3]; δC(75.5 MHz; CDCl3) 165.7
and 164.9 (2 × COC6H4CH3), 143.7 and 138.5–125.9 (Ar-C),
102.9 (C-1), 97.9 (C-1Ј), 84.2, 82.0, 78.1, 75.0, 74.7, 71.2 and
69.6 (C-2, -3, -4, -5, -3Ј, -4Ј, -5Ј), 75.3, 74.5, 73.1, 69.2, 68.8,
67.2, 66.5 and 62.8 (C-6, -6Ј, 3 × PhCH2O, OCH2CH2CH2O
and OCH2CH2Si), 55.0 (C-2Ј), 30.2 (OCH2CH2CH2O), 26.5
[C(CH3)3], 21.5 and 21.4 (2 × COC6H4CH3), 19.0 [C(CH3)3]
and 18.4 (CH2Si); FABMS of C81H91NO15Si2 (M, 1373.6) m/z
1396.7 (M ϩ Na)ϩ.
Ethyl 6-O-(tert-butyldiphenylsilyl)-2-deoxy-3,4-di-O-(p-methyl-
benzoyl)-2-phthalimido-1-thio-â-D-glucopyranoside 7
To a solution of ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthal-
imido-1-thio-β--glucopyranoside27 5 (1.00 g, 2.1 mmol) in
MeOH (20 ml) was added 0.2  NaOMe in MeOH (0.6 ml).
After being stirred at rt for 2 h, the solution was neutralized
with Dowex 50X8 (Hϩ-form), filtered, and concentrated. The
residue was dissolved in dry pyridine (10 ml) and after addition
of DMAP (30 mg), triethylamine (160 µl) and tert-butyl-
chlorodiphenylsilane (TBDPSCl) (0.8 ml, 3.1 mmol) was stirred
overnight at rt. The solution was poured onto ice–water, ex-
tracted with CH2Cl2, and the organic layer was washed with
saturated aq. NaHCO3, dried (MgSO4), and concentrated.
Column chromatography (toluene–EtOAc, 2:1) of the residue
gave compound 6 (0.89 g, 72%); TLC (toluene–EtOAc, 1:1)
Rf 0.58 (6).
To a solution of compound 6 (0.89 g, 1.5 mmol) in dry
pyridine (5 ml) was added dropwise at 0 ЊC a solution of
MBzCl (0.5 ml, 3.8 mmol) in dry CH2Cl2 (5 ml). The solution
was stirred overnight at rt, diluted with CH2Cl2, poured onto
ice–water, extracted with CH2Cl2, and the organic layer was
washed with saturated aq. NaHCO3, dried (MgSO4), and con-
centrated. Column chromatography (toluene–EtOAc, 50:1)
gave amorphous title compound 7 (1.12 g, 90%); TLC (toluene–
EtOAc, 10:1) Rf 0.71 (7); [α]D ϩ18 (c 1, CHCl3) (Found: C,
69.7; H, 5.6. C48H49NO8SSi requires C, 69.6; H, 5.95%); δH(300
MHz; CDCl3) 7.87–7.04 (22 H, m, Phth, 2 × COC6H4CH3 and
2 × Ph), 6.23 (1 H, dd, J2,3 10.4, J3,4 9.4, 3-H), 5.64 (1 H, t, J4,5
9.6, 4-H), 5.63 (1 H, d, J1,2 10.5, 1-H), 4.62 (1 H, t, 2-H), 3.97
(1 H, dt, J5,6 3.5, 5-H), 3.87–3.86 (2 H, m, 6-H2), 2.80–2.59 (2 H,
m, SCH2CH3), 2.34 and 2.27 (each 3 H, 2 s, 2 × COC6H4CH3),
1.26 (3 H, t, J 7.4, SCH2CH3) and 1.04 [9 H, s, C(CH3)3]; δC(75.5
MHz; CDCl3) 165.7 and 164.9 (2 COC6H4CH3), 143.7 and
135.5–123.5 (Ar-C), 80.6, 79.2, 72.1 and 69.3 (C-1, -3, -4, -5),
62.9 (C-6), 54.0 (C-2), 26.5 [C(CH3)3], 23.7 (SCH2CH3), 21.5
and 21.4 (2 × COC6H4CH3), 19.0 [C(CH3)3] and 14.9
(SCH2CH3); FABMS of C48H49NO8SSi (M, 827.3) m/z 850.5
(M ϩ Na)ϩ.
2-(Trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-4-O-{3-[2-deoxy-3,4-
di-O-(p-methylbenzoyl)-2-phthalimido-â-D-glucopyranosyloxy]-
propyl}-â-D-glucopyranoside 9
To a solution of acetyl chloride (3.3 ml) in dry MeOH (50 ml)
was added at rt a solution of compound 8 (1.07 g, 0.8 mmol) in
dry toluene (50 ml). The solution was stirred overnight at rt,
then neutralized with triethylamine, and concentrated. The
residue was dissolved in EtOAc and the solution was washed
with water, dried (MgSO4), and concentrated. Column chrom-
atography (toluene–EtOAc, 5:1 to 3:1) of the residue gave
amorphous title compound 9 (0.81 g, 89%); TLC (toluene–
EtOAc, 5:1) Rf 0.70 (8), 0.27 (9); [α]D Ϫ7 (c 1, CHCl3) (Found:
C, 68.8; H, 6.5. C65H73NO15Si requires C, 68.7; H, 6.5%); δH(300
MHz; CDCl3) 7.83–7.03 (27 H, m, Phth, 2 × COC6H4CH3 and
3 × Ph), 6.24 (1 H, dd, J2Ј,3Ј 10.8, J3Ј,4Ј 9.2, 3Ј-H), 5.48 (1 H, d,
J1Ј,2Ј 8.4, 1Ј-H), 5.44 (1 H, t, J4Ј,5Ј 9.5, 4Ј-H), 4.92 and 4.68 (2 H,
AB system, JA,B 11.1, PhCH2O), 4.81 and 4.63 (2 H, AB system,
JA,B 11.0, PhCH2O), 4.62 and 4.58 (2 H, system, JA,B 12.2,
PhCH2O), 4.48 (1 H, dd, 2Ј-H), 4.33 (1 H, d, J1,2 7.7, 1-H),
2.34 and 2.27 (each 3 H, 2 s, 2 × COC6H4CH3), 1.67–1.59 (2 H,
m, OCH2CH2CH2O), 1.06–1.00 (2 H, m, CH2Si), 0.03 [9 H,
s, Si(CH3)3]; δC(75.5 MHz; CDCl3) 165.8 and 165.6 (2 ×
COC6H4CH3), 144.2–123.4 (Ar-C), 102.9 (C-1), 98.1 (C-1Ј),
84.3, 82.1, 78.1, 75.1, 74.4, 70.7 and 69.8 (C-2, -3, -4, -5, -3Ј, -4Ј,
-5Ј), 75.3, 74.6, 73.4, 68.9, 68.9, 67.2, 66.6 and 61.2 (C-6, -6Ј,
3 × PhCH2O, OCH2CH2CH2O and OCH2CH2Si), 54.8 (C-2Ј),
30.1 (OCH2CH2CH2O), 21.5 and 21.4 (2 × COC6H4CH3) and
18.4 (CH2Si); FABMS of C65H73NO15Si (M, 1135.5) m/z 1158.6
(M ϩ Na)ϩ.
Ethyl (2,3,4,6-tetra-O-acetyl-â-D-galactopyranosyl)-(1→4)-
2,3,6-tri-O-acetyl-1-thio-â-D-glucopyranoside 11
To a solution of (2,3,4,6-tetra-O-acetyl-β--galactopyrano-
syl)-(1→4)-2,3,6-tri-O-acetyl-α--glucopyranosyl
trichloro-
acetimidate33 10 (195 mg, 0.25 mmol) in dry CH2Cl2 (2 ml) was
added ethanethiol (50 µl, 0.67 mmol) and powdered molecular
sieves 4 Å (600 mg) and the suspension was stirred under argon
for 1 h at rt. Then BF3ؒEt2O (250 µl, 2.0 mmol) was added and
the mixture was stirred for 2.5 h at rt. The suspension was
neutralized with triethylamine, diluted with CH2Cl2, filtered
over Celite, and concentrated. Column chromatography
(toluene–EtOAc, 2:1) of the residue gave amorphous title com-
pound 11 (159 mg, 94%); TLC (toluene–EtOAc, 1:1) Rf 0.45
(10), 0.47 (11); [α]D Ϫ4 (c 1, CHCl3) (Found: C, 49.45; H, 6.0.
C28H40O17S requires C, 49.40; H, 5.9%); δH(300 MHz; CDCl3)
5.35 (1 H, dd, J3Ј,4Ј 3.3, J4Ј,5Ј 0.9, 4Ј-H), 5.21 (1 H, t, J1,2 9.5,
J2,3 9.2, 2-H), 5.11 (1 H, dd, J1Ј,2Ј 8.0, J2Ј,3Ј 10.5, 2Ј-H), 4.95
(1 H, dd, 3Ј-H), 4.94 (1 H, t, J3,4 9.8, 3-H), 3.87 (1 H, dt, J5Ј,6Ј
7.0, 5Ј-H), 3.78 (1 H, t, J4,5 9.7, 4-H), 3.62 (1 H, ddd, J5,6a 2.3,
5-H), 2.70–2.65 (2 H, m, SCH2CH3), 2.15–1.96 (21 H, m,
7 × COCH3) and 1.26 (3 H, t, J 7.4, SCH2CH3); δC(75.5 MHz;
CDCl3) 100.9 (C-1Ј), 83.3 (C-1), 76.6, 76.1, 73.7, 70.9, 70.6,
70.2, 69.0 and 66.5 (C-2, -3, -4, -5, -2Ј, -3Ј, -4Ј, -5Ј), 62.2 and
60.7 (C-6, -6Ј), 24.3 (SCH2CH3), 20.7–20.3 (COCH3) and 14.8
2-(Trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-4-O-{3-[6-O-(tert-
butyldiphenylsilyl)-2-deoxy-3,4-di-O-(p-methylbenzoyl)-2-
phthalimido-â-D-glucopyranosyloxy]propyl}-â-D-glucopyrano-
side 8
Compounds 4 (0.56 g, 0.9 mmol) and 7 (1.04 g, 1.3 mmol)
were dissolved in dry toluene (15 ml) and stirred under argon
with powdered molecular sieves 4 Å (5 g) for 1 h at rt. At
Ϫ40 ЊC were added NIS (340 mg, 1.5 mmol) and silver tri-
fluoromethanesulfonate (30 mg, 0.12 mmol) and the suspension
was stirred at 0 ЊC for 1 h. Pyridine (1 ml) was added, the sus-
pension was diluted with CH2Cl2, filtered over Celite, washed
successively with 10% aq. Na2S2O3 and saturated NaHCO3,
dried (MgSO4), and concentrated. Column chromatography
(toluene–EtOAc, 25:1 to 10:1) of the residue gave amorphous
title compound 8 (1.07 g, 85%); TLC (toluene–EtOAc, 5:1) Rf
0.14 (4), 0.77 (7), 0.66 (8); [α]D ϩ2 (c 1, CHCl3); δH(300 MHz;
CDCl3) 7.76–7.04 (37 H, m, Phth, 2 × COC6H4CH3 and
5 × Ph), 6.16 (1 H, dd, J2Ј,3Ј 10.7, J3Ј,4Ј 9.3, 3Ј-H), 5.58 (1 H, t,
3016
J. Chem. Soc., Perkin Trans. 1, 1998

View Article Online
(SCH2CH3); FABMS of C28H40O17S (M, 680.2) m/z 703.4
(M ϩ Na)ϩ.
atography (toluene–EtOAc, 2:3) gave hemiacetal 15 (299 mg,
96%); TLC (toluene–EtOAc, 1:2) Rf 0.70 (14), 0.44 (15).
To a solution of compound 15 (299 mg, 0.39 mmol) in dry
CH2Cl2 (5 ml) was added trichloroacetonitrile (200 µl, 2.0
mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (10 µl). After
being stirred under argon for 2 h at rt, the solution was concen-
trated. Column chromatography (toluene–EtOAc, 3:2) of the
residue gave the imidate 16 (314 mg, 89%); TLC (toluene–
EtOAc, 1:1) Rf 0.20 (15), 0.47 (16); δH(300 MHz; CDCl3) 8.61
2-(Trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-4-O-[3-(3,4,6-tri-O-
acetyl-2-deoxy-2-phthalimido-â-D-glucopyranosyloxy)propyl]-
â-D-glucopyranoside 13
To a solution of silver trifluoromethanesulfonate (52 mg, 0.20
mmol) and tetramethylurea (45 mg, 0.39 mmol) in dry CH2Cl2
(2 ml) was added compound 4 (62 mg, 0.1 mmol), and the
solution was stirred under argon in the presence of powdered
molecular sieves 4 Å (600 mg) for 1 h at rt. Then a solution of
3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β--glucopyranosyl-
bromide34 12 (74 mg, 0.15 mmol) in CH2Cl2 (1 ml) was added
dropwise at Ϫ30 ЊC, and the mixture was stirred overnight at rt,
diluted with CH2Cl2, filtered over Celite, and concentrated.
Purification twice by column chromatography (toluene–EtOAc,
8:1, then heptane–EtOAc, 2:1) gave title disaccharide 13 (78
mg, 76%); TLC (toluene–EtOAc, 1:1) Rf 0.53 (4), 0.52 (12), 0.59
(13); TLC (hexane–EtOAc, 1:1) Rf 0.51 (4), 0.40 (12), 0.56 (13);
[α]D ϩ7 (c 1, CHCl3); δH(300 MHz; CDCl3) 7.79 and 7.67 (each
2 H, 2 m, Phth), 7.35–7.18 (15 H, m, 3 Ph), 5.77 (1 H, dd, J2Ј,3Ј
10.7, J3Ј,4Ј 9.0, 3Ј-H), 5.32 (1 H, d, J1Ј,2Ј 8.4, 1Ј-H), 5.15 (1 H, dd,
J4Ј,5Ј 10.0, 4Ј-H), 4.91 and 4.67 (2 H, AB system, JA,B 11.0,
PhCH2O), 4.78 and 4.60 (2 H, AB system, JA,B 11.0, PhCH2O),
4.57 and 4.53 (2 H, AB system, JA,B 12.2, PhCH2O), 4.31 (1 H,
d, J1,2 7.7, 1-H), 4.30 (1 H, dd, 2Ј-H), 2.08, 2.03 and 1.86 (each
3 H, 3 s, 3 × COCH3), 1.61–1.57 (2 H, m, OCH2CH2CH2O),
1.06–1.00 (2 H, m, CH2Si) and 0.03 [9 H, s, Si(CH3)3]; δC(75.5
MHz; CDCl3) 170.6, 170.0 and 169.3 (3 × COCH3), 138.5–
123.5 (Ar-C), 102.9 (C-1), 97.9 (C-1Ј), 84.3, 82.1, 78.6, 74.8,
71.7, 70.7 and 68.9 (C-2, -3, -4, -5, -3Ј, -4Ј, -5Ј), 75.2, 74.5, 73.2,
69.0, 68.4, 67.3, 66.9 and 61.9 (C-6, -6Ј, 3 × PhCH2O, OCH2-
CH2CH2O and OCH2CH2Si), 54.5 (C-2Ј), 30.0 (OCH2CH2-
CH2O), 20.6, 20.5 and 20.3 (3 × COCH3) and 18.4 (CH2Si);
FABMS of C55H67NO16Si (M, 1025.4) m/z 1048.6 (M ϩ Na)ϩ.
(1 H, s, C᎐NH), 7.87 and 7.75 (each 2 H, 2 m, Phth), 6.44 (d, J
᎐
1,2
3.2, 1-H), 5.75 (1 H, dd, J2Ј,3Ј 10.7, J3Ј,4Ј 9.1, 3Ј-H), 5.46 (1 H, t,
J2,3 10.2, J3,4 9.8, 3-H), 5.34 (1 H, d, J1Ј,2Ј 8.5, 1Ј-H), 5.16 (1 H,
dd, J4Ј,5Ј 10.1, 4Ј-H), 4.95 (1 H, dd, 2-H), 4.27 (1 H, dd, 2Ј-H),
3.36 (1 H, t, J4,5 9.7, 4-H), 2.11, 2.09, 2.06, 2.04, 2.00 and 1.97
(each 3 H, 6 s, 6 × COCH3).
To a solution of compound 16 (314 mg, 0.34 mmol) in dry
CH2Cl2 (3 ml) were added ethanethiol (70 µl, 0.95 mmol) and
powdered molecular sieves 4 Å (900 mg), and the suspension
was stirred under argon for 1 h at rt. BF3ؒEt2O (340 µl, 2.7
mmol) was added at 0 ЊC and the mixture stirred for 2.5 h at
rt, then neutralized with triethylamine, diluted with CH2Cl2,
filtered over Celite, and concentrated. Column chromatography
(toluene–EtOAc, 3:2) of the residue gave amorphous title
compound 17 (245 mg, 87%); TLC (toluene–EtOAc, 1:1) Rf
0.48 (17); [α]D 0 (c 1, CHCl3); δH(300 MHz; CDCl3) 7.86 and
7.76 (each 2 H, 2 m, Phth), 5.75 (1 H, dd, J3Ј,4Ј 9.1, 3Ј-H), 5.33
(1 H, d, J1Ј,2Ј 8.5, 1Ј-H), 5.16 (1 H, t, J4Ј,5Ј 9.3, 4Ј-H), 5.06 (1 H,
t, J2,3 9.7, J3,4 9.3, 3-H), 4.81 (1 H, t, 2-H), 4.40 (1 H, d, J1,2 10.0,
1-H), 3.20 (1 H, t, J4,5 9.4, 4-H), 2.70–2.63 (2 H, m, SCH2CH3),
2.11, 2.08, 2.03, 2.02, 1.97 and 1.85 (each 3 H, 6 s, 6 × COCH3)
and 1.24 (3 H, t, J 7.4, SCH2CH3); δC(75.5 MHz; CDCl3) 170.5,
170.4, 170.0, 169.8, 169.5 and 169.3 (6 × COCH3), 98.0 (C-1Ј),
83.1 (C-1), 76.3, 76.2, 75.7, 71.8, 70.7, 70.3 and 68.9 (C-2, -3,
-4, -5, -3Ј, -4Ј, -5Ј), 69.3, 66.5, 62.7 and 61.9 (C-6, -6Ј and
OCH2CH2CH2O), 54.5 (C-2Ј), 30.0 (OCH2CH2CH2O), 24.2
(SCH2CH3), 20.6–20.3 (COCH3) and 14.8 (SCH2CH3);
FABMS of C37H47NO18S (M, 825.2) m/z 848.4 (M ϩ Na)ϩ.
2-(Trimethylsilyl)ethyl 2,3,6-tri-O-acetyl-4-O-[3-(3,4,6-tri-O-
acetyl-2-deoxy-2-phthalimido-â-D-glucopyranosyloxy)propyl]-
â-D-glucopyranoside 14
2-(Trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-4-O-{3-(2,3,4,6-tetra-
O-acetyl-â-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-â-D-
glucopyranosyl)-(1→6)-(2-deoxy-3,4-di-O-(p-methylbenzoyl)-2-
phthalimido-â-D-glucopyranosyloxy]propyl}-â-D-glucopyrano-
side 18
A solution of compound 13 (37 mg, 0.036 mmol) in acetic acid
(1 ml) was stirred in the presence of 10% Pd–C (20 mg) under H2
overnight at rt. TLC (EtOAc) Rf 0.69 (debenzylated compound).
The solution was diluted with EtOAc, filtered over Celite,
and co-concentrated with toluene. The residue was dissolved in
dry pyridine (2 ml), acetic anhydride (1 ml) was added, and the
mixture was stirred overnight at rt . The solution was co-
concentrated with toluene, and column chromatography
(toluene–EtOAc, 2:1) of the residue gave amorphous title
compound 14 (26 mg, 84%); TLC (toluene–EtOAc, 1:1) Rf 0.55
(14); [α]D Ϫ1 (c 1, in CHCl3); δH(300 MHz; CDCl3) 7.86 and
7.76 (each 2 H, 2 m, Phth), 5.76 (1 H, dd, J2Ј,3Ј 10.7, J3Ј,4Ј 9.1,
3Ј-H), 5.33 (1 H, d, J1Ј,2Ј 8.5, 1Ј-H), 5.17 (1 H, t, J4Ј,5Ј 9.3, 4Ј-H),
5.03 (1 H, t, J2,3 9.6, J3,4 9.4, 3-H), 4.77 (1 H, dd, 2-H), 4.40 (1 H,
d, J1,2 7.9, 1-H), 4.28 (1 H, dd, 2Ј-H), 3.93 and 3.52 (each 1 H,
m, OCH2CH2Si), 3.22 (1 H, t, J4,5 9.4, 4-H), 2.12, 2.10, 2.03,
2.01, 1.97 and 1.89 (each 3 H, 6 s, 6 × COCH3), 0.96–0.82 (2 H,
m, CH2Si) and 0.03 [9 H, s, Si(CH3)3]; δC(75.5 MHz; CDCl3)
99.8 and 98.0 (C-1, -1Ј), 76.5, 74.7, 72.6, 71.7, 71.6, 70.6 and
68.8 (C-2, -3, -4, -5, -3Ј, -4Ј, -5Ј), 69.3, 67.2, 66.4, 62.5 and 61.8
(C-6, -6Ј, OCH2CH2CH2O and OCH2CH2Si), 54.4 (C-2Ј), 30.0
(OCH2CH2CH2O), 20.6–20.3 (COCH3) and 17.7 (CH2Si);
FABMS of C40H55NO19Si (M, 881.3) m/z 904.5 (M ϩ Na)ϩ.
A mixture of compounds 9 (234 mg, 0.21 mmol) and 11 (168
mg, 0.25 mmol) in dry toluene (5 ml) containing powdered
molecular sieves 4 Å (1.5 g) was stirred under argon for 1 h at rt.
Then, NIS (67 mg, 0.30 mmol) and silver trifluoromethane-
sulfonate (7 mg, 0.027 mmol) were added, and the suspension
was stirred for 3 h at rt. Pyridine (1 ml) was added, and the
suspension was diluted with CH2Cl2, and filtered over Celite.
The solution was washed successively with 10% aq. Na2S2O3
and saturated aq. NaHCO3, dried (MgSO4), and concentrated.
Purification twice by column chromatography (toluene–EtOAc,
2:1, then heptane–EtOAc, 1:1) gave title compound 18 (154
mg, 42%); TLC (toluene–EtOAc, 2:1) Rf 0.73 (9), 0.26 (11),
0.39 (18); [α]D Ϫ3 (c 1, CHCl3); δH(300 MHz; CDCl3) 7.80–7.03
(27 H, m, Phth, 2 × COC6H4CH3 and 3 × Ph), 6.15 (1 H, dd,
J2Ј,3Ј 10.7, J3Ј,4Ј 9.2, 3Ј-H), 5.43 (1 H, d, J1Ј,2Ј 8.4, 1Ј-H), 5.34 (1 H,
t, J4Ј,5Ј 9.4, 4Ј-H), 5.33 (1 H, d, J3ٞ,4ٞ 3.6, 4ٞ-H), 5.15 (1 H, t, J1Љ,2Љ
9.0, J2Љ,3Љ 9.0, 2Љ-H), 5.09 (1 H, dd, J1ٞ,2ٞ 7.8, J2ٞ,3ٞ 10.3, 2ٞ-H),
4.53 and 4.44 (each 1 H, 2 d, J 7.7 and 7.8, 1Љ- and 1ٞ-H), 4.44
(1 H, dd, 2Ј-H), 4.31 (1 H, d, J1,2 7.6, 1-H), 2.35 and 2.28 (each
3 H, 2 s, 2 × COC6H4CH3), 2.14–1.96 (21 H, m, 7 × COCH3),
1.68–1.60 (2 H, m, OCH2CH2CH2O), 1.05–1.00 (2 H, m,
CH2Si) and 0.03 [9 H, s, Si(CH3)3]; δC(75.5 MHz; CDCl3) 165.5
and 165.1 (2 × COC6H4CH3), 144.2–125.8 (Ar-C), 102.8, 100.9,
100.4 and 97.8 (C-1, -1Ј, -1Љ, -1ٞ), 84.2, 81.9, 78.2, 76.2, 74.7,
73.5, 72.6, 72.3, 71.3, 70.8, 70.5, 69.8, 68.9 and 66.4 (C-2, -3,
-4, -5, -3Ј, -4Ј, -5Ј, -2Љ, -3Љ, -4Љ, -5Љ, -2ٞ, -3ٞ, -4ٞ, -5ٞ), 75.2, 74.4,
73.2, 69.0, 67.2 and 60.8 (C-6, -6Ј, -6Љ, -6ٞ, OCH2CH2CH2O
Ethyl 2,3,6-tri-O-acetyl-4-O-[3-(3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-â-D-glucopyranosyloxy)propyl]-1-thio-â-D-gluco-
pyranoside 17
Compound 14 (359 mg, 0.41 mmol), as in a mixture of dry
CH2Cl2 (5 ml) and TFA (10 ml), was stirred for 40 min under
argon at rt. Propyl acetate (30 ml) and toluene (60 ml) were
added, and the solution was concentrated. Column chrom-
J. Chem. Soc., Perkin Trans. 1, 1998
3017

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and OCH2CH2Si), 54.7 (C-2Ј), 30.0 (OCH2CH2CH2O), 21.5
and 21.4 (2 × COC6H4CH3), 20.6–20.3 (COCH3) and 18.3
(CH2Si); FABMS of C91H107NO32Si (M, 1753.7) m/z 1776.8
(M ϩ Na)ϩ.
2-(Trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-4-O-(3-{2,3,6-tri-O-
acetyl-4-O-[3-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-â-D-
glucopyranosyloxy)propyl]-â-D-glucopyranosyl}-(1→6)-(2-
acetamido-3,4-di-O-acetyl-2-deoxy-â-D-glucopyranosyloxy)-
propyl)-â-D-glucopyranoside 21
2-(Trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-4-O-(3-{2,3,6-tri-O-
acetyl-4-O-[3-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-â-D-
glucopyranosyloxy)propyl]-â-D-glucopyranosyl}-(1→6)-[2-
deoxy-3,4-di-O-(p-methylbenzoyl)-2-phthalimido-â-D-gluco-
pyranosyloxy]propyl)-â-D-glucopyranoside 19
Treatment of compound 19 (138 mg, 0.072 mmol) according to
the procedure described for the preparation of analogue 20 fol-
lowed by column chromatography (CH2Cl2–acetone, 2:1 to
1:1) gave title product 21 (110 mg, 97%); TLC (CH2Cl2–
acetone, 1:1) Rf 0.73 (21); [α]D Ϫ11 (c 1, CHCl3); δH(300 MHz;
CDCl3) 5.82 (1 H, d, NHCOCH3), 5.43 (1 H, d, J1Ј,2Ј 8.4, 1Ј-H)
and 4.31 (1 H, d, J1,2 7.6, 1-H); δC(75.5 MHz; CDCl3) 138.8–
138.2 and 128.4–127.6 (Ar-C), 103.0, 100.4, 99.8 and 99.5
(C-1, -1Ј, -1Љ, -1ٞ), 84.1, 82.1, 78.1, 76.1, 75.0, 74.5, 72.8, 71.9,
71.6, 71.3, 69.3 and 68.6 (C-2, -3, -4, -5, -3Ј, -4Ј, -5Ј, -2Љ, -3Љ,
-4Љ, -5Љ, -3ٞ, -4ٞ, -5ٞ), 75.4, 74.6, 73.4, 69.4, 68.9, 68.0, 67.3,
65.6, 65.4, 62.8 and 62.0 (C-6, -6Ј, -6Љ, -6ٞ, 3 × PhCH2O,
2 × OCH2CH2CH2O and OCH2CH2Si), 55.1 and 54.9 (C-2Ј,
2ٞ), 29.8 and 29.7 (2 × OCH2CH2CH2O), 23.2 and 23.0
(2 × NHCOCH3), 20.8–20.5 (COCH3) and 18.4 (CH2Si);
FABMS of C76H106N2O31Si (M, 1570.7) m/z 1593.8 (M ϩ Na)ϩ.
A mixture of compounds 9 (275 mg, 0.24 mmol) and 17 (200
mg, 0.24 mmol) in dry toluene (5 ml) containing powdered
molecular sieves 4 Å (1.5 g) was stirred under argon for 2 h at rt.
Then, NIS (72 mg, 0.32 mmol) and silver trifluoromethanesul-
fonate (6 mg, 0.023 mmol) were added, and the mixture was
stirred for 1 h at rt. Additional portions of NIS (55 mg, 0.24
mmol) and silver trifluoromethanesulfonate (10 mg, 0.039
mmol) were added, and the mixture was stirred for 2 h. Work-
up as described for compound 18 and column chromatography
(toluene–EtOAc, 2:1) of the residue gave amorphous title
compound 19 (301 mg, 66%); TLC (toluene–EtOAc, 3:2) Rf
0.72 (9), 0.32 (17), 0.52 (19); [α]D Ϫ4 (c 1, CHCl3); δH(300 MHz;
CDCl3) 7.80–7.03 (31 H, m, 2 × Phth, 2 × COC6H4CH3 and
3 × Ph), 6.15 (1 H, dd, J2Ј,3Ј 10.7, J3Ј,4Ј 9.2, 3Ј-H), 5.75 (1 H, dd,
J2ٞ,3ٞ 10.6, J3ٞ,4ٞ 9.1, 3ٞ-H), 5.43 (1 H, d, J1Ј,2Ј 8.4, 1Ј-H), 5.34
(1 H, t, J4Ј,5Ј 9.6, 4Ј-H), 5.32 (1 H, d, J1ٞ,2ٞ 8.6, 1ٞ-H), 5.16 (1 H,
t, J4ٞ,5ٞ 9.5, 4ٞ-H), 4.46 (1 H, d, J1Љ,2Љ 8.1, 1Љ-H), 4.31 (1 H, d, J1,2
7.7, 1-H), 2.35 and 2.28 (each 3 H, 2 s, 2 × COC6H4CH3), 1.71-
1.59 (4 H, m, 2 × OCH2CH2CH2O), 2.14–1.96 (18 H, m,
6 × COCH3), 1.05–1.00 (2 H, m, CH2Si) and 0.03 [9 H, s,
Si(CH3)3]; δC(75.5 MHz; CDCl3) 170.5, 170.3, 170.0, 169.7,
169.4 and 169.3 (6 × COCH3), 165.5 and 165.1 (2 × COC6-
H4CH3), 144.1–123.5 (Ar-C), 102.8, 100.6, 98.0 and 97.8 (C-1,
-1Ј, -1Љ, -1ٞ), 84.2, 82.0, 78.2, 76.3, 74.8, 74.5, 73.6, 72.7, 71.8,
71.5, 70.8, 70.7, 69.9 and 68.9 (C-2, -3, -4, -5, -3Ј, -4Ј, -5Ј, -2Љ,
-3Љ, -4Љ, -5Љ, -3ٞ, -4ٞ, -5ٞ), 75.2, 74.5, 73.2, 69.4, 69.0, 68.4, 67.2,
66.6, 66.5, 62.5, 61.9 and 60.8 (C-6, -6Ј, -6Љ, -6ٞ, 3 × PhCH2O,
2 × OCH2CH2CH2O and OCH2CH2Si), 54.7 and 54.4 (C-2Ј,
-2ٞ), 21.5 and 21.4 (2 × COC6H4CH3), 20.6–20.3 (COCH3) and
18.4 (CH2Si); FABMS of C100H114N2O33Si (M, 1898.7) m/z
1921.9 (M ϩ Na)ϩ.
2-(Trimethylsilyl)ethyl 2,3,6-tri-O-acetyl-4-O-[3-(2,3,4,6-tetra-
O-acetyl-â-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-â-D-
glucopyranosyl)-(1→6)-(2-acetamido-3,4-di-O-acetyl-2-deoxy-
â-D-glucopyranosyloxy)propyl]-â-D-glucopyranoside 22
A solution of compound 20 (87 mg, 0.057 mmol) in HOAc (2
ml) was stirred in the presence of 10% Pd–C (40 mg) under H2
overnight at rt. The suspension was diluted with MeOH and
filtered over Celite. After co-concentration with toluene, a solu-
tion of the residue in dry pyridine (5 ml) containing acetic
anhydride (2.5 ml) was stirred overnight at rt. Co-concentration
with toluene and column chromatography (CH2Cl2–acetone,
2:1) of the residue gave amorphous title compound 22 (56 mg,
72%); TLC (CH2Cl2–acetone, 3:1) Rf 0.38 (22); [α]D Ϫ18 (c 1,
CHCl3); δC(75.5 MHz; CDCl3) 101.0, 100.4, 99.9 and 99.6 (C-
1, -1Ј, -1Љ, -1ٞ), 76.3, 76.1, 74.8, 73.0, 72.7, 72.6, 72.0, 71.8, 71.4,
70.9, 70.6, 69.4, 69.0 and 66.5 (C-2, -3, -4, -5, -3Ј, -4Ј, -5Ј, -2Љ,
-3Љ, -4Љ, 5 -Љ, -2ٞ, -3ٞ, -4ٞ, -5ٞ), 68.4, 68.2, 67.3, 65.3, 63.1, 61.8
and 60.6 (C-6, -6Ј, -6Љ, -6ٞ, OCH2CH2CH2O and OCH2CH2Si),
55.3 (C-2Ј), 29.6 (OCH2CH2CH2O), 23.2 (NHCOCH3), 20.8–
20.3 (COCH3) and 17.8 (CH2Si); FABMS of C58H87NO34Si (M,
1369.5) m/z 1392.7 (M ϩ Na)ϩ.
2-(Trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-4-O-[3-(2,3,4,6-tetra-
O-acetyl-â-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-â-D-
glucopyranosyl)-(1→6)-(2-acetamido-3,4-di-O-acetyl-2-deoxy-
â-D-glucopyranosyloxy)propyl]-â-D-glucopyranoside 20
2-(Trimethylsilyl)ethyl 2,3,6-tri-O-acetyl-4-O-[3-{2,3,6-tri-O-
acetyl-4-O-[3-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-â-D-
glucopyranosyloxy)propyl]-â-D-glucopyranosyl}-(1→6)-(2-
acetamido-3,4-di-O-acetyl-2-deoxy-â-D-glucopyranosyloxy)-
propyl]-â-D-glucopyranoside 23
To a solution of compound 18 (107 mg, 0.061 mmol) in MeOH
(15 ml) was added a 0.2 M solution of NaOMe in MeOH (2 ml),
and the mixture was stirred for 4 h at rt. The solution was
neutralized with Dowex 50X8 (Hϩ-form), filtered, and concen-
trated. The residue was dissolved in butan-1-ol (15 ml)–
ethylenediamine (3 ml), and the mixture was stirred for 24 h at
80 ЊC, then co-concentrated with toluene. A solution of the
residue in dry pyridine (30 ml) containing acetic anhydride (15
ml) was stirred overnight at rt, then co-concentrated with tolu-
ene. Column chromatography (CH2Cl2–acetone, 3:1) of the
residue gave title product 20 (87 mg, 95%); TLC (CH2Cl2–
acetone, 3:1) Rf 0.63 (20); [α]D Ϫ7 (c 1, CHCl3); δH(300 MHz;
CDCl3) 7.35–7.26 (15 H, m, 3 × Ph), 5.34 (1 H, d, J3ٞ,4ٞ 2.9,
4ٞ-H), 4.63 and 4.38 (each 2 H, 2 d, J 7.7 and 8.1, 1-, 1Ј-, 1Љ- and
1ٞ-H), 2.14–1.96 (30 H, m, 10 × COCH3) and 1.05–1.00 (2 H,
m, CH2Si); δC(75.5 MHz; CDCl3) 138.8–138.3 and 128.4–127.5
(Ar-C), 103.0, 101.0, 100.3 and 99.6 (C-1, -1Ј, -1Љ,1ٞ), 78.2,
76.1, 75.1, 72.8, 72.7, 72.6, 72.0, 71.3, 70.8, 70.6, 69.3, 69.0 and
66.5 (C-2, -3, -4, -5, -3Ј, -4Ј, -5Ј, -2Љ, -3Љ, -4Љ, -5Љ, -2ٞ, -3ٞ, -4ٞ,
-5ٞ), 75.3, 74.6, 73.4, 68.8, 68.2, 67.3, 65.7, 61.8 and 60.6
(C-6, -6Ј, -6Љ, -6ٞ, OCH2CH2CH2O and OCH2CH2Si), 54.9
(C-2Ј), 29.9 (OCH2CH2CH2O), 23.0 (NHCOCH3), 20.7–20.3
(COCH3) and 18.4 (CH2Si); FABMS of C73H99NO31Si (M,
1513.6) m/z 1536.8 (M ϩ Na)ϩ.
Treatment of compound 21 (91 mg, 0.058 mmol) according to
the procedure for the preparation of analogue 22 followed by
column chromatography (CH2Cl2–acetone, 1:1) gave amorph-
ous title product 23 (70 mg, 84%); TLC (CH2Cl2–acetone, 1:1)
Rf 0.46 (23); [α]D Ϫ24 (c 1, CHCl3); δC(75.5 MHz; CDCl3) 100.5,
100.0, 99.9 and 99.6 (C-1, -1Ј, -1Љ, -1ٞ), 76.4, 76.2, 74.8, 74.5,
73.0, 72.9, 72.6, 72.0, 71.8, 71.6, 71.5, 69.4 and 68.7 (C-2, -3,
-4, -5, -3Ј, -4Ј, -5Ј, -2Љ, -3Љ, -4Љ, -5Љ, -3ٞ, -4ٞ, -5ٞ), 68.5, 68.4,
67.3, 65.5, 65.2, 63.1, 62.7 and 62.0 (C-6, -6Ј, -6Љ, -6ٞ, 2 ×
OCH2CH2CH2O and OCH2CH2Si), 55.1 and 55.0 (C-2Ј, -2ٞ),
29.8 (2 × OCH2CH2CH2O), 23.1 (2 × NHCOCH3), 20.7–20.5
(COCH3) and 17.8 (CH2Si); FABMS of C61H94N2O34Si (M,
1426.5) m/z 1449.7 (M ϩ Na)ϩ.
Allyl 2,3,6-tri-O-acetyl-4-O-[3-(2,3,4,6-tetra-O-acetyl-â-D-
galactopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-â-D-gluco-
pyranosyl)-(1→6)-(2-acetamido-3,4-di-O-acetyl-2-deoxy-â-D-
glucopyranosyloxy)propyl]-â-D-glucopyranoside 26
To a solution of compound 22 (80 mg, 0.058 mmol) in dry
CH2Cl2 (1 ml) was added TFA acid (2 ml), and the mixture was
3018
J. Chem. Soc., Perkin Trans. 1, 1998

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stirred under argon for 1 h at rt. Propyl acetate (6 ml) and
toluene (12 ml) were added and the solution was co-
concentrated with toluene. Column chromatography (CH2Cl2–
acetone, 2:1 to 1:3) of the residue gave compound 24 (67 mg,
90%); TLC (CH2Cl2–acetone, 1:1) Rf 0.53 (22), 0.26 (24).
To a solution of compound 24 in dry CH2Cl2 (1 ml) was
added trichloroacetonitrile (100 µl), and at 0 ЊC DBU (10 µl),
and the mixture was stirred for 3 h at rt. Column chrom-
atography (CH2Cl2–acetone, 2:1) gave compound 25 (64 mg,
85%); TLC (CH2Cl2–acetone, 1:1) Rf 0.64 (25); δH(300 MHz;
Allyl 4-O-{3-[â-D-galactopyranosyl)-(1→4)-(â-D-glucopyran-
osyl)-(1→6)-(2-acetamido-2-deoxy-â-D-glucopyranosyloxy]-
propyl}-â-D-glucopyranoside 30
A solution of compound 26 (20 mg, 0.015 mmol) in 0.1 M meth-
anolic NaOMe (5 ml) was stirred overnight at rt. Water (1 ml)
was added, and the solution was stirred for 24 h at rt, neutral-
ized with Dowex 50X8 (Hϩ-form), filtered, and concentrated.
Purification of the residue on Toyopearl HW-40S with water as
eluent gave title compound 30 (10.4 mg, 86%); TLC (butan-1-
ol–water–HOAc, 2:1:1) Rf 0.26 (30); [α]D Ϫ13 (c 0.6, water);
δC(75.5 MHz; CDCl3) 175.3 (NHCOCH3), 134.2 (OCH2-
CDCl ) 8.66 (1 H, s, C᎐NH), 6.49 (1 H, d, J1,2 3.6, 1-H) and 6.00
᎐
3
(1 H, d, NHCOCH3).
CH᎐CH ), 119.6 (OCH CH᎐CH ), 103.8, 103.5, 102.0 and
᎐ ᎐
2 2 2
To a solution of compound 25 in dry CH2Cl2 (0.6 ml) con-
taining molecular sieves 4 Å (150 mg) was added allyl alcohol
(15 µl), and the mixture was stirred for 1 h at rt. Then, at
Ϫ40 ЊC, was added BF3ؒEt2O (5 µl), and the mixture was stirred
for 1 h at Ϫ40 ЊC, followed by 1 h at Ϫ20 ЊC. The solution was
neutralized with triethylamine, filtered over Celite, and concen-
trated. Column chromatography (CH2Cl2–acetone, 2:1 to 1:2)
of the residue gave title compound 26 (36 mg, 61%); TLC
(CH2Cl2–acetone, 1:1) Rf 0.64 (25), 0.71 (26); [α]D Ϫ18 (c 1,
101.9 (C-1, -1Ј, -1Љ, -1ٞ), 79.3, 79.0, 76.4, 76.2, 75.9, 75.7, 75.6,
75.1, 74.6, 74.0, 73.6, 73.4, 71.8, 70.5 and 69.4 (C-2, -4, -5, -3Ј,
-4Ј, -5Ј, -2Љ, -3Љ, -4Љ, -5Љ, -2ٞ, -3ٞ, -4ٞ, -5ٞ), 71.5, 70.4, 69.4, 67.8,
61.8, 61.4 and 60.9 (C-6, -6Ј, -6Љ, -6ٞ, OCH2CH2CH2O and
OCH CH᎐CH ), 56.4 (C-2Ј), 30.3 (OCH CH CH O) and 23.0
᎐
2
2
2
2
2
(NHCOCH3); FABMS of C32H55NO22 (M, 805.3) m/z 806.3
(M ϩ H)ϩ; m/z 828.3 (M ϩ Na)ϩ.
Allyl 4-O-[3-{4-O-[3-(2-acetamido-2-deoxy-â-D-glucopyranosyl-
oxy)propyl]-â-D-glucopyranosyl}-(1→6)-(2-acetamido-2-deoxy-
â-D-glucopyranosyloxy)propyl]-â-D-glucopyranoside 31
CHCl ); δ (75.5 MHz; CDCl ) 133.3 (OCH CH᎐CH ), 117.4
᎐
3
C
3
2
2
(OCH CH᎐CH ), 101.0, 100.4, 99.6 and 99.3 (C-1, -1Ј, -1Љ, -1ٞ),
᎐
2
2
76.3, 76.1, 74.6, 73.0, 72.7, 72.6, 71.9, 71.7, 71.3, 70.8, 70.5,
69.3, 69.0 and 66.5 (C-2, -3, -4, -5, -3Ј, -4Ј, -5Ј, -2Љ, -3Љ, -4Љ, -5Љ,
-2ٞ, -3ٞ, -4ٞ, -5ٞ), 69.9, 68.4, 68.3, 65.3, 63.0, 61.8 and 60.6
Treatment of compound 29 (38 mg, 0.028 mmol) according to
the procedure described for the preparation of analogue 30 gave
title compound 31 (23.3 mg, 92%); TLC (butan-1-ol–water–
HOAc, 2:1:1) Rf 0.31 (31); [α]D Ϫ18 (c 0.6, water); δC(75.5
(C-6, -6Ј, -6Љ, -6ٞ, OCH CH CH O and OCH CH᎐CH ), 55.2
᎐
2
2
2
2
2
(C-2Ј), 29.8 (OCH2CH2CH2O), 23.2 (NHCOCH3) and 20.7–
20.3 (COCH3); FABMS of C56H79NO34 (M, 1309.4) m/z 1310.4
(M ϩ H)ϩ; m/z 1332.4 (M ϩ Na)ϩ.
MHz; CDCl ) 175.3 (NHCOCH ), 134.0 (OCH CH᎐CH ),
᎐
3 3 2 2
119.4 (OCH CH᎐CH ), 103.4, 101.8, 101.7 and 101.6 (C-1,
᎐
2
2
-1Ј, -1Љ, -1ٞ), 78.8, 76.5, 76.3, 76.1, 75.8, 75.5, 74.5, 74.4, 73.9,
73.8, 70.6 and 70.3 (C-2, -3, -4, -5, -3Ј, -4Ј, -5Ј, -2Љ, -3Љ, -4Љ, -5Љ,
-3ٞ, -4ٞ, -5ٞ), 71.2, 70.2, 69.2, 67.6, 67.4, 61.4 and 61.2 (C-6,
Allyl 2,3,6-tri-O-acetyl-4-O-[3-{2,3,6-tri-O-acetyl-4-O-[3-(2-
acetamido-3,4,6-tri-O-acetyl-2-deoxy-â-D-glucopyranosyloxy)-
propyl]-â-D-glucopyranosyl}-(1→6)-(2-acetamido-3,4-di-O-
acetyl-2-deoxy-â-D-glucopyranosyloxy)propyl]-â-D-gluco-
pyranoside 29
-6Ј, -6Љ, -6ٞ, 2 × OCH CH CH O, OCH CH᎐CH ), 56.2 (C-2Ј,
᎐
2
2
2
2
2
-2ٞ), 30.0 (2 × OCH2CH2CH2O) and 22.9 (2 × NHCOCH3);
FABMS of C37H64N2O23 (M, 904.4) m/z 905.4 (M ϩ H)ϩ.
To a solution of compound 23 (99 mg, 0.069 mmol) in dry
CH2Cl2 (1 ml) was added TFA (2 ml), and the mixture was
stirred under argon for 1 h at rt. Propyl acetate (6 ml) and
toluene (12 ml) were added and the solution was co-
concentrated with toluene. Column chromatography (CH2Cl2–
acetone, 1:1 to 1:2) of the residue gave compound 27 (76 mg,
84%); TLC (CH2Cl2–acetone, 1:1) Rf 0.46 (23), 0.26 (27).
To a solution of compound 27 in dry CH2Cl2 (1 ml) was
added trichloroacetonitrile (100 µl), and at 0 ЊC DBU (10 µl),
and the mixture was stirred for 2 h at rt, then concentrated.
Column chromatography (CH2Cl2–acetone, 1:1) of the residue
gave compound 28 (69 mg, 82%); TLC (CH2Cl2–acetone, 1:1)
Allyl 4-O-{3-(â-D-galactopyranosyl)-(1→4)-(â-D-glucopyran-
osyl)-(1→6)-[â-D-galactopyranosyl-(1→4)]-(2-acetamido-2-
deoxy-â-D-glucopyranosyloxy)propyl}-â-D-glucopyranoside 32
To a solution of compound 30 (9.6 mg, 11.9 µmol) in sodium
cacodylate buffer (50 m, pH 7.5; 5 m MnCl2) containing
bovine serum albumin (0.7 mg) and NaN3 (0.02%) were added
alkaline phosphatase (6 U), UDP-galactose (9 mg, 14.7 µmol)
and β-1,4-galactosyltransferase (2 U). The reaction mixture
(600 µl) was incubated for 3 h at 37 ЊC. Then water (100 ml) was
added, and UDP-galactose was removed using a Dowex 1X8
(ClϪ-form) column with water as eluent. The eluate was concen-
trated, applied on a Toyopearl HW-40S column, and eluted
with 5 m aq. NH4HCO3 at a flow rate of 13 ml hϪ1. The
appropriate fractions were freeze-dried to give title compound
32 (9.8 mg, 85%); TLC (butan-1-ol–water–HOAc, 2:1:1)
R 0.54 (28); δ (300 MHz; CDCl ) 8.66 (1 H, s, C᎐NH), 6.49
᎐
f
H
3
(1 H, d, J1,2 3.6, 1-H), 5.94 and 5.89 (each 1 H, 2 d,
NHCONCH3), 4.75 and 4.72 (each 1 H, 2 d, J 8.6 and 8.8,
1Ј- and 1ٞ-H) and 4.51 (1 H, d, J1Љ,2Љ 7.9, 1Љ-H).
1
Rf 0.26 (30), 0.15 (32); [α]D Ϫ15 (c 0.4, water); H NMR data
To a solution of compound 28 in dry CH2Cl2 (0.6 ml) con-
taining molecular sieves 4 Å (150 mg) was added allyl alcohol
(15 µl), and the mixture was stirred under argon for 1 h at rt.
Then, at Ϫ40 ЊC, was added BF3ؒEt2O (5 µl), and the mixture
was stirred for 1 h at Ϫ40 ЊC, followed by 1 h at Ϫ20 ЊC. The
solution was neutralized with triethylamine, filtered over Celite,
and concentrated. Column chromatography (CH2Cl2–acetone,
2:1 to 1:1) of the residue afforded title compound 29 (38 mg,
59%); TLC (1:1 CH2Cl2–acetone) Rf 0.52 (29); [α]D Ϫ20 (c 1,
are given in Table 1; δC(75.5 MHz; CDCl3) 175.3 (NHCOCH3),
134.2 (OCH CH᎐CH ), 119.6 (OCH CH᎐CH ), 103.8, 103.6,
᎐
᎐
2
2
2
2
103.3, 102.0 and 101.9 (C-1, -1Ј, -1Љ, -1ٞ, -1٣), 79.4, 79.1, 78.7,
76.4, 76.2, 76.1, 75.9, 75.6, 75.1, 74.3, 74.0, 73.5, 73.4, 73.2, 71.8
and 69.4 (C-2, -3, -4, -5, -3Ј, -4Ј -,5Ј, -2Љ, -3Љ, -4Љ, -5Љ, -2ٞ, -3ٞ,
-4ٞ, -5ٞ, -2٣, -3٣, -4٣, -5٣), 71.5, 70.4, 68.2, 67.9, 61.9, 61.4
and 60.9 (C-6, -6Ј, -6Љ, -6ٞ, -6٣, OCH2CH2CH2O, OCH2-
CH᎐CH ), 55.9 (C-2Ј), 30.3 (OCH CH CH O) and 23.0
᎐
2
2
2
2
(NHCOCH3); FABMS of C38H65NO27 (M, 967.4) m/z 968.3
(M ϩ H)ϩ; m/z 990.3 (M ϩ Na)ϩ.
CHCl ); δ (75.5 MHz; CDCl ) 133.3 (OCH CH᎐CH ), 117.4
᎐
3
C
3
2
2
(OCH CH᎐CH ), 100.5, 99.9, 99.6 and 99.3 (C-1, -1Ј, -1Љ, -1ٞ),
᎐
2
2
76.3, 76.2, 74.6, 74.5, 73.0, 72.9, 72.7, 72.0, 71.6, 71.4, 69.3 and
68.6 (C-2, -3, -4, -5, -3Ј, -4Ј, -5Ј, -2Љ, -3Љ, -4Љ, -5Љ, -3ٞ, -4ٞ, -5ٞ),
69.9, 68.6, 68.4, 65.5, 65.2, 63.0, 62.8 and 62.0 (C-6, -6Ј, -6Љ, -6ٞ,
Allyl 4-O-[3-{4-O-[3-(â-D-galactopyranosyl)-(1→4)-(2-acet-
amido-2-deoxy-â-D-glucopyranosyloxy)propyl]-â-D-glucopyran-
osyl}-(1→6)-[â-D-galactopyranosyl-(1→4)]-(2-acetamido-2-
deoxy-â-D-glucopyranosyloxy)propyl]-â-D-glucopyranoside 33
To a solution of compound 31 (9.4 mg, 10.4 µmol) in sodium
cacodylate buffer (50 m, pH 7.5; 5 m MnCl2) containing
bovine serum albumin (0.7 mg) and NaN3 (0.02%) were added
2 × OCH CH CH O, OCH CH᎐CH ), 55.2 and 55.1 (C-2Ј,
᎐
2
2
2
2
2
-2ٞ), 29.8 (2 × OCH2CH2CH2O), 23.2 (2 × NHCOCH3) and
20.8–20.5 (COCH3); FABMS of C59H86N2O34 (M, 1366.5) m/z
1367.4 (M ϩ H)ϩ; m/z 1389.4 (M ϩ Na)ϩ.
J. Chem. Soc., Perkin Trans. 1, 1998
3019

View Article Online
alkaline phosphatase (7 U), UDP-galactose (20.6 mg, 34.0
µmol), and galactosyltransferase (2 U). The reaction mixture
(800 µl) was incubated for 7 h at 37 ЊC. Work-up as described
for compound 32 and purification twice on Toyopearl HW-
40S gave title compound 33 (10.2 mg, 80%); TLC (butan-1-ol–
water–HOAc, 2:1:1) Rf 0.31 (31), 0.17 (33); [α]D Ϫ10 (c 0.4,
water); 1H NMR data are given in Table 2; δC(75.5 MHz;
for recording the NMR spectra of compounds 32 and 33 and
Mrs. A. C. H. T. M. van der Kerk-van Hoof for recording the
FAB mass spectra.
References
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CDCl ) 175.3 (NHCOCH ), 134.2 (OCH CH᎐CH ), 119.6
᎐
3
3
2
2
(OCH CH᎐CH ), 103.7, 103.6, 103.4, 102.0, 101.9 and 101.8
᎐
2
2
(C-1, -1Ј, -1Љ, -1ٞ, -1٣, -1Љٞ;), 79.4 79.0, 78.7, 76.4, 76.3, 76.2,
76.1, 76.0, 75.9, 75.6, 74.3, 74.0, 73.9, 73.4, 73.3, 73.2, 71.8 and
69.4 (C-2, -3, -4, -5, -3Ј, -4Ј, -5Ј, -2Љ, -3Љ, -4Љ, -5Љ, -3ٞ, -4ٞ, -5ٞ,
-2٣, -3٣, -4٣,-5٣, -2Љٞ;, -3Љٞ, -4Љٞ, -5Љٞ), 71.4, 70.4, 68.2, 67.8,
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2
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2
2
2
6 J. B. Robbins, R. Austrian, C.-J. Lee, S. C. Rastogi, G. Schiffman,
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CH2CH2O), 23.1 (2 × NHCOCH3); FABMS of C49H84N2O33
(M, 1228.5) m/z 1229.4 (M ϩ H)ϩ; m/z 1251.3 (M ϩ Na)ϩ.
3-(2-Aminoethylthio)propyl 4-O-{3-(â-D-galactopyranosyl)-
(1→4)-(â-D-glucopyranosyl)-(1→6)-[â-D-galactopyranosyl-
(1→4)]-(2-acetamido-2-deoxy-â-D-glucopyranosyloxy)propyl}-
â-D-glucopyranoside 34
The allyl glycoside 32 (11.1 mg, 11.5 µmol) was dissolved in aq.
cysteamine hydrochloride (6.6 mg, 57.5 µmol in 300 µl), and the
mixture was irradiated under UV-light for 3 h at rt. The product
was purified by size-exclusion chromatography on a Toyopearl
HW-40S column, eluted with 0.1  aq. NH4OAc at a flow rate
of 13 ml hϪ1. Product-containing fractions were lyophilized and
deionized on a Dowex 1X8 (OHϪ-form) column with water as
eluent to give title glycoside 34 (9.4 mg, 78%); TLC (butan-1-
ol–water–HOAc, 2:1:1) Rf 0.17 (32), 0.08 (34); δH(300 MHz;
CDCl3) 4.57–4.52 (3 H, 1Ј-, 1Љ- and 1٣-H), 4.45 and 4.43 (each
1 H, 2 d, J 7.4 and 7.7, 1- and 1ٞ-H), 4.29 (1 H, d, J 10.5, 6Ј-H),
3.09, 2.80 and 2.69 (each 2 H, 3 t, OCH2CH2CH2SCH2-
CH2NH2) and 2.05 (3 H, s, NHCOCH3); δC(75.5 MHz; CDCl3)
176.2 (NHCOCH3), 104.6, 104.4, 104.1, 103.9 and 102.8 (C-1,
-1Ј, -1Љ, -1ٞ, -1٣), 80.1, 79.9, 79.4, 77.2, 77.0, 76.9, 76.8, 76.4,
76.0, 75.1, 74.9, 74.3, 74.2, 74.0, 72.6 and 70.2 (C-2, -3, -4,
-5, -3Ј, -4Ј, -5Ј, -2Љ, -3Љ, -4Љ, -5Љ, -2ٞ, -3ٞ, -4ٞ, -5ٞ, -2٣, -3٣, -4٣,
-5٣), 71.3, 70.4, 69.0, 68.6, 62.7, 62.7, 62.2 and 61.8 (C-6, -6Ј,
-6Љ, -6ٞ, -6٣ OCH2CH2CH2O and OCH2CH2CH2S), 56.8
(C-2Ј), 40.4, 31.7, 30.5, 30.4 and 28.8 (OCH2CH2CH2O and
OCH2CH2CH2SCH2CH2NH2) and 23.2 (NHCOCH3);
FABMS of C40H72N2O27S (M, 1044.4) m/z 1045.3 (M ϩ H)ϩ.
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3-(2-Aminoethylthio)propyl 4-O-[3-{4-O-[3-(â-D-galactopyran-
osyl)-(1→4)-(2-acetamido-2-deoxy-â-D-glucopyranosyl)oxy-
propyl]-â-D-glucopyranosyl}-(1→6)-[â-D-galactopyranosyl-
(1→4)]-(2-acetamido-2-deoxy-â-D-glucopyranosyloxy)propyl]-
â-D-glucopyranoside 35
Treatment of compound 33 (28.9 mg, 23.5 µmol) according
to the procedure described for the preparation of analogue 34
gave title glycoside 35 (26.1 mg, 85%); TLC (butan-1-ol–water–
HOAc, 2:1:1) Rf 0.15 (33), 0.06 (35); δC(75.5 MHz; CDCl3)
175.3 (2 × NHCOCH3), 103.7, 103.6, 103.4, 103.0, 101.9 and
101.8 (C-1, -1Ј, -1Љ, -1ٞ, -1٣, -1ٞٞ), 79.3, 79.0, 78.6, 76.4, 76.3,
76.2, 76.1, 76.0, 75.9, 75.6, 74.3, 74.0, 73.9, 73.3, 73.2, 71.8 and
69.3 (C-2, -3, -4, -5, -3Ј, -4Ј, -5Ј, -2Љ, -3Љ, -4Љ, -5Љ, -3ٞ, -4ٞ, -5ٞ,
-2٣,-3٣, -4٣, -5٣, -2Љٞ, -3Љٞ -4Љٞ, -5Љٞ), 70.4, 69.5, 68.1, 67.8,
67.7, 61.9, 61.3 and 60.9 (C-6, -6Ј, -6Љ, -6ٞ, -6٣, -6Љٞ, 2 × OCH2-
CH CH O, OCH CH᎐CH ), 55.9 (C-2Ј, -2ٞ), 39.1, 29.5, 29.0
᎐
2
2
2
2
36 U. Brodbeck and K. E. Ebner, J. Biol. Chem., 1966, 241, 762.
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and 27.9 (OCH2CH2CH2SCH2CH2NH2), 30.2 (2 × OCH2CH2-
CH2O) and 23.0 (2 × NHCOCH3); FABMS of C51H91N3O33S
(M, 1305.5) m/z 1306.4 (M ϩ H)ϩ.
39 R. T. Lee and Y. C. Lee, Carbohydr. Res., 1974, 37, 193.
Acknowledgements
This work was supported by the European Union programs
CARENET-1 (grant ERB CHRX CT940442) and VACNET
(grant ERB BIO 4CT960158). We thank Dr. P. H. Kruiskamp
Paper 8/04272A
Received 5th June 1998
Accepted 17th July 1998
3020
J. Chem. Soc., Perkin Trans. 1, 1998