Chemical synthesis of globotriose and galabiose: Relative stabilities of their complexes with Escherichia coli Shiga-like toxin-1 as determined by denaturation-titration with guanidinium chloride

Article abstract of DOI:10.1039/a801429i

Globotriose [α-D-Gal-(1→4)-β-D-Gal-(1→4)-D-Glc] is the carbohydrate moiety of the globotriosyl ceramide (Gb₃), also known as the germinal centre B-cell differentiation antigen CD77, a glycolipid present on the plasma membrane of certain mammalian cells. In Gb₃, globotriose functions as the cell-surface receptor for Shiga toxin and for the Shiga-like toxins (verocytotoxins). Here we report the chemical synthesis of globotriose and the corresponding terminal disaccharide, galabiose [α-D-Gal-(1→4)-β-D-Gal]. Globotriose and galabiose are attached via a linker to CNBr-activated Sepharose to generate affinity matrices that permit the one-step purification of recombinant Shiga-like toxin-1 from crude E. coli homogenates. Toxin is released from either of the immobilised saccharides by elution with 6 M guanidinium chloride. After dilution of the denaturant, the released toxin had full catalytic activity. Denaturation-titration experiments show that the bound toxin is released from galabiose-Sepharose at 2.3 M guanidinium chloride, while its release from globotriose-Sepharose requires a higher concentration of 4.8 M. These results indicate that the glucose component of globotriose contributes ～2.6 kcal mol^-1 to the binding energy relative to galabiose.

Full text of DOI:10.1039/a801429i

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Chemical synthesis of globotriose and galabiose: relative stabilities of
their complexes with Escherichia coli Shiga-like toxin-1 as determined
by denaturation-titration with guanidinium chloride
Dieter Müller,a Gabin Vic,a Peter Critchley,a David H. G. Crout,a,*
Nicholas Lea,b Lynne Robertsb and J. Michael Lordb
a Department of Chemistry, University of Warwick, Coventry, UK CV4 7AL
b Department of Biological Sciences, University of Warwick, Coventry, UK CV4 7AL
Globotriose [á-D-Gal-(1→4)-â-D-Gal-(1→4)-D-Glc] is the carbohydrate moiety of the globotriosyl
ceramide (Gb3), also known as the germinal centre B-cell differentiation antigen CD77, a glycolipid
present on the plasma membrane of certain mammalian cells. In Gb3, globotriose functions as the
cell-surface receptor for Shiga toxin and for the Shiga-like toxins (verocytotoxins). Here we report the
chemical synthesis of globotriose and the corresponding terminal disaccharide, galabiose [á-D-Gal-
(1→4)-â-D-Gal]. Globotriose and galabiose are attached via a linker to CNBr-activated Sepharose to
generate affinity matrices that permit the one-step purification of recombinant Shiga-like toxin-1 from
crude E. coli homogenates. Toxin is released from either of the immobilised saccharides by elution with
6 M guanidinium chloride. After dilution of the denaturant, the released toxin had full catalytic activity.
Denaturation-titration experiments show that the bound toxin is released from galabiose-Sepharose at
2.3 M guanidinium chloride, while its release from globotriose-Sepharose requires a higher concentration
of 4.8 M. These results indicate that the glucose component of globotriose contributes ~2.6 kcal mol؊1 to
the binding energy relative to galabiose.
free receptor or receptor immobilised on an appropriate inert
Introduction
non-toxic matrix could potentially be used in the case of E. coli
0157 infections to intercept SLTs before they reach target cells
in the kidney. As a preliminary to such studies, we now describe
the chemical synthesis of globotriose and galabiose, and show
that the immobilised carbohydrates are capable of specifically
binding SLT-1 from E. coli 0157.
The plasma membrane glycolipid globotriosyl ceramide
(Gb3) functions as the receptor on sensitive cells responsible for
the binding and subsequent internalisation of Shiga toxin
(ST), produced by the bacterium Shigella dysenteriae, and
the Shiga-like toxins (SLTs, also known as verocytotoxins)
produced by enterohaemorrhagic strains of Escherichia coli.1–4
ST and SLTs consist of a single catalytically active polypeptide
of 32 kDa (the A subunit) non-covalently associated with
a pentamer of identical Gb3-binding polypeptides (the B
subunits) each of 7.7 kDa. During endocytic uptake the A
chain is cleaved at a furin-sensitive site to generate a catalytic
A1 domain which remains disulfide-bonded to a B chain-
interacting A2 domain. The A1 fragment can subsequently
enter the cytosol from the endoplasmic reticulum lumen.
Within the cytosol, A1 acts as an N-glycosidase that cleaves
a specific adenine residue from a highly conserved loop in 28S
ribosomal RNA. The adenine residue that is removed by the
toxin normally plays an essential role during protein synthesis
by binding elongation factors. Ribosomes containing toxin-
depurinated 28S RNA are therefore unable to synthesise
proteins, which in turn leads rapidly to cell death.5–7 Recently,
Gb3 has been shown to act as a receptor for binding of
Burkholderia (Pseudomonas) cepacia to epithelial cells via an
adhesin in cystic fibrosis patients. Infection by B. cepacia can
give rise to rapidly fatal deterioration with pneumonia and
septicaemia, the so-called ‘cepacia syndrome’.7,8
Recent outbreaks of food poisoning involving the SLT-
producing E. coli strain 0157:H7 have focused attention on
the diseases caused by this organism. SLTs are involved in
the etiology of haemorrhagic colitis and the more serious
haemolytic uraemic syndrome (HUS), where they bind to and
enter the microvascular endothelial cells of the gastrointestinal
tract and the kidney respectively. Intestinal and kidney cells are
vulnerable to the toxins since, unlike most human cell types,
they have a significant cell surface content of Gb3. The high
specificity of the carbohydrate moiety of Gb3 for SLTs suggests
that it may have potential as a therapeutic agent. For example,
Chemical synthesis
Syntheses of globotriose or derivatives have been reported by a
number of groups.9–16 The objective of our synthesis was to
prepare the trisaccharide thioglycoside building block 10
(Scheme 1). This was expected to be chemically stable but easy
to activate for the preparation of a variety of derivatives.
Thiophenyl β-lactoside 4 was prepared from lactose 1 in three
high-yielding steps via the polyacetates 2 and 3.9 Formation
of the isopropylidene derivative of glycoside 4, followed by
benzoylation without isolation, was carefully controlled (see
Experimental section) to favour formation of the 4,6- (kinetic)
derivative 5, rather than the 3,4- (thermodynamic) derivative 6
that was formed with longer reaction times in the isopropylide-
nation reaction. The yield of the 4,6-isopropylidene derivative
5 was favoured by using an excess of 2,2-dimethoxypropane
and a controlled amount of acid catalyst. Benzoylation to the
fully protected intermediate 5 followed by removal of the iso-
propylidene group gave the intermediate 7. This was selectively
benzoylated on the 6Ј-hydroxy group by using benzoyl cyanide.
By careful control of the reaction conditions, the required
6-benzoylated product 8 was obtained in 90% yield.
The acceptor 8 was coupled with the glycosyl fluoride
donor 9 as in an analogous reaction described by Nicolaou9
to give the protected target trisaccharide glycoside 10 in 68–
95% yield. The yield was dependent on the ratio of donor to
acceptor. With a ratio of 3.4:1 the product was obtained in 95%
yield, based on acceptor. However, in practice, it was more
economical to use a ratio of 1:1 as the excess of the more
valuable acceptor 8 could be recovered quantitatively by
chromatography of the product mixture.
J. Chem. Soc., Perkin Trans. 1, 1998
2287

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OR"
OH
R'O
RO
HO
HO
OR
OH
i
O
O
O
O
O
RO
O
X
HO
OR
OH
OH
RO
OH
1
2 R = R' = R" = Ac, X = α-OAc
3 R = R' = R" = Ac, X = β-SPh
4 R = R' = R" = H, X = β-SPh
5 R = Bz, R'R" = Me2C, X = β-SPh
7 R = Bz, R' = R" = H, X = β-SPh
8 R = R" = Bz, R' = H, X = β-SPh
ii
iii
OBz
O
iv
v
OBz
O
O
vi
O
O
BzO
SPh
OBz
OBz
6
OR
RO
OBn
O
BnO
BnO
O
RO
vii
+
8
RO
OR'
O
OR'
BnO
F
O
O
X
R'O
O
R'O
9
OR'
OR'
10 R = Bn, R' = Bz, X = β-SPh
11 R = Bn, R' = Bz, X = β-O[CH2]6NHCO2Bn
12 R = R' = H, X = β-O[CH2]6NH2
viii
iii, x
Scheme 1 Reagents: i, Ac2O, DMAP, pyridine; ii, PhSSiMe3, ZnI2; iii, MeONa, MeOH; iv, MeC(OMe)2Me, TsOH; v, TFA; vi, PhCOCN, NEt3; vii,
SnCl2, AgOTf; viii, HO[CH2]6NHCO2Bn, AgOTf, Br2; ix, cyclohexene, 20% Pd(OH)2–C
We were interested to study the interaction between the B5
pentamer and the corresponding disaccharide derivative α--
Gal-(1→4)-β--GalO[CH2]6NH-Sepharose (galabioseO[CH2]6-
NH-Sepharose). Such a study would help to illuminate the
question of docking the B monomer with the carbohydrate
acceptor. It would also have a practical outcome in showing
whether or not the disaccharide would provide adequate
binding for purification purposes. This was a likely outcome
since globotriose immobilised on amino-Fractogel18 had been
shown to be effective for the purification of the toxin. In this
material, the integrity of the reducing glucose unit must have
been destroyed. The disaccharide analogue was synthesised as
shown in Scheme 2 by following a procedure closely analogous
to that used for the synthesis of the globotriose preparation.
In practice, it proved to be as effective as the immobilised
trisaccharide for the toxin’s purification.
Biological results
Release of Shiga toxin from affinity columns is routinely carried
Fig. 1 Binding of E. coli Shiga-like toxin 1 to immobilised globotriose.
Silver-stained SDS-PAGE of various protein fractions. Lanes 1: total E.
coli proteins applied to the column; Lanes 2 and 3: E coli proteins that
do not bind to the globotriose matrix; Lanes 4 and 5: E. coli Shiga-like
toxin 1 eluted from the matrix in 6  guanidinium chloride. Upper
arrow: A chain; lower arrow: B chain.
out by elution with 6  guanidinium chloride. Following a
different procedure, experiments were carried out on binding
and release of SLT-1 B chain produced in E. coli strain JM105
transformed with pSLTwt (a plasmid coding the SLT-1 operon
under control of the lac promoter). In these experiments, the
concentration of guanidinium chloride in the eluent was
increased stepwise from 0  to 6  and the concentration at
which toxin was released was determined by estimation of
protein in the eluate. The results are shown in Fig. 2a,b. Also
shown, in Fig. 2c, is the elution profile for a commercial sample
of trisaccharide immobilised on Fractogel. The elution profiles
clearly show that toxin is released from the disaccharide column
at a concentration significantly lower than that required to
release it from the trisaccharide column. The results obtained
with the commercial material (Fig. 2c) were very similar to
those obtained with the disaccharide column (Fig. 2b), con-
sistent with the suggestion, above, that the recognition unit
in this preparation is the terminal disaccharide component of
the original globotriose.
For coupling to a solid support a 6-aminohexanol spacer arm
was chosen. This was converted into the benzoyloxycarbonyl
derivative and coupled to the trisaccharide thioglycoside 10 by
in situ glycosyl bromide formation and activation with silver
trifluoromethanesulfonate (triflate).17 By this means the spacer
arm was attached in 84% yield to give exclusively the β-glyco-
sidic product 11. A two-step deprotection sequence gave the
unprotected globotriose with an attached spacer arm, com-
pound 12. This was coupled to CNBr-activated Sepharose 4B.
The immobilised trisaccharide was highly effective in selec-
tively binding Shiga toxin (Fig. 1). Lanes 1–3 show the total
periplasmic protein mixture and the mixture eluted from
the column in 0.5  NaCl in phosphate-buffered saline (PBS),
respectively. Lanes 4 and 5 show the protein mixture sub-
sequently eluted with 6  guanidinium chloride (pH 6.7).
The interaction between guanidinium chloride and proteins
has been extensively studied, particularly in relation to protein
folding and unfolding.19 Tanford has described an approach to
2288
J. Chem. Soc., Perkin Trans. 1, 1998

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∆GD = ∆GDH O ϩ Σαiniδgtr,i
(1)
2
OAc
O
OR"
AcO
AcO
i
R'O
RO
i
solution of the denaturant, ∆GDH O is the corresponding free
energy of unfolding in water, δgtr,i is the free energy of transfer
of a group of type i from water to denaturant, ni is the total
number of groups of type i in the protein and αi is the average
fractional change in the degree of exposure of groups of
type i on unfolding. Since individual αi values are not readily
accessible, an average value ‰, is usually used, leading to the
unfolding equation (2). Values of δgtr have been determined
O
2
OAc
X
OAc
OR
13
14 R = R' = R" = Ac, X = SEt
15 R = R' = R" = H, X = SEt
16 R = Bz, R'R" = Me2C, X = SEt
17 R = R" = Bz, R' = H, X = SEt
ii
iii, iv
v, vi
OR
RO
O
∆GD = ∆GDH O ϩ ‰Σni δgtr,i
(2)
2
RO
vii
9
+
17
i
RO
OR'
O
for transfer of the peptide group and amino acid side chains
from water to guanidinium chloride solutions of different
concentrations. If the populations, ni, of specific amino acids in
O
X
R'O
OR'
a given protein, are known, ∆GDH O, the free energy of unfolding
2
18 R = Bn, R' = Bz, X = SEt
viii
ii, x
in water, can be calculated if ‰ and ∆GD for a given denaturant
at a specified concentration can be determined. Using the free
energies of transfer from amino acids given by Tanford and
from the known amino acid sequence of verotoxin B subunits,
19 R = Bn, R' = Bz, X = β-O[CH2]6NHCO2Bn
20 R = R' = H, X = β-O[CH2]6NH2
Scheme
2
Reagents: i, EtSH, SnCl4; ii, MeONa, MeOH; iii,
MeC(OMe)2Me, TsOH; iv, BzCl, pyridine; v, TFA; vi, BzCN, Et3N; vii,
AgOTf, SnCl2; viii, HO[CH2]6NHCO2Bn, AgOTf, Br2; ix, cyclohexene,
10% Pd–C
the terms Σni δgtr,i in equation (2) can be calculated for the
i
concentrations of guanidinium chloride corresponding to the
peaks of the elution profiles in Fig. 2a,b. These are 19.08 kcal
molϪ1 (for 2.25  guanidinium chloride) and 32.11 kcal molϪ1
for 5.28  guanidinium chloride† (Fig. 2a). If it is assumed
that the peak values of the guanidinium chloride concen-
trations for the trisaccharide (Fig. 2a) and disaccharide
(Fig. 2b) correspond to the mid-points of the denaturation
curves, then ∆GD = 0 [equation (2)] for both cases. Accordingly
the difference in free energies of unfolding in water, ∆∆GDH O
,
2
with simultaneous release of carbohydrate, of B subunits
bound to disaccharide and trisaccharide respectively, is given
by equation (3).
∆∆GDH O = ‰(32.1 Ϫ 19.1) = 13‰
(3)
2
The overall equilibria involved in the binding and release of
the B chain are shown in Fig. 3, where BN represents the B chain
k1
BN
+
GT
BN •GT
k–1
k–2
k–3
k3
k2
BU
+
GT
k–1
k1
k2
k–2
k–3
k3
= K1,
= K2,
= K3
Fig. 3 Equilibria between immobilised globotriose, native Shiga toxin
B chain (BN) and unfolded Shiga toxin B chain (BU)
in its native state, BNؒGT its complex with globotriose, GT, and
BU the unfolded B chain. Because this is a thermodynamic
cycle, we can write equation (4) where the subscripts corre-
Ϫ∆G3 = ∆G1 Ϫ ∆G2
(4)
spond to those of the equilibrium constants of Fig. 3. For the
corresponding equilibria for disaccharide (galabiose) binding,
indicated by primes, we can write equation (5). However,
Ϫ∆G3Ј = ∆G1Ј Ϫ ∆G2Ј
(5)
Fig. 2 Denaturation-titration of Shiga toxin B chain bound to (a)
globotriose-Sepharose, (b) galabiose-Sepharose, (c) globotriose-amino-
Fractogel
Ϫ∆G3 = Ϫ∆G3Ј, since both correspond to the unfolding of the
B chain in the absence of bound carbohydrate. Accordingly
equations (4) and (5) lead to equation (6). This leads to the
the estimation of the free energy of unfolding in terms of data
obtained from solubility studies on amino acids and peptides
[equation (1)],20,21 where ∆GD is the free energy of unfolding in a
† 1 cal = 4.184 J.
J. Chem. Soc., Perkin Trans. 1, 1998
2289

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∆G1 Ϫ ∆G1Ј = ∆G2 Ϫ ∆G2Ј
(6)
each other was significantly greater than when it was bound to
galabiose.
conclusion that the free-energy difference for unfolding-release
of carbohydrate between globotriose and galabiose complexes
is equal to the difference between the binding energies of the
two carbohydrates. The unfolding-carbohydrate release term is
In the absence of X-ray crystallographic data for globotriose
bound to B pentamer, Nyholm et al.24 have studied the inter-
action by molecular modelling. According to these studies,
the glucose component of globotriose interacts only weakly
with B monomer via a putative hydrogen bond from the
6-OH group to the carbonyl group of Gly60. Although the
addition of glucose in going from galabiose to globotriose
might alter the conformational stability of the galabiose com-
ponent of globotriose, the addition of one stabilising hydrogen
bond (worth ~1 kcal molϪ1 net in binding energy) would be
consistent with the change in binding energy estimated above
from the denaturation-elution studies.
Given the weak binding of globotriose to B pentamer
(Ks = 1–2 × 10Ϫ3) and the weaker binding of galabiose, it is
surprising that both are so effective in immobilised form
for the purification of the protein. It seems likely that the
microenvironment of the bound galabiose and globotriose
may be important in this respect. This possibility deserves
further study, particularly in relation to the influence of
microenvironment on the binding of proteins to membrane-
associated carbohydrate receptors in vivo.
∆G2 Ϫ ∆G2Ј which corresponds to ∆∆GDH O in equation (3). This
2
was calculated as ‰ × 13.0 kcal molϪ1. The value of ‰ for this
system has not been determined. However, values reported in
the literature for globular proteins vary about a median value of
0.3, typically rising as high as 0.5 and going down to 0.17.22 The
value for verotoxin B chain is likely to be at the lower end of this
range for the following reason. The parameter ‰ is a measure of
the fractional increase in exposure of a group in going from the
native to the unfolded state. For individual peptide groups and
side chains, ‰i, in general, will be larger for groups and side
chains buried inside a protein than for those on the surface. For
a sphere, the ratio of surface area to volume varies as 3/r where
r is the radius. As the radius decreases, the ratio of surface
area to volume increases. It follows that for roughly spherical
globular proteins, the ratio surface groups to interior groups
will increase as the size of the protein decreases. Accordingly, in
general, it can be predicted that the smaller the protein, the
smaller will be the value of ‰ unless the protein contains a high
proportion of amino acids with side chains for which δgtr is
small or negligible. If a value of 0.2 is assumed for verotoxin B
Experimental
chain unfolding. ∆∆GDH O = 13.0 × 0.2 = 2.6 kcal molϪ1. Using
2
NMR spectra were recorded on a Bruker WH250 (250 MHz,
1H; 62.5 MHz, 13C) or a WH400 (400 MHz, 1H; 100 MHz, 13C)
spectrometer. Chemical shifts are reported in parts per million
(δ) relative to tetramethylsilane; J-values are given in Hz.
Optical rotations were determined with an Optical Activity
LTD AA-1000 polarimeter at 589 and 546 nm; [α]R-values are
given in units of 10Ϫ1 deg cm2 gϪ1. Mps were measured with a
Gallenkamp Melting Point Apparatus in an open tube and are
uncorrected. All reactions were carried out under nitrogen and
anhydrous conditions with dry, freshly distilled solvents where
necessary. Yields are usually taken after purification and refer
to chromatographically pure materials unless otherwise noted.
All reactions were monitored by TLC on 0.25 mm silica gel
60 plates (60F-254) (Merck) with UV light and/or basic aq.
KMnO4 (2 g KMnO4 and 5 g K2CO3 solved in 100 cm3 distilled
water). Moisture- and air-sensitive operations were generally
carried out in a glove bag under dry nitrogen. Solvents were
evaporated off under reduced pressure at 45 ЊC bath temper-
ature. Column chromatography was performed following the
‘flash’-method using silica gel 60 (Merck). Light petroleum had
boiling range 40–60 ЊC.
microcalorimetry, it has been determined that the free energy of
binding of soluble globotriose to verotoxin B chain is Ϫ3.6 kcal
mol .
Ϫ1 23 The binding energy for galabiose can thus be estimated
as 1 kcal molϪ1. This value is clearly too low and suggests that
unfolding of the B chain should have an ‰-value less than 0.2.
However, it should be noted that the conditions under which
the microcalorimetric measurements were made (β-methyl
glycoside of globotriose and B pentamer in solution) and those
of the present study (soluble B pentamer and immobilised
globotriose/galabiose) were different. The microcalorimetric
measurement was of binding of globotriose to B pentamer.
However, in the experiments with immobilised globotriose/
galabiose it appears more probable that the binding observed
was between single B monomers and carbohydrate for the
following reasons. First, it would be expected that dissociation
of B pentamer into subunits would be promoted at concen-
trations of guanidinium chloride different from those required
to unfold the protein with simultaneous release of carbo-
hydrate. In this case, release of B chains bound to immobilised
globotriose/galabiose would be expected to occur in two bursts,
one corresponding to pentamer dissociation with release of
four monomer units, and the other to unfolding with simul-
taneous release from globotriose of the remaining monomer.
This was not seen. It is possible that pentamer dissociation and
unfolding required similar concentrations of denaturant so that
protein would be released on a single burst. However, this
conclusion is ruled out by the fact that markedly different con-
Phenyl 2,3-di-O-benzoyl-4,6-isopropylidene-â-D-galactopyrano-
syl-(1→4)-2,3,6,tri-O-benzoyl-1-thio-â-D-glucopyranoside 5
To a stirred solution of lactose glycoside 4 (3.0 g, 6.9 mmol)9
and toluene-p-sulfonic acid (TsOH) (98 mg, 0.35 mmol) in dry
DMF (20 cm3) was added 2,2-dimethoxypropane (3.6 cm3, 29.3
mmol). After 90 min the mixture was quenched by the addition
of triethylamine (1 cm3), concentrated, and dried under reduced
pressure for 2 h. The residue was re-dissolved in dry pyridine
(20 cm3). Benzoyl chloride (6 cm3) was added at 0 ЊC. The
cooling bath was removed after 30 min and the mixture
was stirred at rt for a further 2.5 h. The mixture was diluted
with ethyl acetate (100 cm3) and CH2Cl2 (100 cm3), washed
(successively) five times with 0.2  aq. copper() sulfate and
once with distilled water. The organic layer was dried (MgSO4)
and the solvent was evaporated under reduced pressure. The
resulting oil was dried under reduced pressure and purified by
flash chromatography (8 × 20 cm; toluene–ethyl acetate [19:1])
to give derivative 5 (4.70 g, 68%), Rf 0.23 (toluene–ethyl acetate
[9:1]); mp 122 ЊC; δH(400 MHz; CDCl3) 1.15 (3 H, s, CH3), 1.23
(3 H, s, CH3), 2.86 (1 H, br d, H-5Ј), 3.37 (1 H, dd, J 1.8 and
12.7, Hb-6Ј), 3.49 (1 H, dd, J 2.7 and 12.7, Ha-6Ј), 3.90 (1 H,
ddd, J 2.1, 5.1 and 9.4, H-5), 4.11 (1 H, dd, J 9.4 and 9.1, H-4),
centrations of denaturant were required to release
B
chains from bound galabiose and guanidinium. The experiment
with galabiose showed that B chain could be unfolded
in the presence of 2.25  guanidinium chloride. Clearly, B5
pentamer must be dissociated at or below this concen-
tration. However, in the experiment with bound globotriose
there was negligible release of B chain with 2.25  guanidinium
chloride. It follows that, in both experiments, B monomer
only was bound. The possibility of multivalent attachment
is ruled out by the average spacing between activated sites
in the CNBr-activated Sepharose used to immobilise the
carbohydrate. This was too large to permit binding of two
or more carbohydrate molecules at a distance apart small
enough to permit simultaneous binding to a B5 pentamer.
The only other possible conclusion would be that when B
pentamer was bound to globotriose, binding of subunits to
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J. Chem. Soc., Perkin Trans. 1, 1998

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4.25 (1 H, apparent d, J 3.6, H-4Ј), 4.34 (1 H, dd, J 12.0 and 5.1,
Hb-6), 4.68 (1 H, dd, J 12.0 and 2.1, Ha-6), 4.75 (1 H, d, J 7.9,
H-1Ј), 4.90 (1 H, d, J 10.2, H-1), 5.04 (1 H, dd, J 10.4 and 3.6,
H-3Ј), 5.70 (1 H, dd, J 10.2 and 7.9, H-2), 5.84 (1 H, dd, J 7.9
and 9.1, H-3), 7.00–7.60 (18 H, m, ArH) and 7.85–8.0 (12 H, m,
ArH); δC(100 MHz; CDCl3) 18.9 (CH3), 28.3 (CH3), 61.5 (C-6Ј),
62.4 (C-6), 65.8 (C-4Ј), 66.7 (C-5Ј), 69.5 (C-2Ј), 70.6 (C-2), 72.6
(C-3Ј), 74.9 (C-3), 76.3 (C-4), 76.7 (C-5), 85.5 (C-1), 98.7
(CMe2), 101.1 (C-1Ј), 127.9, 128.0, 128.1, 128.2, 128.2, 128.3,
128.6, 128.7, 128.8, 129.0, 129.1, 129.4, 129.5, 129.6, 129.7 and
129.8 (Ar CH), 131.4, 132.8, 132.9, 133.0, 133.1 and 133.1
(ArC) and 164.7, 164.9, 165.2, 165.4 and 165.9 (CO); m/z (FAB)
1041 (M ϩ 2Na, 0.1%), 980 (0.2) 885 (0.5), 411 (6.8), 353 (0.6),
323 (0.3), 289 (0.8), 231 (1.9), 165 (1.1), 154 (5.6), 149 (1.3), 136
(6.8), 123 (1.3), 105 (100), 77 (22) and 52 (8.6).
(Ar CH), 131.8, 132.7, 133.0, 133.1, 133.2 and 133.3 (Ar C),
164.9, 165.1, 165.5, 165.2 (br) and 165.9 (CO); m/z (FAB-CIϩ)
1081 (M ϩ Na, 0.2%), 949 (1.5), 705 (0.3), 579 (0.8), 475 (10.6),
353 (4.1), 231 (1.0), 154 (9.3), 137 (5.5), 105 (100) and 77 (18.8).
Phenyl 2,3,4,6-tetra-O-benzyl-á-D-galactopyranosyl-(1→4)-
2,3,6,tri-O-benzoyl-â-D-galactopyranosyl-(1→4)-2,3,6-tri-O-
benzoyl-1-thio-â-D-glucopyranoside 10
A 100 cm3 two necked flask was charged with glycoside 8 (1.0 g,
0.94 mmol), silver triflate (481 mg, 1.87 mmol), tin() chloride
(365 mg, 1.92 mmol) and freshly activated 4 Å molecular sieves
(3.0 g). The mixture was dried for 2 h under high vacuum (<0.1
mbar)‡ and the flask was filled again with dry nitrogen. Dry
diethyl ether (12 cm3) and dry dichloromethane (6 cm3) were
added and the mixture was cooled to 0 ЊC (ice-bath). The
galactopyranosyl fluoride 99 (554 mg, 1.13 mmol) as a solution
in dry dichloromethane (4 cm3) was added and the reaction
mixture was stirred for 3 h at 0 ЊC. When the reaction had
finished (TLC), ethyl acetate (25 cm3) was added. The suspen-
sion was filtered through Celite and the filter pad was washed
thoroughly with dichloromethane. The organic layer was
washed successively with saturated aq. NaHCO3 (3 × 50 cm3)
and distilled water (50 cm3). The organic phase was dried
(MgSO4) and the solvent was evaporated off under reduced
pressure. Purification by flash chromatography (toluene–ethyl
acetate [19:1]) gave protected trisaccharide 10 (1.02 g, 69%).
The unchanged acceptor 8 (313 mg, 31%) was recovered during
the purification. For protected trisaccharide 10 (Found: C, 72.2;
H, 5.6. C94H84O21S requires C, 71.38; H, 5.35%); Rf 0.44
(toluene–ethyl acetate [9:1]); mp 76 ЊC; [α]D23 ϩ53.3 (c 1.00,
CHCl3); [α]25346 ϩ61.9 (c 1.00, CHCl3); δH(250 MHz; CDCl3) 2.98
(1 H, dd, J 8.5 and 4.9), 3.35 (1 H, t, J 8.9), 3.45–3.55 (1 H,
m), 3.70 (1 H, t, J 6.48), 3.82–4.95 (20 H, m), 5.05 (1 H, dd,
J 10.74 and 2.87), 5.29 (1 H, d, J 3.47), 5.34 (1 H, dd, J ~9.7
and 9.7), 5.74 (1 H, dd, J 10.71 and 7.81), 5.81 (1 J, dd, J ~9.2
and 9.2), 7.02–7.62 (45 H, m), 7.75–7.85 (2 H, m, ArH)
and 7.90–8.05 (8 H, m, ArH); δC(62.5 MHz; CDCl3) 61.9, 62.6,
67.2, 69.7, 70.7, 72.5, 72.8, 73.2, 73.9, 74.3, 74.6, 74.8, 74.9,
75.3, 75.7, 76.2, 76.9, 78.9, 85.6 and 93.4 (internal C), 101.0 and
101.1 (C-1 and -1Ј), 127.1, 127.2, 127.2, 127.3, 127.5, 127.6,
127.7, 127.8, 127.9, 127.9, 128.0, 128.0, 128.1, 128.1, 128.2,
128.2, 128.3, 128.3, 128.4, 128.6, 128.9, 129.4, 129.5, 129.6,
129.6, 129.7, 129.8, 132.9, 133.0 and 133.0 (Ar CH), 131.6,
137.9, 138.1, 138.3, 138.5, 138.6, 138.7 and 138.8 (Ar C), 164.9,
165.0, 165.1, 165.5, 165.6 and 166.3 (CO); m/z (FAB-CIϩ) 1605
(M ϩ Naϩ, 25.2%), 1473 (Mϩ Ϫ SPh, 11.2), 949 (51.5), 829
(5.0), 705 (27.1), 565 (55.2), 503 (14.5), 475 (100), 431 (8.6), 353
(37.2), 335 (16.6) and 271 (31.0) {Found: m/z [FAB (M ϩ Na)],
1603.5154. C94H84NaO21S requires m/z 1603.5099}.
Phenyl 2,3-di-O-benzoyl-â-D-galactopyranosyl-(1→4)-2,3,6,tri-
O-benzoyl-1-thio-â-D-glucopyranoside 7
To a cold (0 ЊC) solution of compound 5 (4.7 g, 4.72 mmol) in
THF (20 cm3) and distilled water (7.3 cm3) was added TFA (17
cm3). After the mixture had been stirred for 15 min a solid
precipitated. The cooling bath was removed and additional
THF (15 cm3) was added. The reaction was monitored by TLC.
After 45 min the mixture was poured into saturated aq.
NaHCO3 and the product was extracted with ethyl acetate
(4 × 20 cm3). The combined organic layers were dried (MgSO4)
and the solvents were evaporated off under reduced pressure.
The crude solid were purified by flash chromatography with
toluene–ethyl acetate 2:1 as eluent to give glycoside 7 (3.90 g,
87%), mp 211 ЊC; Rf 0.23 (toluene–ethyl acetate [2:1]); 0.42
(toluene–ethyl acetate [1:1]); [α]D23 ϩ 88.4 (c 1.00, CHCl3);
δH(250 MHz; CDCl3) 3.20–3.43 (3 H, m), 3.87–3.96 (1 H, m,
H-5), 4.10 (1 H, dd, J ~9.4 and 9.4), 4.19 (1 H, d, J 3.2), 4.42
(1 H, dd, J 5.5 and 11.9, Hb-6), 4.65 (1 H, dd, J 2.0 and 11.9,
Ha-6), 4.77 (1 H, d, J 8.1, H-1Ј), 4.90 (1 H, d, J 9.9, H-1), 5.09
(1 H, dd, J 3.2 and 10.5), 5.39 (1 H, dd, J ~9.5 and 9.5), 5.78–
5.80 (2 H, m), 7.03–7.68 (20 H, m, ArH) and 7.85–8.02 (10 H,
m, ArH); m/z (FAB-CIϩ) 978 (M ϩ Naϩ, 0.2%), 845 (1.3), 475
(1.6), 371 (8.0), 353 (1.7), 249 (4.0), 231 (1.0), 201 (1.0), 189
(1.2), 165 (1.4), 154 (5.2), 136 (5.6), 105 (100) and 77 (20).
Phenyl 2,3,6-tri-O-benzoyl-â-D-galactopyranosyl-(1→4)-2,3,6,tri-
O-benzoyl-1-thio-â-D-glucopyranoside 8
To a solution of glycoside 7 (2.01 g, 2.09 mmol) in dry DMF
(30 cm3) was added dry trimethylamine (0.35 cm3, 2.5 mmol).
After cooling of this mixture to Ϫ20 ЊC (solid CO2 in CCl4),
benzoyl cyanide (0.25 cm3, 2.09 mmol) was added. The cooling
bath was replaced by an ice-bath after 15 min. The reaction was
quenched with methanol (1 cm3) after a total reaction time of
30 min. The reaction mixture was diluted with ethyl acetate–
dichloromethane (1:1) (400 cm3) and washed with distilled
water (4 × 75 cm3). The organic layer was dried (MgSO4) and
the solvents were evaporated off. The crude solid was purified
by flash chromatography with ethyl acetate–toluene (1:9) as
eluent to give glycoside 8 (1.97 g, 90%), mp 131 ЊC (Found: C,
68.1; H, 4.9. C60H50O16S requires C, 68.04; H, 4.76%); [α]D23
ϩ51.9 (c, 1.026, CHCl3); Rf 0.19 (toluene–ethyl acetate 9:1);
δH(400 MHz; CDCl3) 3.61 (1 H, dd, J 6.5 and 11.2, Hb-6Ј), 3.71
(1 H, t, J 6.5, Ha-6Ј), 3.92 (1 H, ddd, J 1.8, 5.4 and 9.7, H-5),
4.07 (1 H, dd, J 6.2 and 11.3, H-5Ј), 4.10 (1 H, d, J 9.7, H-4),
4.15 (1 H, br d, J 3.7, H-4Ј), 4.47 (1 H, dd, J 5.4 and 12.1, Hb-6),
4.61 (1 H, dd, J 1.8 and 12.1, Ha-6), 4.77 (1 H, d, J 7.9, H-1Ј),
4.94 (1 H, d, J 10.0, H-1), 5.16 (1 H, dd, J 3.3 and 10.3, H-3Ј),
5.41 (1 H, dd, J ~9.0 and 9.0, H-2), 5.75 (1 H, dd, J 7.9 and 10.3,
H-2Ј), 5.80 (1 H, dd, J ~9.0 and 10.0, H-3), 7.06–7.61 (23 H,
m, ArH) and 7.88–8.00 (12 H, m, ArH); δC(100 MHz; CDCl3)
61.7 and 62.6 (C-6 and -6Ј), 66.6, 70.0, 70.3 and 72.5 (C-2, -3,
-2Ј and -3Ј), 74.0 and 74.1 (C-5 and -5Ј), 76.1 and 76.8 (C-4
and -4Ј), 85.6 (C-1), 101.1 (C-1Ј), 127.9, 128.1, 128.2, 128.3,
128.5, 128.7, 128.8, 128.9, 129.0, 129.3, 129.4, 129.5 and 129.7
Benzyl N-(6-hydroxyhexyl)carbamate
A solution of 6-aminohexanol (3.67 g, 31.3 mmol) in aq.
sodium hydroxide [2 ; 15.8 cm3 (31.3 mmol NaOH)] was
cooled to 0 ЊC (ice-bath). To this mixture was added benzyl
chloroformate (4.7 cm3, 31.3 mmol) over a period of 10 min.
The cooling ice-bath was removed and the reaction mixture was
stirred for a further 1 h. Distilled water (100 cm3) was added
and the product was extracted with dichloromethane (3 × 150
cm3). The combined organic fractions were washed successively
with HCl (1 ; 100 cm3), saturated aq. NaHCO3 (100 cm3) and
distilled water (100 cm3). The organic phase was dried (MgSO4)
and the solvent was evaporated off under reduced pressure.
Crystallisation (ethyl acetate–light petroleum) gave the title
carbonate (6.82 g, 87%) as a solid, mp 81 ЊC (Found: C, 67.0; H,
8.4; N, 5.6. C14H21NO3 requires C, 66.91; H, 8.42; N, 5.57%); Rf
0.23 (ethyl acetate–light petroleum [1:1]); δH(250 MHz; CDCl3)
1.31–1.63 (8 H, m, 4 × CH2), 3.21 (2 H, t, J 6.7, CH2N), 3.64
‡ 1 bar = 105 Pa.
J. Chem. Soc., Perkin Trans. 1, 1998
2291

View Article Online
(2 H, t, J 6.4, CH2OH), 4.79 (1 H, br s, NH), 5.11 (2 H, s,
CH2Ph) and 7.28–7.41 (5 H, m, ArH); δC(62.5 MHz; CDCl3)
25.1, 26.2, 29.8 and 32.4 (4 × CH2), 40.7 (NCH2), 62.4 (COH),
66.4 (OCPh), 127.9 and 128.3 (Ar CH), 136.4 (Ar C) and 156.3
(CO).
C-1, -1Ј and -1Љ) and 126.7, 127.4, 127.8, 127.9, 128.0, 128.1,
128.1, 128.2, 128.3, 128.4, 128.4, 128.6, 128.7, 129.7, 129.8,
130.7, 136.4, 136.8, 137.1 and 137.7 (Ar C); m/z (FAB-CIϩ)
1121 (M ϩ Naϩ, 3.2%), 1099 (M ϩ Hϩ, 3.2), 1098 (M, 4.1), 391
(27.2), 377 (14.5), 197 (6.3), 181 (20.4), 149 (100) and 121 (6.7)
(C60H75NO18 = 1098.26).
6-(Benzyloxycarbonylamino)hexyl 2,3,4,6-tetra-O-benzyl-á-D-
galactopyranosyl-(1→4)-2,3,6-tri-O-benzoyl-â-D-galactopyrano-
syl-(1→4)-2,3,6-tri-O-benzoyl-â-D-glucopyranoside 11
6-Aminohexyl á-D-galactopyranosyl-(1→4)-â-D-galactopyrano-
syl-(1→4)-â-D-glucopyranoside 12
A 50 cm3 two-necked flask was charged with protected tri-
saccharide 10 (1.0 g, 0.63 mmol), benzyl N-(6-hydroxyhexyl)-
carbamate (694 mg, 2.76 mmol), silver triflate (2.2 g, 8.7 mmol)
and 4 Å molecular sieves (4.0 g). After drying of the solid mix-
ture under high vacuum for 60 min, dry dichloromethane (15
cm3) was added. The mixture was cooled to 0 ЊC (ice-bath) and
9.2 cm3 of previously prepared and cooled bromine solution
(100 mm3 of Br2 in 10 cm3 of dichloromethane) was added in
portions over a period of 5 min. After being stirred for 2 h at
0 ЊC the mixture was filtered through Celite and the filter pad
was thoroughly rinsed with CH2Cl2 (250 cm3). The organic
layer was washed successively with saturated aq. NaHCO3
(3 × 100 cm3) and distilled water (100 cm3). The solution was
dried (MgSO4) and evaporated under reduced pressure. Purifi-
cation by flash chromatography with toluene–ethyl acetate
(12:1) as eluent followed by a second flash chromatographic
purification with toluene–ethyl acetate (19:1) as eluent gave tri-
saccharide glycoside 11 (914 mg, 84%) as a solid, mp 59 ЊC
(Found: C, 70.61; H, 5.9; N, 0.9. C102H99NO24 requires C, 71.11;
6-(Benzyloxycarbonylamino)hexyl 2,3,4,6-tetra-O-benzyl-α--
galactopyranosyl-(1→4)-β--galactopyranosyl-(1→4)-β--
glucopyranoside (200 mg, 0.182 mmol) was dissolved in
methanol (4 cm3). Pd(OH)2/C (20%; 600 mg) and cyclohexene
(4 cm3) were added. The mixture was boiled under reflux for
4.5 h and then was filtered (Celite). The filter pad was washed
thoroughly with both methanol and distilled water. The filtrate
was evaporated under reduced pressure and the residue was
purified by flash chromatography with CH3OH–aq. ammonia
(4:1) as eluent to give glycoside 12 (70.0 mg, 60%), mp 185 ЊC
(Found: m/z, 604.2801. C24H46NO16 requires m/z, 604.2803);
Rf 0.20 (CH3OH–conc. NH3 [4:1]); δH(250 MHz; CDCl3) 1.15–
1.65 (8 H, m, CH2), 2.40 (2 H, m), 2.83 (1 H, m), 3.14–3.28
(2 H, m), 3.44–3.98 (18 H, m), 4.28 (1 H, t, J 6.5), 4.40 (2 H, t,
J 7.3), 4.71 (1 H, d, J 7.3, H-1Љ) and 4.82 (1 H, d, J 11.62,
H-1Ј); δH(400 MHz; D2O) 1.36 (4 H, m, CH2CH2), 1.59 (4 H,
m, CH2CH2), 4.86 (1 H, d, J 3.8, H-1), 2.87 (2 H, t, J 7.2,
CH2NH2), 3.25 (1 H, m, H-2), 3.51–4.06 (18 H, m), 4.31 (1 H, t,
J 6.3, H-5Ј), 4.44 (1 H, d, J 8.0, H-1), 4.47 (1 H, d, J 7.8, H-1Ј)
and 4.91 (1 H, d, J 3.8, H-1Љ); δC(62.5 MHz, D2O) 25.2, 26.0,
28.3, 29.1 and 40.3 (CH2), 60.7 (C-6), 61.0 (C-6Ј), 61.1 (C-6Љ),
69.2 (C-2Љ), 69.6 (C-4Љ), 69.8 (C-3Љ), 71.1 (OCH2), 71.4 (C-5Љ),
71.5 (C-5 or -2Ј), 72.8 (C-3Ј), 73.6 (C-2), 75.2 (C-3), 75.4 (C-2Ј or
-5), 76.1 (C-5Ј), 78.0 (C-4Ј), 79.3 (C-4), 100.9 (C-1Љ), 102.6 (C-1)
and 103.9 (C-1Ј); m/z (FAB-CIϩ) 604 (M ϩ Hϩ, 47.5%), 370
(8.1), 307 (5.4), 299 (6), 278 (21), 259 (15), 245 (16), 225 (6), 215
(16), 207 (35.5), 199 (8), 186 (100), 167 (27.5), 153 (17), 137 (9),
131 (9.5), 118 (15), 115 (46) and 107 (10.5).
H, 5.79; N, 0.81%); Rf 0.22 (toluene–ethyl acetate [9:1]; 0.72
23
546
toluene–ethyl acetate [2:1]); [α]D23 ϩ47.7 (c 0.64, CHCl3); [α]
ϩ56.9 (c 0.64, CHCl3); δH(250 MHz; CDCl3) 1.05–1.60 (8 H, m,
CH2), 2.87–3.07 (2 H, m), 3.30–3.47 (2 H, m), 3.67 (1 H, m),
3.72–4.90 (24 H, m), 4.98–5.18 (2 H, m, CO2CH2Ph), 5.36 (2 H,
m), 5.71–5.88 (2 H, m), 7.05–7.62 (45 H, m, ArH), 7.79–7.85
(2 H, m, ArH) and 7.90–8.07 (8 H, m, ArH); δC(62.5 MHz;
CDCl3) 25.2, 26.0, 29.0, 29.4, 40.7, 61.9, 62.3, 66.3, 67.1, 69.6,
69.7, 69.7, 72.2, 72.5, 72.8, 72.9, 73.1, 73.2, 74.3, 74.7, 75.3,
75.5, 75.7, 76.2 and 78.9 (aliphatic and internal carbon atoms),
101.1, 101.0, 100.8 (C-1, -1Ј and -1Љ), 127.1, 127.1, 127.2, 127.3,
127.4, 127.8, 127.9, 127.9, 127.9, 128.0, 128.1, 128.1, 128.1,
128.2, 128.3, 128.4, 128.8, 128.9, 129.3, 129.4, 129.4, 129.5,
129.6, 129.7, 132.9, 132.9, 132.9 and 133.0 (Ar CH), 136.5,
138.0, 138.3, 138.7 and 138.8 (Ar C) and 164.8, 165.0, 165.1,
165.4, 165.6 and 166.3 (CO); m/z (FAB-CIϩ) 1762 (M ϩ K,
37%), 1723 (M ϩ Na, 100), 1723 (M, 14), 1589 (12), 1472 (10),
1291 (27.5), 1247 (8.2), 1201 (8.0), 1183 (6.7) and 1039 (13.7).
Coupling of 6-aminohexyl á-D-galactopyranosyl-(1→4)-â-D-
galactopyranosyl-(1→4)-â-D-glucopyranoside 12 to CNBr-
activated Sepharose 4B
Dry Sepharose (1 g) was swollen in aq. HCl (1 m) for 15 min.
The acidic solution was removed by suction without allowing
the gel to dry. The gel was transferred into a previously
prepared solution of glycoside 12 (9.5 mg) in aq. NaHCO3
(0.1  NaHCO3 and 0.5  NaCl, pH 10.3; 15 cm3). The mixture
was shaken for 4 h at 25 ЊC (110 rpm). The coupling solution
was filtered by suction and the gel was again placed in aq.
NaHCO3 (0.1  NaHCO3 and 0.5  NaCl, pH 10.3; 15 cm3).
Ethanolamine was added to deactivate the gel. The mixture
was shaken for 2 h, the solvent was removed by suction, and
the gel was washed successively with aq. HCl (1 m) and aq.
NaCl (0.5 ). It was stored in aq. NaCl (0.5 ) containing
NaN3 to prevent bacterial contamination.
6-(Benzyloxycarbonylamino)hexyl 2,3,4,6-tetra-O-benzyl-á-D-
galactopyranosyl-(1→4)-â-D-galactopyranosyl-(1→4)-â-D-
glucopyranoside
A solution of trisaccharide glycoside 11 (800 mg, 0.46 mmol) in
methanol (7 cm3) was treated with sodium methoxide (102 mg,
1.88 mmol). The mixture was stirred for 5 h at rt, neutralised
with Amberlyst 15 ion-exchange resin, and filtered through
a pad of Celite. The solvent was evaporated under reduced
pressure. The product was purified by flash chromatography
with dichloromethane–methanol (15:1) as eluent to give 6-
Ethyl 2,3,4,6-tetra-O-acetyl-1-thio-â-D-galactopyranoside 14
The peracetylated galactopyranoside 13 (Sigma) (6.14 g, 15.74
mmol) was dissolved in ClCH2CH2Cl (200 cm3) containing 4 Å
molecular sieves (5 g) and ethanethiol (2.31 cm3, 31.25 mmol).
The mixture was stirred at Ϫ30 ЊC for 1 h, SnCl4 (2.00 cm3,
17.02 mmol) was added, and the reaction medium was allowed
to warm to Ϫ17 ЊC over a period of 1 h. The reaction was
quenched by addition of saturated aq. NaHCO3 (150 cm3).
The mixture was stirred for 1 h and filtered through a pad of
Celite. The layers were separated and the aqueous layer was
extracted with CH2Cl2 (150 cm3). The organic layers were
combined, washed with water (200 cm3), dried (MgSO4), and
evaporated. Crystallisation (Et2O–light petroleum) afforded
thioglycoside 14 as a crystalline solid (4.1 g, 80%); mp 77–78 ЊC
(Found: C, 48.94; H, 6.2. C16H24O9S requires C, 49.00; H,
benzyloxycarbonylamino)hexyl
2,3,4,6-tetra-O-benzyl-α--
galactopyranosyl-(1→4)-β--galactopyranosyl-(1→4)-β--
glucopyranoside (423 mg, 84%), mp 40 ЊC; Rf 0.16 (CH2Cl2–
CH3OH [15:1]); [α]D23 Ϫ7.5 (c 0.54, CHCl3); [α]25346 Ϫ9.6 (c 0.54,
CHCl3); δH(250 MHz; CDCl3) 1.20–1.68 (8 H, m, 4 × CH2),
2.99 (2 H, t, J 6.5, CH2NCO), 3.17–4.28 (22 H, m), 4.32 (1 H, d,
J 10.76, CH2Ph), 4.40 (1 H, d, J 10.75, CH2Ph), 4.54 (1 H, d,
J 11.34, H-1), 4.67 (1 H, d, J 11.92, H-1Ј), 4.75 (1 H, d, J 3.75,
H-1Љ), 4.79 (2 H, s, COOCH2Ph), 4.90 (1 H, d, J 11.91), 4.95
(1 H, d, J 11.3), 5.09 (2 H, s) and 7.20–7.48 (25 H, m, ArH);
δC(62.5 MHz; CDCl3) 25.3, 26.1, 29.2, 29.6, 29.6, 40.7, 60.0,
62.4, 62.5, 65.7, 66.4, 69.8, 69.8, 71.4, 72.6, 73.4, 73.7, 73.8,
74.0, 74.3, 74.4, 74.8, 75.4, 100.8, 102.5 and 103.9 (last three
2292
J. Chem. Soc., Perkin Trans. 1, 1998

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6.12%); Rf 0.41 (light petroleum–Et2O [2:8]); [α]D28 Ϫ8.6
(c 0.98, CHCl3); δH(250 MHz; CDCl3) 1.22 (3 H, t, J 7.4,
CH3CH2), 1.92 (3 H, s, COCH3), 1.98 (3 H, s, COCH3), 2.00
(3 H, s, COCH3), 2.09 (3 H, s, COCH3), 2.56–2.74 (2 H, m,
SCH2CH3), 3.84–3.90 (1 H, m), 4.00–4.14 (2 H, m), 4.43 (1 H,
d, J 9.8, H-1), 4.98 (1 H, dd, J 10.0 and 3.3, Ha-6), 5.17 (1 H,
dd, J ~9.9 and 9.9) and 5.36 (1 H, dd, J 3.3 and 1.0, H-4);
δC(62.5 MHz; CDCl3) 14.7 (SCH2CH3), 20.5, 20.6, 20.7 and
24.3 (SCH2CH3), 61.3 (C-6), 67.0, 67.1, 71.7, 74.2, 83.9 (C-1)
and 169.5, 170.0, 170.1 and 170.3 (CO).
toluene) afforded the selectively deprotected glycoside 17 (1.05 g,
96%) as a solid, mp 121–122 ЊC (Found: C, 64.9; H, 5.25.
C29H28O8S requires C, 64.94; H, 5.25%); Rf 0.57 (toluene–
EtOAc [7:3]); [α]D32 ϩ59.4 (c 1.02, CHCl3); δH(250 MHz; CDCl3)
1.32 (3 H, t, J 7.4, SCH2CH3), 2.70–2,86 (2 H, m, SCH2CH3),
4.10 (1 H, dd, J ~6.4 and 6.4, H-4), 4.36–4.40 (1 H, m,
H-5), 4.54–4.72 (2 H, m, H2-6), 4.75 (1 H, d, J 9.8, H-1), 5.40
(1 H, dd, J 2.9 and 9.8, H-3), 5.83 (1 H, dd, J ~9.8 and 9.8,
H-2), 7.32–7.61 (10 H, m, ArH) and 7.94–8.06 (5 H, m, ArH);
δC(100 MHz; CDCl3) 14.8 (SCH2CH3), 24.1 (SCH2CH3), 62.7
(C-6), 67.3, 67.7, 74.9 and 75.9 (C-2, -3, -4 and -5), 83.8 (C-1),
128.2, 128.3, 128.8, 129.2, 129.3, 129.6 and 129.7 (ArCH),
133.1, 133.2 and 133.3 (ArC) and 165.3, 165.6 and 166.3 (CO).
Ethyl 1-thio-â-D-galactopyranoside 15
To a solution of thioglycoside 14 (4.03 g, 10.28 mmol) in
MeOH (100 cm3) was added a catalytic amount of NaOMe
(55.5 mg, 1.02 mmol). The mixture was kept for 17 h at rt,
neutralised with Amberlyst 15, filtered and concentrated under
reduced pressure to yield deprotected thioglycoside 15 quanti-
tatively as a syrup, [α]D24 Ϫ23.2 (c 0.59, MeOH); δH(250 MHz;
D2O) 1.03 (3 H, t, J 7.4, SCH2CH3), 2.44–2.60 (2 H, m,
SCH2CH3), 3.27–3.57 (5 H, m), 3.73 (1 H, d, J 3.1, H-4) and
4.24 (1 H, d, J 9.5, H-1); δC(62.5 MHz; D2O) 15.3 (SCH2CH3),
25.0 (SCH2CH3), 61.9 (C-6), 69.6, 70.4, 74.7, 79.7 and 86.4
(C-1); m/z (FAB) 247 (M ϩ Naϩ).
Ethyl 2,3,4,6-tetra-O-benzyl-á-D-galactopyranosyl-(1→4)-2,3,6-
tri-O-benzoyl-1-thio-â-D-galactopyranoside 18
To glycoside 17 (0.33 g, 0.61 mmol) were added silver triflate
(0.43 g, 1.70 mmol), tin() chloride (0.24 g, 1.31 mmol) and
activated molecular sieves (4 Å). The mixture was dried for 2 h
under reduced pressure, cooled to 0 ЊC, and dissolved in Et2O
(12 cm3)–CH2Cl2 (6 cm3). A solution of glycosyl fluoride 9 (0.46
g, 0.86 mmol) in Et2O (3 cm3) was added. The mixture was
stirred for 3.5 h at 0 ЊC, EtOAc (50 cm3) was added, and the
suspension was filtered through a pad of Celite. The organic
layer was washed three times with saturated aq. NaHCO3
(30 cm3) and once with brine (30 cm3), dried (MgSO4), and
evaporated under reduced pressure. Flash chromatography
(40% Et2O in light petroleum) gave disaccharide glycoside 18
(0.33 g, 51%) as an amorphous solid, mp 44–45 ЊC (Found: C,
71.2; H, 5.8. C63H62O13S requires C, 71.47; H, 5.85%); Rf 0.46
(silica, Et2O–light petroleum [1:1]); [α]D24 ϩ61.5 (c 0.72, CHCl3);
δH(100 MHz; CDCl3) 1.25 (3 H, t, J 7.4, SCH2CH3), 2.69–2.88
(2 H, m, SCH2CH3), 2.89–2.92 (1 H, m, Ha-6Ј), 3.38 (1 H, t,
J 8.8, Hb-6Ј), 4.00–4.10 (5 H, m, H-2Ј, -4Ј, -5Ј and CH2Ph), 4.15
(1 H, dd, J 2.5 and 10.2, H-3Ј), 4.27–4.34 (1 H, m, H-5), 4.44
(1 H, d, J 2.8, H-4), 4.47 (1 H, d, J 11.0, CH2Ph), 4.68–4.87
(8 H, m, H-1, H2-6, 3 × CH2Ph), 4.94 (1 H, d, J 3.4, H-1Ј), 5.27
(1 H, dd, J 2.8 and 10.7, H-3), 5.84 (1 H, dd, J ~10.0 and 10.0,
H-2), 7.12–7.59 (30 H, m, ArH) and 7.88–8.01 (5 H, m, ArH);
δC(100 MHz; CDCl3) 14.9 (SCH2CH3), 23.6 (SCH2CH3), 62.3
(C-6), 67.3 (C-6Ј), 67.8 (C-2), 69.6 (C-5), 72.5 (CH2Ph), 72.7
(CH2Ph), 73.8 (CH2Ph), 74.7 (C-5Ј), 74.8 (CH2Ph), 75.2 (C-3),
75.4 (C-4), 75.8 (C-4Ј), 76.3 (C-3Ј), 78.8 (C-2Ј), 83.5 (C-1), 100.6
(C-1Ј), 127.2, 127.2, 127.3, 127.8, 127.9, 128.0, 128.1, 128.1,
128.2, 128.3, 128.3, 128.9, 129.3, 129.5, 129.7, 133.0, 133.0 and
133.1 (ArCH), 138.1, 138.2, 138.5 and 138.7 (ArC) and 165.2,
165.9 and 166.2 (CO).
Ethyl 2,3-di-O-benzoyl-4,6-O-isopropylidene-1-thio-â-D-
galactopyranoside 16
To a solution of thioglycoside 15 (1.00 g, 4.46 mmol) in DMF
(9 cm3) were added a catalytic amount of TsOH (138.9 mg, 0.73
mmol) and 2,2-dimethoxypropane (2.18 cm3, 17.83 mmol).
After 90 min the reaction mixture was quenched by the addition
of triethylamine (1.3 cm3), evaporated under reduced pressure
and dried overnight under reduced pressure. The residue
was dissolved in pyridine (15 cm3), and benzoyl chloride (1.55
cm3, 13.39 mmol) was added dropwise at 0 ЊC. After 30 min
the reaction mixture was allowed to warm to rt and stirred
for a further 3.5 h. The reaction mixture was diluted with
ethyl acetate (50 cm3) and washed with water (3 × 50 cm3).
The organic layer was dried (MgSO4), and evaporated under
reduced pressure. Flash chromatography (5% EtOAc in
toluene) afforded the protected glycoside 16 (1.00 g, 50%) as a
foam (Found: C, 63.6; H, 5.9. C25H28O7S requires C, 63.57;
H, 5.92%); Rf 0.57 (toluene–EtOAc [7:3]); [α]D29 ϩ87.5 (c 1.15,
CHCl3); δH(250 MHz; CDCl3) 1.30 (3 H, t, J 7.4, SCH2CH3),
1.37 (3 H, s, CH3), 1.47 (3 H, s, CH3), 2.34–2.96 (2 H, m,
SCH2CH3), 3.58 (1 H, br s, H-5), 4.04–4.06 (2 H, m, H2-6), 4.58
(1 H, dd, J 0.8, 3.4, H-4), 4.66 (1 H, d, J 9.8, H-1), 5.28 (1 H, dd,
J 3.4 and 9.8, H-3), 5.90 (1 H, t, J 9.8, H-2), 7.22–7.50 (7 H, m,
ArH) and 7.95–8.00 (3 H, m, ArH); δC(100 MHz; CDCl3) 14.6
(SCH2CH3), 18.4 (CH3), 22.8 (SCH2CH3), 28.9 (CH3), 62.6
(C-6), 66.7 (C-4), 67.1 (C-5), 69.6 (C-2), 73.8 (C-3), 82.5 (C-1),
98.8 (CMe2), 128.0, 128.1, 128.8, 129.1, 129.4, 129.6 and 129.7
(ArCH), 132.9 and 131.3 (ArC) and 165.2 and 169.9 (CO).
6-(Benzyloxycarbonylamino)hexyl 2,3,4,6-tetra-O-benzyl-á-D-
galactopyranosyl-(1→4)-2,3,6-tri-O-benzoyl-1-thio-â-D-galacto-
pyranoside 19
To glycoside 18 (0.3 g, 0.28 mmol) were added benzyl 6-
hydroxyhexyl carbamate (0.31 g, 1.24 mmol), silver triflate
(1.07 g, 4.19 mmol) and activated molecular sieves (4 Å). The
mixture was dried under reduced pressure for 3 h and cooled to
0 ЊC. Dichloromethane (10 cm3) was added and a solution of
bromine (83 mm3 in CH2Cl2 [10 cm3]) was added dropwise
over a period of 10 min. The mixture was stirred for 2 h, the
suspension was filtered through a pad of Celite, and the filter
pad was washed with CH2Cl2 (3 × 30 cm3). The organic layer
was washed successively with saturated aq. NaHCO3 (3 × 30
cm3) and water (30 cm3), dried (MgSO4), and evaporated under
reduced pressure. Flash chromatography (10% EtOAc in
toluene) afforded glycoside 19 (0.28 g, 83%) as a foam, Rf 0.35
(PhMe–EtOAc [9:1]) (Found: C, 72.1; H, 5.95; N, 1.1.
C75H77NO16 requires C, 72.25; H, 6.09; N, 1.12%); [α]D27 ϩ54.7
(c 0.61, CHCl3); δH(400 MHz; CDCl3) 1.12–1.22 (6 H, m,
O[CH2]2CH2CH2CH2CH2NH), 1.40–1.53 (2 H, m, OCH2CH2-
[CH2]4NH), 2.88–2.91 (1 H, m, Ha-6Ј), 2.94–3.00 (1 H, m,
Hb-6Ј), 3.29–3.38 (1 H, m, Ha-6), 3.47–3.54 (1 H, m, OCH2-
Ethyl 2,3,6-tri-O-benzoyl-1-thio-â-D-galactopyranoside 17
To a solution of glycoside 16 (0.92 g) in THF (11 cm3) at 0 ЊC
were added water (2.5 cm3) and TFA (7 cm3). After 50 min
the reaction was quenched by addition of saturated aq.
NaHCO3 (4 cm3) and the product was extracted with EtOAc
(2 × 60 cm3). The combined organic layers were washed with
brine (20 cm3), dried (MgSO4), and evaporated under reduced
pressure. The residue was co-evaporated twice with toluene
and dried overnight under reduced pressure. The residue was
dissolved in DMF (15 cm3) and the solution was cooled
to Ϫ20 ЊC. Triethylamine (0.33 cm3) and benzoyl cyanide (0.24
cm3) were added and the mixture was stirred for 15 min at
Ϫ20 ЊC and for a further 20 min at 0 ЊC. The reaction was
quenched with MeOH (1 cm3). The resulting mixture was
diluted with EtOAc (50 cm3), washed successively with water
(20 cm3) and brine (20 cm3), dried (MgSO4), and evaporated
under reduced pressure. Flash chromatography (10% EtOAc in
J. Chem. Soc., Perkin Trans. 1, 1998
2293

View Article Online
[CH2]5NH), 3.90–4.08 (5 H, m, H-5, -2Ј, CH2Ph, OCH2-
[CH2]5NH), 4.10 (1 H, br s, H-4Ј), 4.18 (1 H, dd, J 2.5 and 10.2,
H-3Ј), 4.32–4.35 (1 H, m, H-5Ј), 4.40 (1 H, d, J 2.5, H-4), 4.45 (2
H, d, J 11.0, CH2Ph), 4.61 (1 H, m, NH), 4.66 (1 H, d, J 7.6, H-
1), 4.71–4.88 (7 H, m, Hb-6, 2 × CH2Ph, O[CH2]5CH2NH), 4.92
(1 H, d, J 3.4, H-1Ј), 5.07 (2 H, s, NHCOOCH2Ph), 5.22 (1 H,
dd, J 2.8 and 10.5, H-3), 5.74 (1 H, dd, J 7.6 and 10.6, H-2),
7.10–7.59 (35 H, m, ArH) and 7.89–8.05 (5 H, m, ArH); δC(100
MHz; CDCl3) 15.9 (CH2), 18.9 (CH2), 29.1 (CH2), 29.5 (CH2),
40.7 (CH2NH), 62.3 (CH2Ph), 66.4 (C-6), 67.3 (C-6Ј), 69.5
(C-5Ј), 69.6 (OCH2), 69.8 (C-2), 72.3 (CH2Ph), 72.5 (C-5), 72.7
(NHCO2CH2Ph), 73.8 (CH2Ph), 74.0 (C-3), 74.6 (C-4Ј), 74.8
(CH2Ph), 75.5 (C-4), 75.7 (C-2Ј), 78.8 (C-3Ј), 101.0 (C-1Ј),
101.4 (C-1), 127.2, 127.2, 127.3, 127.4, 127.8, 127.9, 128.1,
128.1, 128.2, 128.3, 128.3, 129.4, 129.5, 129.7, 132.9 and 133.0
(ArCH), 136.5, 138.1, 138.6 and 138.7 (ArC), 156.1 (CO2NH)
and 165.1, 165.9 and 166.3 (CO).
1 m), incubated on ice for 10 min, and pelleted at 10 000 g for
20 min. Columns of Gb3-Sepharose, Gb2-Sepharose and Gb3-
Fractogel (1 cm3) were equilibrated with NaCl solution (0.5 ) in
phosphate-buffered saline (PBS; NaCl, 137 m; KCl, 2.7 m;
Na2HPO4, 10.1 m; KH2PO4, 1.8 m; pH 7.4). Periplasm (3
cm3) was loaded on to each column. The columns were washed
with 40 column volumes of NaCl (0.5 ) in PBS. Fractions were
eluted from the columns with a stepwise gradient from 0 to 6 
of guanidinium chloride. Thus guanidinium chloride (pH 6.7,
0.5 cm3) was placed on the column and pumped through at 0.4
cm3 minϪ1. Fractions (1 cm3) were collected at 2.5 min intervals.
As the guanidinium chloride was taken into the column, it was
replaced by a second aliquot of 1  denaturant and so on in
steps of 0.5  denaturant up to 6 . Protein in the eluted
fractions was measured from the absorbance at 280 nm.
Fractions corresponding to peaks in the elution profile were
removed for refractometry analysis to determine the concen-
tration of guanidinium chloride by comparison with standard
curves. The remainder was dialysed against PBS. Aliquots
(10 mm3) from each sample were examined by sodium dodecyl
sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) with
silver staining to confirm the identity of the eluted protein.
6-Aminohexyl á-D-galactopyranosyl-(1→4)-â-D-galactopyrano-
side 20
To a solution of glycoside 19 (0.2 g, 0.16 mmol) in MeOH
(8 cm3) at rt was added MeONa (9 mg). The mixture was stirred
for 20 h, neutralised with Amberlyst 15 ion exchange resin,
filtered, and evaporated under reduced pressure, and the
residue was dried for 3 h under reduced pressure. A single spot
was visualised by TLC, Rf 0.67 (CH2Cl2–MeOH [15:1]). The
residue was dissolved in EtOH (5 cm3), 10% Pd(OH)2 on carbon
(0.18 g) and cyclohexene (76 mm3, 0.74 mmol) were
added, and the mixture was boiled under reflux for 1.75 h
before being allowed to cool to rt and filtered through a pad of
Celite. Purification by flash chromatography (20% aq. NH3 in
MeOH) afforded disaccharide glycoside 20 (49.1 mg, 70%)
(Found: C, 48.7; H, 7.7; N, 3.0. C18H35NO11 requires C, 49.01;
H, 7.93; N, 3.17%); Rf 0.21 (aq. NH3–MeOH [2:8]); δH(400
MHz; D2O) 1.36–1.39 (4 H, m, 2 × CH2), 1.60–1.61 (4 H, m,
2 × CH2), 2.92 (1 H, t, J 7.4, CH2NH2), 3.49 (1 H, dd, J 7.7 and
7.7, H-2), 3.61–3.92 (10 H, m, H-3, -5, -2Ј, -3Ј, H2-6, H2-6Ј,
OCH2), 3.99 (2 H, d, J 3.0, H-4 and -4Ј), 4.33 (1 H, dd, J ~5.6
and 5.6, H-5Ј), 4.41 (1 H, d, J 7.7, H-1) and 4.92 (1 H, d, J 3.8,
H-1Ј); δC(100 MHz; D2O) 25.3 (CH2), 25.9 (CH2) 27.6 (CH2),
29.2 (CH2), 40.1 (CH2NH), 60.7 (C-6 or -6Ј), 61.1 (C-6Ј or -6),
69.3 (C-2Ј), 69.6 (C-4Ј), 69.8 (C-3Ј), 71.0 (OCH2), 71.4 (C-5Ј),
71.6 (C-2), 73.1 (C-3), 75.7 (C-5), 77.7 (C-4), 100.8 (C-1Ј) and
103.5 (C-1).
Acknowledgements
We thank the Ciba Geigy Jubiläumsstiftung (to D. M.) and
the EPSRC for financial support, and the EPSRC Mass
Spectrometry Service, Swansea for mass spectra.
References
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galactopyranoside 20 to CNBr-activated Sepharose 4B
Freeze-dried CNBr-activated Sepharose 4B (1 g) was swollen
for 15 min in HCl (1 ; 200 cm3) and then was washed succes-
sively with HCl (1 m; 200 cm3) and aq. NaHCO3 (0.1 ,
pH 8.3; 5 cm3). The gel was immediately transferred into a
previously prepared solution of glycoside 20 (7 mg) in aq.
NaHCO3 (0.1 , pH 8.3; 10 cm3) and shaken (110 rpm) for
4 h at rt. The gel was transferred to a solution of ethanolamine
(0.1 ) in aq. NaHCO3 (0.1 , pH 8.3; 10 cm3) and the mixture
was shaken for 2 h. The blocking solution was removed by
filtration and the gel was washed successively with the following
solutions: aq. NaHCO3 (0.1  pH 8.3; 100 cm3), aq. NaOAc
(0.1 , pH 4.0; 200 cm3) and aq. NaHCO3 (0.1 , pH 8.3; 100
cm3) and stored in the last solution.
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of the lac promoter) in E. coli strain JM 105 was grown in 1 dm3
of Luria broth to an absorbance A of 0.6 at 600 nm. Cells
were induced for 3 h with a final concentration of isopropyl
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Paper 8/01429I
Received 19th February 1998
Accepted 12th May 1998
2294
J. Chem. Soc., Perkin Trans. 1, 1998