Identification and synthesis of a trisaccharide produced from lactose by transgalactosylation

Article abstract of DOI:10.1016/S0008-6215(99)00309-2

Enzymatic transgalactosylation of lactose by means of Streptococcus thermophilus, subspecies DN-001065, led to a mixture of D-galactose (~4%), D-glucose (~15%), lactose (~51%), minor disaccharides (6%), trisaccharides (~20%) and tetrasaccharides (3%). The major trisaccharide (~16%) was identified by NMR spectroscopy and chemical synthesis as being the known β-D-galactopyranosyl-(1→3)-β-D-galactopyranosyl-(1→4)-D-glucose (3'-β-D-galactopyranosyl-lactose). It was purified from a mixture of peracetylated oligosaccharides by column chromatography followed by deacetylation. For the first time, 3'-β-D-galactopyranosyl-lactose has been obtained on the 1 g scale, by resorting to simple techniques and equipment. NMR spectra have been unambiguously assigned. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(99)00309-2

www.elsevier.nl/locate/carres
Carbohydrate Research 325 (2000) 202–210
Note
Identification and synthesis of a trisaccharide produced
from lactose by transgalactosylation
Ve´ronique Perrin a, Bernard Fenet b, Jean-Pierre Praly a,*, Francis Lecroix c,
Chi Dung Ta d
a Uni6ersite´ Claude-Bernard Lyon I, CPE-Lyon, Baˆtiment 308, 43 boule6ard du 11 No6embre 1918,
F-69622 Villeurbanne, France
b Centre de RMN UCB-CPE, 43 boule6ard du 11 No6embre 1918, F-69622 Villeurbanne, France
c Ble´dina s.a., rue Re´mi Goetgheluck, F-59114 Steen6oorde, France
d Danone s.a., Centre de Recherches Jean The`6es, BP 16, 6 rue Edouard Vaillant, F-91207 Athis-Mons, France
Received 1 July 1999; revised 22 November 1999; accepted 24 November 1999
Abstract
Enzymatic transgalactosylation of lactose by means of Streptococcus thermophilus, subspecies DN-001065, led to
a mixture of
(ꢀ20%) and tetrasaccharides (3%). The major trisaccharide (ꢀ16%) was identified by NMR spectroscopy and
chemical synthesis as being the known b- -galactopyranosyl-(1“3)-b- -galactopyranosyl-(1“4)- -glucose (3%-b-
galactopyranosyl-lactose). It was purified from a mixture of peracetylated oligosaccharides by column chromatogra-
-galactopyranosyl-lactose has been obtained on the 1 g scale,
D
-galactose (ꢀ4%),
D
-glucose (ꢀ15%), lactose (ꢀ51%), minor disaccharides (6%), trisaccharides
D
D
D
D-
phy followed by deacetylation. For the first time, 3%-b-
D
by resorting to simple techniques and equipment. NMR spectra have been unambiguously assigned. © 2000 Elsevier
Science Ltd. All rights reserved.
Keywords: Transgalactosylation; b-
lactose
D
-Galactopyranosyl-(1“3)-b-
D
-galactopyranosyl-(1“4)-
D
-glucose; 3%-b-D-Galactopyranosyl-
1. Introduction
biocatalyzed transformations and molecular
recognition [1]. The fact that some oligosac-
charides are not metabolized by humans, but
are selectively fermented by bacteria having a
beneficial incidence on the health of humans
and animals, has led to the concept of Ecolog-
ical Health Control Products [2], and to the
development of gluco- [2], galacto- [3], and
fructo- [4] oligosaccharides. Along this line,
enzymatic modification of sucrose has wide
applications [5], in particular because glycosy-
lation employing biosystems opens new av-
Carbohydrates, because of their particular
structural features, comprise a vast number of
isomers that may have quite different proper-
ties in spite of apparent similarities. These
differences may be understood on the basis of
chemical reactivity and, particularly as far as
one considers life-related aspects, in terms of
* Corresponding author. Tel.: +33-472-431161; fax: +33-
478-898914.
E-mail address: jean-pierre.praly@univ-lyon1.fr (J.-P.
Praly)
enues
for
regio-
and
stereo-specific
preparations of complex oligosaccharides [6]
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00309-2

V. Perrin et al. / Carbohydrate Research 325 (2000) 202–210
203
of interest in food products [7]. Some of them,
such as the fructo-oligosaccharides Actilight,
are resistant to the action of human digestive
enzymes, so that they can reach the lower
digestive tract without being absorbed. Those
that can be utilized there by Bifidobacteria as
an energy source, thus favouring their prolif-
eration, are of particular interest. Bacteria of
this class contribute to maintaining the state
of the intestinal bacterial flora, which is con-
sidered as having a major impact on human
health. In humans, the dietetic benefits of
intestinal Bifidobacteria are as follows: (a)
maintenance of a normal intestinal balance;
(b) improvement of lactose tolerance and di-
gestibility of milk products; (c) antitumori-
genic activity; (d) reduction of serum
cholesterol levels; (e) synthesis of B-complex
vitamins and (f) enhanced absorption of di-
etary calcium [8,9]. For these reasons, galacto-
oligosaccharides, considered as promoters for
the growth of Bifidobacteria and Lactobacilli
in the large intestine, have been investigated
thoroughly [9,10], also in connection with the
modified viscosity of dairy produce [11]. An-
other reason for studying oligosaccharides
stems from attempts made by the industry to
bring the composition of infant formula closer
to that of human milk [12]. In this context, we
became involved in a project aiming at the
production of lactose-derived oligosaccharides
by enzyme-mediated transgalactosylation [13],
with the focus on the structural identification
and purification on the gram scale, for stan-
dard purposes, of the major trisaccharide
present in the crude mixture.
cused our attention on the predominant trisac-
charide with the following objectives: (i) its
isolation from the oligosaccharide mixture for
structure determination, (ii) its preparation on
the 1 g scale by either chemical synthesis or
separation from the raw mixture. Although
structural analyses of oligosaccharides rely on
both long-known [14] and continuously im-
proving methods [15] with the help of new
techniques in mass spectroscopy and high-field
NMR [16], we decided to tackle the problem
by applying methodologies familiar to the car-
bohydrate synthetic chemist, which might pos-
sibly allow the isolation of the desired
trisaccharide in significant amounts from the
mixture, with quite simple equipment.
To this end, the aqueous syrupy mixture
was first concentrated to dryness under dimin-
ished pressure before acetylation under stan-
dard conditions (acetic anhydride, pyridine)
and the resulting mixture of peracetylated
oligosaccharides was subjected to column
chromatography on silica gel with solvent
mixtures of increasing polarity. From the
weights of the collected fractions, the distribu-
tions of the different mono-, di-, tri-, and
tetrasaccharides were 23.8, 57.2, 17.7 and
1.3%, respectively. The fractions containing
the major peracetylated trisaccharide, visible
as a single spot on TLC plates, were pooled
and examined by NMR spectroscopy. As ex-
pected, the isolated material turned out to be
an anomeric mixture (a:b ratio ꢀ3:7). There-
fore, we preferred to achieve the transforma-
tion of 1a,b stereoselectively, via the
corresponding peracetylated bromide 2 [17]
into the O-methyl glycoside 3, taking advan-
tage of the anchimeric assistance of the O-
acetyl group at the C-2 position of the
2. Results and discussion
D-glucose unit. The trisaccharide 3, prepared
by this two-step sequence (HBr–AcOH, then
MeOH, AgOTf, sym-collidine) was a single
anomer, as shown, in particular, by the pres-
ence of one methoxy resonance at 3.48 ppm.
The same two-step protocol was applied to the
oligosaccharide mixture, which was therefore
converted, without purification of the interme-
diate bromides, to the corresponding peracetyl-
ated O-methyl glycosides. Their separation
by column chromatography on silica gel af-
forded homogeneous fractions, which were ac-
An aqueous mixture of oligosaccharides
(ꢀ35% w/w), obtained from lactose by Strep-
tococcus thermophilus-induced transgalacto-
sylation using DN-001065 subspecies [Collec-
tion Nationale de Cultures de Microorganis-
mes, Institut Pasteur, 25, rue du Docteur
Roux, F-75724 Paris, France], was shown by
HPLC to contain
D
-glucose (ꢀ15%) and
D-
galactose (ꢀ4%), lactose (ꢀ51%) and minor
disaccharides (ꢀ6%), various trisaccharides
(ꢀ21%) and tetrasaccharides (3%). We fo-

204
V. Perrin et al. / Carbohydrate Research 325 (2000) 202–210
curately weighted. The calculated distribution
of the O-methyl mono-, di-, tri-, and tetra-sac-
charides (21.3, 59.1, 16.4 and 3.2%) was simi-
lar to that found for the peracetates. The
observed fit supported the conclusion that the
chemical treatments applied were mild enough
so as to prevent any cleavage of O-glycosidic
bonds. Therefore, the structure of the ob-
tained methyl trisaccharide 3, giving NMR
spectra simpler than those of 1a,b, was deter-
mined, to deduce the sugar sequence in the
major trisaccharide precursor 4a,b.
proposed by Adel et al. [18] was modified so
as to include relays. Considering first the
anomeric proton H-1, the standard 1D COSY
experiment provided the antiphase multiplet
H-2 and allowed the measurement of the pas-
sive coupling constant. A new transfer period
was added to the 1D COSY sequence with a
transfer adjusted to 1/2J where J was equal to
J2,3. The success of this pulse sequence stems
from the fact that the COSY transfer can
occur in spite of the B1 Hz coupling between
H-4 and H-5 in the
D
-galacto units. Hence,
this technique, which does not require 13C-la-
belled molecules [19], appears to be a method
of choice for polysaccharide structural deter-
mination when superimposed signals cannot
be distinguished by classical 2D NMR tech-
niques. Afterwards,
a
classical gradient
HMBC sequence was applied so as to achieve
the sequencing of the sugar units by establish-
ing unambiguously which carbon atoms were
engaged in the glycosidic bonds. Each pyra-
1H and 13C NMR (1D, 2D) spectroscopy
led to the conclusion that the isolated per-
acetylated O-methyl trisaccharide 3 was
4
nosyl unit in 3 was found in the C1-D chair
conformation and the stereochemistry of the
three glycosidic bonds was established as b on
the basis of the ꢀ8 Hz vicinal couplings
observed for each of the anomeric protons.
Therefore, the major enzymatically produced
methyl (2,3,4,6-tetra-O-acetyl-b-
ranosyl)-(1“3)-(2,4,6-tri-O-acetyl-b-
topyranosyl)-(1“4)-2,3,6-tri-O-acetyl-b-
glucopyranoside. The presence of one -glu-
copyranose unit and two -galactopyranose
D-galactopy-
D
-galac-
D
-
D
D
1
trisaccharide 4 was likely to be b-
ranosyl-(1“3)-b- -galactopyranosyl-(1“4)-
-glucose (3%-b- -galactopyranosyl-lactose).
D-galactopy-
units, which was suggested by the H NMR
spectrum, was confirmed by COSY, 1D
COSY with multistep relays, HSQC and
HMBC experiments. The 2D gradient COSY
experiment allowed a complete assignment of
D
D
D
From the isolated amount of the peracetylated
O-methyl trisaccharide 3, the proportion of
compound 4 in the mixture was estimated to
be ꢀ16%.
the protons of the
However, this task was not straightforward
for the two -galactose units due to the small
D
-glucopyranose unit.
The chemical synthesis of 4 (Scheme 1) was
attempted following a known procedure [20],
D
coupling constant between their H-4 and H-5
protons and because of the similar chemical
using 2,3,4,6-tetra-O-acetyl-a-
nosyl bromide (5) [17], and benzyl 4-O-(2,6-di-
O-acetyl-b- -galactopyranosyl)-2,3,6-tri-O-
acetyl-b- -glucopyranoside (7) [20]. However,
D-galactopyra-
shifts of the H-4 proton in each
D-galacto
D
unit. In the sugar series, the 1D TOCSY ex-
periment is generally initiated by selective ex-
citation of the anomeric proton, allowing a
complete assignment of the spin system for
long spin-lock times. However, in the present
case, the magnetization was not transferred
D
in our hands, the glycosidation step was found
to be unsatisfactory. Therefore, 2,3,4,6-tetra-
O-acetyl-a- -galactopyranose trichloroacetim-
D
idate (6) [21], prepared from the corre-
sponding peracetates by selective 1-O-deacetyl-
ation [22], was envisaged as the glycosyl
donor. Coupling of 6 and 7 afforded trisac-
charide 8 (28% yield), which was converted to
9 by acetylation. In spite of the enhanced
reactivity of the equatorial hydroxy group at
from H-4 to H-5 in the
D-galacto units, prob-
ably because of a competition between
TOCSY and ROESY effects. Fortunately, a
set of multistep relayed 1D COSY experi-
ments was successfully carried out (see Fig. 1).
The pulse sequence used, derived from that

V. Perrin et al. / Carbohydrate Research 325 (2000) 202–210
205
1
Fig. 1. The H NMR spectrum of 3 is shown, in part, at the top. The selectively excited H-1% and H-1%% protons of the
D
-Galp
units are indicated by rectangles with dashed and normal lines. The corresponding 1D relayed COSY set of spectra, which are
respectively represented below, in dashed and normal frames, show, successively, the transfer of magnetization from the anomeric
proton to the ring protons, in each unit.

206
V. Perrin et al. / Carbohydrate Research 325 (2000) 202–210
Scheme 1.
C-3% in 7, under homogeneous glycosidation
conditions [23,24], the coupling was not effi-
cient, as observed recently by others [25].
Compound 4 was obtained from 9 in two
high-yielding steps by deacetylation and hy-
drogenolysis, as previously described [20].
Finally, since the chemical synthesis of 4
required optimization, separation of the mix-
ture of peracetylated oligosaccharides was car-
ried out by column chromatography with
mobile phases of increasing polarity. Pure
compound 1a,b was subjected to deacetylation
(MeOH, H2O, NEt3) to yield quantitatively 4
as a white crystalline material (ꢀ1 g) [20].
The NMR data obtained for 4 were identical
(ꢀ3%). The trisaccharide fraction mainly con-
-galactopyranosyl-lac-
tains the known 3%-b-
D
tose, not mentioned in related investigations
[9,11]. This compound results from preferential
transfer of a b-
lactose [10], which was also reported to enzy-
matically undergo transfer of b- -Galp unit to
the 4% [3,9,29], and 6% [9,30] positions, mainly
[10]. For the first time, 3%-b- -galactopyra-
D
-Galp unit to the 3% position of
D
D
nosyl-lactose has been obtained on the 1 g
scale, by resorting to simple techniques and
equipment, and previous assignments, based
on comparison, of its 13C NMR spectrum have
been unambiguously confirmed.
to that reported for 3%-b- -galactopyranosyl-
D
lactose, isolated from the milk of the tammar
wallaby and the grey kangaroo [16,26–28],
and found as a component of human milk
[12].
In conclusion, our work unambiguously es-
tablishes that S. thermophilus-induced modifi-
cations of lactose, using subspecies DN-
3. Experimental
General methods.—Melting points were de-
termined with a Bu¨chi capillary apparatus and
were not corrected. Optical rotations were de-
termined with a Perkin–Elmer 241 polarime-
1
ter. H and 13C NMR spectra were recorded
001065, lead to mixtures containing
D
-galac-
tose (ꢀ4%) and -glucose (ꢀ15%), lactose
D
with Bruker AC 200/AM 300/DRX500 instru-
ments for solutions in CDCl3, with Me4Si as
the internal reference or in D2O. Reactions
and minor disaccharides (ꢀ57% altogether),
trisaccharides (ꢀ20%) and tetrasaccharides

V. Perrin et al. / Carbohydrate Research 325 (2000) 202–210
207
6:2:7 v/v) and 1:1 EtOAc–CH2Cl2 to give
mixtures of peracetylated monosaccharides
(Rf ꢀ0.73 in 6:2:7 EtOAc–petroleum ether–
CH2Cl2, 106 mg, ꢀ24%), and disaccharides
(Rf ꢀ0.53, 255 mg, ꢀ57%), trisaccharides (Rf
ꢀ0.16, 79 mg, ꢀ18%), and tetrasaccharides
(Rf ꢀ0.04, 6 mg, ꢀ1.3%).
were monitored by TLC on Silica Gel 60 F254
(E. Merck) plates exposed to H2SO4 (10% in
1:1 EtOH–water) spray followed by charring
(ꢀ250 °C). Spraying successively a fluores-
cein solution in abs EtOH (0.1% w/v), then a
1:1 mixture of H2O2 (30% in water)–AcOH
followed by charring was used to detect
bromine-containing compounds, which ap-
peared as pink-coloured spots. Column chro-
matography was performed using Silica Gel
Geduran Si 60 (E. Merck). Solvents were dis-
tilled before use. Water was distilled twice.
Enzymatic transgalactosylation of lactose.—
The mixture of trans-galacto-oligosaccharides
was produced by Ble´dina s.a. (Steenvoorde,
France). Enzyme production was based on a
reported procedure [31], using S. thermophilus
strains [13], subspecies DN-001065 [Collection
Nationale de Cultures de Microorganismes,
Institut Pasteur, 25, rue du Docteur Roux,
F-75724 Paris, France] grown for ꢀ6 h at
ꢀ50 °C. Then, an aq concd soln of lactose
was incubated with the obtained culture
medium. After centrifugation for removal of
Streptococcus, the aq mixture of oligosaccha-
rides (ꢀ35% w/w) was shown by HPLC (25
cm×4.6 mm Supercosil LC NH2 5 m column,
3:1 CH3CN–water as the mobile phase) to
Methyl (2,3,4,6-tetra-O-acetyl-i-
topyranosyl)-(1“3)-(2,4,6-tri-O-acetyl-i-
galactopyranosyl)-(1“4)-2,3,6-tri-O-acetyl-
i- -glucopyranoside (3).—To a solution of a
D-galac-
D
-
D
dried mixture of oligosaccharides (833 mg) in
Ac2O (4.2 mL) was added at rt 33% HBr in
AcOH (0.83 mL). After stirring for 22 h at rt,
another portion of HBr in AcOH (4.2 mL)
was added and stirring was maintained for 9 h
at rt. The mixture was concd under reduced
pressure to a syrup from which traces of Ac2O
were removed by co-evaporation with toluene
[17]. The residue (1.7 g) was dissolved in
MeOH (5 mL) and the solution, cooled to
0 °C was treated successively with AgOTf (687
mg, 2.67 mmol) and sym-collidine (0.29 ml,
2.2 mmol). After stirring for 5 h, the mixture,
quenched by adding satd aq Na2CO3 and
NaCl solns (10 mL each), was filtered, after
addition of CH2Cl2 (15 mL) to dissolve the
organic materials. The organic phase was
washed with water (3×15 mL), dried with
Na2SO4 and evaporated. The crude material
(1.57 g) was purified by chromatography with
a column (length: ꢀ50 cm; external diameter:
25 mm) eluted with EtOAc–petroleum ether–
CH2Cl2 mixtures (gradient: 4:3:7“7:2:6, v/v)
to give peracetylated methyl glycosides:
monosaccharides (Rf ꢀ0.71 in 7:2:6 EtOAc–
petroleum ether–CH2Cl2, 118 mg, ꢀ21%),
disaccharides (Rf ꢀ0.50, 328 mg, ꢀ59%),
trisaccharide 3 (Rf ꢀ0.17, 91 mg, ꢀ16%), and
tetrasaccharides (Rfꢀ0.02, 18 mg, ꢀ3%).
3: Mp 164–165 °C; [h]D −5.4° (c 1, CHCl3).
1H NMR (300.13 MHz, CDCl3): l 5.36 (dd, 2
H, J3%,4%=J3%%,4%% 3.4, J4%,5%=J4%%,5%% 0.8 Hz, H-4%
and H-4%%), 5.18 (dd, 1 H, J2,3 9.3, J3,4 9.2 Hz,
H-3), 5.09 (dd, 1 H, J1%,2% 8.0, J2%,3% 10.2 Hz,
H-2%), 5.06 (dd, 1 H, J1%%,2%% 7.8, J2%%,3%% 10.6 Hz,
H-2%%), 4.92 (dd, 1 H, H-3%%), 4.88 (dd, 1 H, J1,2
7.9 Hz, H-2), 4.52 (d, 1 H, H-1%%), 4.46 (dd, 1
H, J5,6a 2.0, J6a,6b 11.9 Hz, H-6a), 4.39 (d, 1 H,
H-1), 4.34 (d, 1 H, H-1%), 4.18 (dd, 1 H, J5%%,6%%a
6.1, J6%%a,6%%b 11.2 Hz, H-6%%a), 4.14–4.11 (m, 3
contain
D
-glucose (ꢀ15%) and
D-galactose
(ꢀ4%), lactose (ꢀ51%) and minor disaccha-
rides (ꢀ6%), various trisaccharides (21%) and
tetrasaccharides (3%). The major tri-, and te-
trasaccharides amounted to ꢀ18 and 1.5%,
respectively.
(2,3,4,6-Tetra-O-acetyl-i-
syl)-(1“3)-(2,4,6-tri-O-acetyl-i-
pyranosyl)-(1“4)-1,2,3,6-tetra-O-acetyl-
D
-galactopyrano-
D
-galacto-
D-
glucopyranose (1).—After concn to dryness of
the aq mixture of oligosaccharides resulting
from enzymatic modification of lactose, an
aliquot (1.19 g) in pyridine (3 mL, 37.1 mmol)
and Ac2O (5.5 mL, 58.3 mmol) was stirred for
4 days at rt. Excess Ac2O was quenched by
addition of ice–water (30 mL). The aq layer
was extracted with CH2Cl2 (3×20 mL); the
organic phase was washed with water (3×30
mL), dried (Na2SO4) and concd. The crude
material was purified by chromatography with
a column (length: ꢀ50 cm; external diameter:
35 mm) eluted with mixtures of EtOAc–
petroleum ether–CH2Cl2 (gradient: 3:3:7“

208
V. Perrin et al. / Carbohydrate Research 325 (2000) 202–210
chromatography on silica gel (1:1 CH2Cl2–
H, H-6b, H-6%a, H-6%%b), 4.03 (dd, 1 H, J5%,6%b
6.7, J6%a,6%b 11.5 Hz, H-6%b), 3.86 (ddd, 1 H,
J5%%,6%%b 7.1 Hz, H-5%%), 3.78 (dd, 1 H, H-3%),
3.79–3.76 (m, 2 H, H-5%, H-4), 3.60 (ddd, 1 H,
J4,5 7.4, J5,6b 4.9 Hz, H-5), 3.48 (s, 3 H,
OCH3), 2.17, 2.13, 2.13, 2.10, 2.08, 2.06, 2.04,
Et2O) to yield benzyl (2,3,4,6-tetra-O-acetyl-b-
D
-galactopyranosyl)-(1“3)-(2,6-di-O-acetyl-
b -
acetyl-b-
D
- galactopyranosyl) - (1“4) - 2,3,6 - tri - O-
-glucopyranoside (8) (220 mg, 28%)
D
[20]: 13C NMR (75.47 MHz, CDCl3): l 170.82,
170.61, 170.33, 170.18, 170.12, 170.00, 169.63,
169.31, 168.62 (CꢀO), 136.75, 128.47, 128.03,
127.75 (phenyl), 101.55 (C-1), 100.57 (C-1%%),
99.19 (C-1%), 79.62 (C-3%), 75.68 (C-4), 72.89
(C-5), 72.45 (C-3), 72.00 (C-2), 71.60 (C-5%),
71.06 (C-2%), 70.77 (CH2Ph), 70.63 (C-5%%),
70.52 (C-3%%), 68.38 (C-4%), 68.20 (C-2%%), 66.84
(C-4%%), 62.71 (C-6), 62.31 (C-6%), 61.19 (C-6%%),
20.87, 20.82, 20.75, 20.69, 20.67, 20.63, 20.60,
20.56, 20.54 (OCOCH3).
13
2.03, 2.02, 1.96 (10 s, 30 H, acetyl). C NMR
(75.47 MHz, CDCl3): l 170.52, 170.37, 170.33,
170.14, 169.81, 169.75, 169.67, 169.16, 168.51
(CꢀO), 101.33 (C-1), 100.97 (C-1%%), 100.70
(C-1%), 75.87 (C-4), 75.66 (C-3%), 72.64 (C-5),
72.52 (C-3), 71.49 (C-2), 71.25 (C-5%), 70.90
(C-2%), 70.72 (C-5%%), 70.55 (C-3%%), 68.49 (C-4%),
68.44 (C-2%%), 66.63 (C-4%%), 62.08 (C-6), 61.63
(C-6%), 60.84 (C-6%%), 57.00 (OCH3), 20.84,
20.73, 20.70, 20.68, 20.65, 20.65, 20.63, 20.58,
20.49, 20.49 (OCOCH3). Anal. Calcd for
C39H54O26 (938.84): C, 49.89; H, 5.80; O,
44.31. Found: C, 49.93; H, 5.95; O, 44.33.
A mixture of peracetylated tri- and tetra-
saccharides (275 mg) was treated with Ac2O
(1.4 mL) and 30% HBr in AcOH (1.7 mL) for
8 h at rt to give a mixture of the correspond-
ing bromides as a gum (296 mg). This was
reacted for 5 h at 0 °C with MeOH (0.6 mL)
in the presence of silver triflate (84 mg) and
sym-collidine (35 mL). Trisaccharide 3 (127
mg) was obtained by column chromatogra-
phy, as before.
The product 8 (150 mg), after acetylation
with Ac2O (1.1 mL) and pyridine (0.75 mL)
was chromatographed on silica gel (3:1 then
2.5:1.5 CH2Cl2–Et2O) to give 9 (111 mg, 71%)
1
[20]: H NMR (300.13 MHz, CDCl3): l 7.34–
7.25 (m, 5 H, Ph), 5.35 (d, 2 H, J3%,4%=J3%%,4%% 3.2
Hz, H-4% and H-4%%), 5.14 (dd, 1 H, J2,3 9.3, J3,4
9.1 Hz, H-3), 5.08 (dd, 1 H, J1%,2% 8.0, J2%,3% 10.1
Hz, H-2%), 5.05 (dd, 1 H, J1%%,2%% 7.8, J2%%,3%% 10.3
Hz, H-2%%), 4.96 (dd, 1 H, J1,2 7.9 Hz, H-2),
4,91 (dd, 1 H, H-3%%), 4.86 (d, 1 H, J 12.3 Hz,
CH2Ph), 4.59 (d, 1 H, J 12.3 Hz, CH2Ph), 4.51
(d, 1 H, H-1%%), 4.48 (d, 1 H, H-1%), 4.46 (m, 1
H, H-6a), 4.34 (d, 1 H, H-1), 4.16 (dd, 1 H,
J5%%,6%%a 6.1, J6%%a,6%%b 11.1 Hz, H-6%%a), 4.14–4.06
(m, 3 H, H-6b, H-6%a, H-6%%b), 4.02 (dd, 1 H,
J5%,6%b 6.7, J6%a,6%b 11.2 Hz, H-6%b), 3.86 (broad t,
1 H, J5%%,6%%b 6.8 Hz, H-5%%), 3.80–3.73 (m, 3 H,
H-3%, H-4, H-5%), 3.60 (ddd, 1 H, J4,5 7.6, J5,6a
1.9, J5,6b 5.2 Hz, H-5), 2.16, 2.15, 2.12, 2.08,
2.07, 2.06, 2.02, 2.01, 1.99, 1.96 (10 s, 30 H,
acetyl). 13C NMR (75.47 MHz, CDCl3): l
170.55, 170.55, 170.39, 170.37, 170.16, 169.85,
169.82, 169.60, 169.20, 168.54 (CꢀO), 136.69,
128.48, 128.05, 127.75 (phenyl), 101.07 (C-1),
100.72 (C-1%%), 99.09 (C-1%), 75.91 (C-4), 75.79
(C-3%), 72.82 (C-5), 72.59 (C-3), 71.38 (C-2),
71.02 (C-5%), 70.86 (C-2%), 70.77 (CH2Ph),
70.72 (C-5%%), 70.65 (C-3%%), 68.63 (C-4%), 68.58
(C-2%%), 66.78 (C-4%%), 62.22 (C-6), 61.76 (C-6%),
60.96 (C-6%%), 20.89, 20.78, 20.73, 20.72, 20.68,
20.68, 20.63, 20.61, 20.53, 20.53 (OCOCH3).
Benzyl
topyranosyl)-(1“3)-(2,4,6-tri-O-acetyl-i-
galactopyranosyl)-(1“4)-2,3,6-tri-O-acetyl-
i- -glucopyranoside (9) [20].—To a solution
of benzyl 4-O-(2,6-di-O-acetyl-b- -galactopyr-
anosyl)-2,3,6-tri-O-acetyl-b- -glucopyrano-
side (7) [20] (514 mg, 0.8 mmol) in anhyd
(2,3,4,6-tetra-O-acetyl-i-
D
-galac-
D-
D
D
D
,
CH2Cl2 (15 mL) were added 4 A molecular
sieves (415 mg) and 2,3,4,6-tetra-O-acetyl-a-
D-
galactopyranosyl trichloroacetimidate (6) [21]
(591 mg, 1.2 mmol) and stirring was continued
for 1 h at rt. The mixture was cooled to
−18 °C and treated with Me3SiOTf (15 mL,
0.08 mmol). After 5 h at −18 °C, the reaction
was quenched by addition of a satd aq
NaHCO3 soln (10 mL) before warming up to
rt. After separation of the aq layer, it was
extracted with CH2Cl2 (3×10 mL). The com-
bined organic layers were washed with water
until neutral and dried (Na2SO4). After con-
centration, the residue was purified by column
(length: ꢀ50 cm; external diameter: 25 mm)
i-
topyranosyl-(1“4)-
chemical synthesis: a solution of 9 (101 mg) in
D
-Galactopyranosyl-(1“3)-i-
D
-galac-
D
-glucose (4).—(a) By

V. Perrin et al. / Carbohydrate Research 325 (2000) 202–210
209
spectra were identical to those described
above. Anal. Calcd for C18H32O16·H2O
(522.45): C, 41.38; H, 6.56; O, 52.06. Found:
C, 41.28; H, 6.75; O, 51.78.
MeOH (5 ml) containing a catalytic amount
of NaOMe was stirred for 48 h at rt. The soln
was evaporated under reduced pressure and
the residue was dissolved in water. The aq
soln was then neutralized with Amberlite
IRC-50 (H+) and evaporated under reduced
pressure to give benzyl b-
D
-galactopyranosyl-
Acknowledgements
(1“3)-b- -galactopyranosyl-(1“4)-b-
D
D-glu-
copyranoside (60 mg, 100%). A solution of
this compound (60 mg) in 80% aq EtOH (26
mL) was hydrogenolysed in the presence of
Pd/C (10%) for 24 h. The catalyst was re-
moved by filtration, and the solvent was evap-
orated under reduced pressure to yield 4 (50
mg, 98%, a/b ratio ꢀ35:65). 1H NMR
(300.13 MHz, D2O) [12]: l 5.23 (d, 1 H, J1,2
4.7 Hz, H-1 a anomer), 4.68 (d, 1 H, J1,2 7.9
Hz, H-1 b anomer), 4.62 (d, 1 H, J1,2 7.1 Hz),
4.52 (d, 1 H, J1,2 7.6 Hz), 4.2–3.5 (m, 18 H).
13C NMR (75.47 MHz, D2O): l 104.88 (C-1%%),
103.12 (C-1%), 96.34 (C-1 b-anomer), 92.39
(C-1 a anomer), 82.45 (C-3%), 78.93 (C-4 a
anomer), 78.81 (C-4 b anomer), 75.62 (C-5%%),
75.54 (C-5%), 75.34 (C-5 b anomer), 74.92 (C-3
b anomer), 74.40 (C-2 b anomer), 73.09 (C-
3%%), 71.97 (C-2 a anomer), 71.74 (C-3 a
anomer), 71.60 (C-2%%), 70.75 (C-2%), 70.65 (C-5
a anomer), 69.15 (C-4%%), 68.98 (C-4%), 61.53
(C-6%, C-6%%), 60.69 (C-6 b anomer), 60.57 (C-6
a anomer). The assignment of the carbon
This research was supported by a contract
with Danone s.a. (contract CNRS-Danone no.
0711255).
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D
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