The synthesis of 2-, 3-, 4- and 6-O-α-d-glucopyranosyl-d-galactopyranose, and their evaluation as nutritional supplements for pre-term infants

Article abstract of DOI:10.1016/j.carres.2007.04.022

Four methods have been screened for the synthesis of some α-d-glucopyranosides, with the recently reported (Mukaiyama) combination of 2,3,4,6-tetra-O-benzyl-α-d-glucopyranosyl iodide and triphenylphosphine oxide being the most successful, especially in th

Full text of DOI:10.1016/j.carres.2007.04.022

Carbohydrate Research 342 (2007) 1793–1804
The synthesis of 2-, 3-, 4- and
6-O-a-D-glucopyranosyl-D-galactopyranose, and their evaluation
as nutritional supplements for pre-term infants
Peter J. Meloncelli,a Tracey M. Williams,b Peter E. Hartmannb and Robert V. Sticka,*
aChemistry M313, School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia,
Crawley, WA 6009, Australia
bBiochemistry M310, School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia,
Crawley, WA 6009, Australia
Received 30 March 2007; received in revised form 20 April 2007; accepted 25 April 2007
Available online 1 May 2007
Abstract—Four methods have been screened for the synthesis of some a-D-glucopyranosides, with the recently reported (Mukai-
yama) combination of 2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl iodide and triphenylphosphine oxide being the most successful,
especially in the diastereoselectivity exhibited. The a-D-glucopyranosides so obtained have been deprotected to yield 2-, 3-, 4-
and 6-O-a-D-glucopyranosyl-D-galactopyranose. Only the last disaccharide showed any hydrolysis by a-glycosidases but this success
was not emulated by mucosal extracts from the small intestine of the pig.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Disaccharides; Synthesis; Diastereoselectivity; Halide catalysis; Lactose; Supplement; Nutritional; Infants; Pre-term
1. Introduction
nately, these supplements provide the infant with only
D-glucose, which often elicits a diabetic-like response.12
It occurred to us that an alternative source of carbo-
hydrate for pre-term infants might be found in a diaste-
reoisomer of lactose, namely 4-O-a-D-galactopyranosyl-
D-glucopyranose; perhaps the enzymes present in the
small intestine could accommodate and hydrolyze the
a-D-galactosidic linkage.13 An even better design would
be 4-O-a-D-glucopyranosyl-D-galactopyranose, a mole-
cule that should be stable in aqueous solution, capable
of hydrolysis by enzymes present in the small intestine,
and still able to provide the necessary D-glucose and
D-galactose for the growing infant. It should be noted
here that we never considered a disaccharide based so-
lely on D-glucose as it is well recognized, though perhaps
not completely understood, that D-galactose is an essen-
tial nutrient for development in the young.14–16
Lactose (4-O-b-D-galactopyranosyl-D-glucopyranose) is
the disaccharide present in mammalian milk and,
through the action of lactase, is digested by the infant
to form D-galactose and D-glucose necessary for healthy
development.1 Pre-term infants lack the levels of lactase
to utilize lactose and so must be supplemented with
other sources of carbohydrate.2–6 The direct feeding of
monosaccharides is not possible as the subsequent in-
crease in osmolality results in diarrhoea in the infant.7,8
One of the present solutions is to provide nutritional
carbohydrate in the form of maltodextrins, for example,
Polyjoule (D-glucopyranose linked mainly a-(1!4)).9
Pre-term infants obviously possess high enough levels
of enzymes (the glucoamylase–maltase complex) that
are able to cleave a-D-glucosidic linkages.10,11 Unfortu-
This paper therefore describes various methods for
the synthesis of 4-O-a-D-glucopyranosyl-D-galactopyra-
nose, as well as the regioisomeric 2-, 3- and 6-O-a-D-
glucopyranosyl-D-galactopyranose. The paper concludes
*
Corresponding author. E-mail: rvs@chem.uwa.edu.au
0008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.04.022

1794
P. J. Meloncelli et al. / Carbohydrate Research 342 (2007) 1793–1804
with an investigation of the four isomers as potential
candidates for use as nutritional supplements for pre-
term infants.
With the failure of alcohols 3 and 4 to undergo glycos-
ylation under the conditions of halide catalysis, we
turned our attention to the more common methods of
glycosylation, namely those employing trichloroacetimi-
dates and thioglycosides. Thus, treatment of b-D-gluco-
pyranosyl trichloroacetimidate 9 (Scheme 3) with
alcohols 2–5 under standard conditions20 gave disaccha-
ride derivatives 10–13 (Scheme 4; Table 1).
2. Discussion
2.1. Synthesis of targets
Treatment of thioglycoside 14 with alcohols 2–5 under
conditions described by Crich21 and by van Boom22
again gave disaccharide derivatives 10–13 (Table 2).
At this stage of our work we noted two publications
by Mukaiyama and Kobashi that announced even fur-
ther improvements to the halide catalysis protocol,
whereby triphenylphosphine oxide is added to the a-gly-
cosyl iodide before the addition of the acceptor.23,24 This
combination purportedly forms a very reactive b-phos-
phonium ion, ensuring high a-selectivity in the subse-
quent glycosylation step; the phosphine oxide also acts
The synthesis of the a-D-glycosidic linkage has occupied
chemists for years, from Fischer’s early successes with
simple alcohol acceptors,17 to the seminal work on
‘halide catalysis’ by Lemieux.18 As new glycosyl donors
were developed (trichloroacetimidates, thioglycosides),
so were they applied to the synthesis of the a-D-linkage,
with the common characteristic being the presence of a
non-participating protecting group at O2. We were nat-
urally attracted to the halide catalysis method and noted
the recent significant improvements made by Lam and
Gervay-Hague.19 Therefore, we treated 6-O-acetyl-
2,3,4-tri-O-benzyl-a-D-glucopyranosyl iodide 1 with the
four D-galacto acceptors 2–5 (Scheme 1). Only alcohols
2 and 5 were sufficiently reactive, forming a-D-gluco-
sides 6 and 7, respectively (the actual a/b ratio was
97:3 in both instances), together with significant
amounts of alkene 8 (Scheme 2). The formation of this
alkene necessitated the use of an excess of donor 1 and
generally detracted from the method.
BnO
BnO
BnO
O
O
BnO
OC(NH)CCl3
SPh
BnO
BnO
BnO
BnO
9
14
Scheme 3.
Ph
O
OH
O
BnO
HO
HO
BnO
AcO
OBn
O
OBn
O
O
O
O
O
BnO
BnO
O
O
O
BnO
BnO
BnO
AllO
I
OCH3
OCH3
HO
OCH3
1
2
3
4
5
Scheme 1.
Ph
O
AcO
BnO
O
O
O
O
a
+
+
1
2
AllO
BnO
OCH3
BnO
OBn
BnO
BnO
O
8
AcO
6
OBn
BnO
OBn
OAc
O
O
O
b
+
+
1
5
8
7
O
O
O
O
i
i
˚
˚
Scheme 2. Reagents: (a) Bu4NI, EtPr2N, 4 A ms, benzene, 70%; (b) Bu4NI, EtPr2N, 4 A ms, benzene, 90%.

P. J. Meloncelli et al. / Carbohydrate Research 342 (2007) 1793–1804
1795
Ph
OBn
BnO
BnO
OBn
OBn
O
BnO
O
OBn
O
O
O
BnO
BnO
O
O
O
O
O
BnO
BnO
O
OBn
O
AllO
BnO
O
BnO
BnO
BnO
OCH3
O
OCH3
O
BnO
O
O
BnO
BnO
BnO
BnO
O
OCH3
BnO
10
11
12
13
Scheme 4.
Table 1. Glycosylation of alcohols 2–5 with trichloroacetimidate 9 and
Table 3. Glycosylation of alcohols 2–5 with iodide 15 and triphenyl-
trimethylsilyl trifluoromethanesulfonate in ether
phosphine oxide in chloroform
Acceptor
Product
Yield (%)
a:b
Acceptor
Product
Yield (%)
a:b
2
3
4
5
10
11
12
13
82
72
72
71
85:15
75:25
65:35
80:20
2
3
4
5
10
11
12
13
70
86
57
90
90:10
95:5
93:7
94:6
graphy, the diastereoselectivity was again initially deter-
mined by 1H NMR spectroscopy, and subsequently
confirmed by debenzylation and acetylation of the reac-
tion mixture, which facilitated the separation of the a-
and b-anomers; for 13, where the anomers were again
inseparable by chromatography, the diastereoselectivity
was determined by direct analysis of the 1H NMR
spectrum.
From an operational point of view, we chose to pre-
pare disaccharides 6 and 7 using Gervay-Hague’s halide
catalysis protocol, and disaccharides 11 and 12 using the
trichloroacetimidate method. For the latter method, it
was always possible, at some stage, to separate the de-
sired a-D-anomer from the smaller amount of contami-
nating b-D-anomer. In fact, having the pure undesired
anomer helped to confirm the structure of the a-D-ano-
mer. Finally, disaccharide derivatives 6, 11, 12 and 7
were converted into the target disaccharides by simple
deprotection procedures (Schemes 6–9).
Table 2. Glycosylation of alcohols 2–5 with thioglycoside 14 in
dichloromethane, promoted by either 1-benzenesulfinylpiperidine (A)
or diphenyl sulfoxide (B), in combination with 2,4,6-tri-tert-butylpyr-
imidine and trifluoromethanesulfonic anhydride
Acceptor
Product
Yield (%)
a:b
A
B
A
B
2
3
4
5
10
11
12
13
71
65
74
65
76
75
85
85
45:55
70:30
70:30
35:65
45:55
75:25
75:25
40:60
as a weak base to neutralize the liberated hydrogen
iodide (Scheme 5). Treatment of 2,3,4,6-tetra-O-benz-
yl-a-D-glucopyranosyl iodide 15 with alcohols 2–5 in
the presence of triphenylphosphine oxide again formed
disaccharide derivatives 10–13 in good yield but also
with high diastereoselectivity (Table 3).
Although glycosyl iodide 15 is a little more tedious to
prepare and handle,25 it is more reactive than its coun-
terpart 1; also, the near neutral reaction conditions
avoided the formation of any alkene by-product.
The diastereoselectivity for the formation of 6, 7 and
2.2. Evaluation of 2-, 3-, 4- and 6-O-a-D-glucopyranosyl-
D-galactopyranose as nutritional supplements for pre-term
infants
1
10 was determined from an analysis of the H NMR
spectrum of the reaction mixtures, and confirmed for
10 by isolation of the a- and b-anomers; for 11 and
12, where the anomers were inseparable by chromato-
With the four synthetic disaccharides in hand, we were
now in a position to examine their hydrolysis with a range
of glycoside hydrolases. Initially, we chose commercially
BnO
BnO
BnO
BnO
BnO
Ph3PO
ROH
O
O
O
BnO
BnO
HOPPh3I
+
BnO
OPPh3I
BnO
BnO
BnO
BnO
OR
I
15
Scheme 5.

1796
P. J. Meloncelli et al. / Carbohydrate Research 342 (2007) 1793–1804
HO
AcO
OH
O
OAc
O
AllO
BnO
AcO
BnO
a
b
c
O
O
6
OCH3
OCH3
BnO
BnO
BnO
BnO
O
O
AcO
AcO
AcO
AcO
HO
OAc
O
OAc
O
OH
O
AcO
AcO
AcO
AcO
HO
HO
d
e
O
O
O
OAc
OH
OCH3
O
AcO
AcO
HO
AcO
AcO
HO
O
O
AcO
AcO
HO
Scheme 6. Reagents: (a) AcOH/H2O, 78%; (b) (i) (Ph3P)3RhCl, EtOH; (ii) 3 M HCl; (iii) Ac2O, pyridine, DMAP, 84%; (c) (i) H2, Pd/C, MeOH; (ii)
Ac2O, pyridine, DMAP, 90%; (d) Ac2O, H2SO4, 95%; (e) NaOMe, MeOH.
AcO
O
AcO
O
OAc
O
OAc
O
a
b
AcO
AcO
11
AcO
AcO
OAc
AcO
AcO
OCH3
AcO
AcO
O
O
(plus β-anomer)
AcO
AcO
HO
OH
O
c
O
HO
HO
OH
HO
HO
O
HO
Scheme 7. Reagents: (a) (i) H2, Pd/C, MeOH; (ii) Ac2O, pyridine, DMAP, 64%; (b) Ac2O, H2SO4, 94%; (c) NaOMe, MeOH.
AcO
AcO
AcO
AcO
O
O
AcO
AcO
a
b
AcO
AcO
O
12
OAc
O
O
OAc
O
(plus β-anomer)
AcO
AcO
AcO
AcO
OAc
OCH3
HO
HO
O
HO
c
HO
O
OH
O
HO
HO
OH
Scheme 8. Reagents: (a) (i) H2, Pd/C, MeOH; (ii) Ac2O, pyridine, DMAP, 55%; (b) Ac2O, H2SO4; (c) NaOMe, MeOH.
OH
OH
OH
HO
OH
OH
O
HO
HO
OH
OH
O
O
O
a
b
7
O
O
O
O
HO
O
O
HO
Scheme 9. Reagents: (a) H2, Pd/C, MeOH, 89%; (b) CF3CO2H, H2O.

P. J. Meloncelli et al. / Carbohydrate Research 342 (2007) 1793–1804
1797
available a-amylase, isomaltase, a-glucosidase and
a-galactosidase, and a standard colorimetric glucose
assay.26 The four disaccharides were unaffected by a-
amylase, a-glucosidase and a-galactosidase (the last as
expected) but isomaltase caused some minor hydrolysis
of 2-O-a-D-glucopyranosyl-D-galactopyranose and sig-
nificant hydrolysis of 6-O-a-D-glucopyranosyl-D-galac-
topyranose. This last result was enough to encourage
us to investigate the possible hydrolysis with mamma-
lian enzymes; a porcine source was available and appro-
priate.27–29
Dahlqvist has developed a method, based on a color-
imetric change in o-dianisidine, to determine the disac-
charidase activity of intestinal mucosa samples.30 We
decided to adopt this method for our purposes but used
ABTS [2,20-azino-di(3-ethylbenzothiazoline-6-sulfonic
acid)] as the colorimetric agent; other minor improve-
ments were also made to the determination. Thus, the
small intestinal mucosa of a 12 week old pig was col-
lected at 50 cm intervals, to provide data of disacchari-
dase activity throughout the entire length of the small
intestine. The mucosae were prepared as homogenates,
following the method of Dahlqvist; Table 4 summarizes
the results.
low levels. These observations were shown to have rele-
vance in the human model with similar patterns ob-
served by Raul et al.5
3. Conclusion
The synthesis of the four a-D-glucopyranosides was best
approached through the halide catalysis procedure,
utilizing an a-D-glucopyranosyl iodide as the donor,
preferably in the presence of triphenylphosphine oxide.
Although this method was operationally more difficult
than those employing the more established trichloroace-
timidate and thioglycoside donors, the compensation
was in the excellent diastereoselectivity obtained.
Lactose still remains as the most easily hydrolyzed
disaccharide in the mammalian small intestine for the
supply of both D-glucose and D-galactose. The only syn-
thetic disaccharide to show any promise as an alterna-
tive was 6-O-a-D-glucopyranosyl-D-galactopyranose.
4. Experimental
4.1. Synthesis of the four disaccharides
It is evident that there was significant maltase, sucrase
and lactase activity along most of the small intestine; the
same is true of isomaltase activity but fewer points were
assayed. Most disappointing was the lack of significant
hydrolysis of 6-O-a-D-glucopyranosyl-D-galactopyra-
nose by any of the enzymes present in the small intes-
tine. The small amount of hydrolysis that was
observed was attributed to the action of sucrase.
The results here provide an overall picture of disac-
charidase activity along the small intestine of the pig.
Maltase activity appears to be quite high and constant
throughout the length of the small intestine. Sucrase
activity on the other hand peaks around the area of
the jejunum and then declines to approximately 50%
of its peak level. Lactase activity appears to peak quite
early in the small intestine and then quickly declines to
4.1.1. General methods. General experimental proce-
dures have been described previously.31
4.1.2. Methyl 3-O-allyl-4,6-O-benzylidene-a-D-galacto-
pyranoside (2). Methyl 3-O-allyl-a-D-galactopyran-
oside32 (3.08 g, 13.2 mmol) in DMF (30 mL) was
treated with benzaldehyde diethyl acetal (3.55 g, 19.7
mmol) and CSA (100 mg) and the solution stirred
(35 ꢁC, 36 h). The solution was neutralized with Et3N
and concentrated; flash chromatography (EtOAc/petrol,
1:1) gave alcohol 2 (2.88 g, 68%) as a colourless solid,
1
mp 159–161 ꢁC. [a]D +216.0; H NMR (500 MHz): dH
3.47 (s, CH3), 3.66 (d, J = 0.6, H4), 3.75 (dd, J2,3
=
10.0, J1,2 3.7, H2), 4.09 (dd, J6,6 = 12.4, J5,6 = 1.7,
H6), 4.16–4.31 (m, 5H, CH2O, H3, H5, H6), 4.96 (d,
Table 4. Summary of pig intestinal disaccharidase activity
Intestinal Section
Maltasea (U/g)
Sucrase (U/g)
Lactase (U/g)
Disaccharidase acting on
Isomaltase (U/g)
a-Glcp-(1!6)-Gal (U/g)
1
2
3
4
5
6
7
8
9
48
136
142
143
146
145
152
155
151
91
3.4
20.4
13.6
32.7
36.4
39.9
27.2
31.0
24.4
19.9
19.8
22.1
16.6
48.9
39.7
48.9
41.9
22.0
18.1
10.1
9.3
0.70
1.20
1.06
1.51
1.86
1.34
1.50
1.36
1.08
0.42
1.18
1.12
19.8
17.6
27.3
39.9
10.1
3.7
10
11
12
1.5
1.0
5.4
142
141
a Maltase activity (on maltose) of a mucosal homogenate, expressed as lmol Lꢀ1 minꢀ1 gꢀ1 protein.

1798
P. J. Meloncelli et al. / Carbohydrate Research 342 (2007) 1793–1804
H1), 5.21–5.23, 5.32–5.36 (2 · m, CH2CH), 5.56 (s,
PhCH), 5.93–6.03 (m, CH2CH), 7.32–7.39, 7.52–7.54
(2 · m, Ph); 13C NMR (125.8 MHz): dC 55.62 (CH3),
62.82 (C5), 67.88, 73.64, 76.01 (C2, C3, C4), 69.50,
70.51 (CH2O, C6), 100.18, 101.00 (PhCH, C1), 117.54
(CH2CH), 126.26–137.76 (Ph), 134.97 (CH2CH). Anal.
Calcd for C17H22O6: C, 63.3; H, 6.9. Found: C, 63.4;
H, 6.7.
warmed (0 ꢁC). The solution was then treated with
Et3N (100 lL) and filtered, the filtrate concentrated
and subjected to flash chromatography (EtOAc/petrol,
1:2) to give the disaccharide as a colourless oil. The
results of the four separate glycosidations are presented
in Table 2.
4.1.6. General procedure for glycosylations with the iodide
15/triphenylphosphine oxide. 1-O-Acetyl-2,3,4,6-tetra-
O-benzyl-D-glucopyranose36 (290 mg, 0.51 mmol) in
4.1.3. General procedure (A) for glycosylations with
iodide 1. The acceptor alcohol (0.32 mmol) in dry
C6H6 (2 mL) was treated with EtNPri2 (130 mg,
˚
dry CH2Cl2 (5 mL) was stirred with 4 A molecular sieves
(300 mg) (0 ꢁC, 1 h). Iodotrimethylsilane (122 mg,
0.608 mmol) was then added and the mixture stirred
(30 min) before being concentrated and the excess re-
agent removed by azeotropic distillation with PhMe
(4 · 10 mL). The residue, presumably iodide 15, in dry
CHCl3 (2 mL) was treated with Ph3PO (280 mg,
1.0 mmol) and the acceptor alcohol (0.22 mmol) and
the mixture stirred (rt, 14 h). The mixture was filtered,
the filtrate concentrated and subjected to flash chroma-
tography (EtOAc/petrol, 1:2) to give the disaccharide.
The results of the four separate glycosidations are pre-
sented in Table 3.
˚
0.96 mmol), Bu4NI (350 mg, 0.96 mmol) and 4 A mole-
cular sieves (300 mg) and the mixture stirred (rt, 2 h).
Iodide 119 (800 mg, 1.28 mmol) in C6H6 (3 mL) was
added and the mixture refluxed (6 h). The mixture was
filtered, the filtrate concentrated and subjected to
flash chromatography (EtOAc/petrol, 1:2) to give the
disaccharide.
4.1.4. General procedure (B) for glycosylations with
trichloroacetimidate 9. The trichloroacetimidate 933
(37 mg, 0.055 mmol) and the acceptor alcohol
˚
(0.046 mmol) in dry ether (2 mL) were stirred with 4 A
molecular sieves (50 mg) (rt, 3 h). The mixture was
cooled (ꢀ40 ꢁC), trimethylsilyl trifluoromethanesulfo-
nate (20 lL) added and the mixture allowed to warm
slowly (rt). The mixture was then treated with Et3N
(100 lL) and filtered, the filtrate was concentrated and
subjected to flash chromatography (EtOAc/petrol, 1:2)
to give the disaccharide as a colourless oil. The results
of the four separate glycosidations are presented in
Table 1.
4.1.7. Methyl 2-O-(6-O-acetyl-2,3,4-tri-O-benzyl-a-D-
glucopyranosyl)-3-O-allyl-4,6-O-benzylidene-a-D-galacto-
pyranoside (6). Using general procedure (A), acceptor
alcohol 2 (100 mg) gave disaccharide 6 (172 mg, 70%)
as a colourless solid and as predominantly the a-anomer
(a:b, 97:3). Mp 134–136 ꢁC; [a]D +111.5; 1H NMR
(60 MHz): dH 2.00 (s, CH3CO), 3.43–3.45 (m, H5),
3.45 (s, CH3O), 3.54 (dd, J2,3 = 9.6, J1,2 = 3.6, H2),
3.65 (s, H4), 3.92 (dd, J5 ,6 = 10.2, 3.5, H50), 4.06–4.09
(m, 2H, H3, H6), 4.16–4.19 (m, 3H, CH2O, H6), 4.21–
4.31 (m, 5H, H20, H30, H40, H60), 4.56, 4.85 (AB,
J = 11.2, PhCH2), 4.69, 4.78 (AB, J = 12.0, PhCH2),
4.80, 5.00 (AB, J = 10.8, PhCH2), 4.90 (d, H1), 4.96
0
0
4.1.5. General procedure for glycosylations with thiogly-
coside 14. (a) Diphenyl sulfoxide/trifluoromethanesulf-
onic anhydride promotion. Thioglycoside 1434 (55 mg,
0.095 mmol), 2,4,6-tri-tert-butylpyrimidine35 (65 mg,
0.265 mmol) and Ph2SO (54 mg, 0.265 mmol) in dry
CH2Cl2 (2 mL) were treated with Tf2O (45 lL,
0.265 mmol) and the solution was stirred (ꢀ60 ꢁC,
10 min). The acceptor alcohol (0.142 mmol) in dry
CH2Cl2 (1 mL) was added and the solution warmed
(0 ꢁC). The solution was then treated with Et3N (100
lL) and filtered, the filtrate concentrated and subjected
to flash chromatography (EtOAc/petrol, 1:2) to give
the disaccharide as a colourless oil. The results of the
four separate glycosidations are presented in Table 2.
(b) 1-Benzenesulfinylpiperidine/trifluoromethanesulfonic
anhydride promotion. Thioglycoside 14 (84.7 mg, 0.146
mmol), 1-benzenesulfinylpiperidine (30.5 mg, 0.146
mmol) and 2,4,6-tri-tert-butylpyrimidine (65 mg,
0.265 mmol) in dry CH2Cl2 (2 mL) were treated with
Tf2O (45 lL, 0.265 mmol) and the solution was stirred
(ꢀ60 ꢁC, 10 min). The acceptor alcohol (0.219 mmol)
in dry CH2Cl2 (1 mL) was added and the solution
(d, J1 ,2 = 3.4, H10), 5.11–5.15, 5.27–5.35 (2 · m,
CH2CH), 5.54 (s, PhCH), 5.91–5.94 (m, CH2CH),
7.23–7.38, 7.49–7.51 (2 · m, Ph); 13C NMR
(150.9 MHz): dC 20.55 (CH3CO), 55.17 (CH3O), 62.43
(C4), 62.94 (CH2O), 68.53 (C20), 69.26 (C6), 71.02
(C60), 71.33, 74.02, 74.26 (C30, C40, C50), 72.87, 74.38,
75.52 (3C, PhCH2), 77.40 (C5), 79.40 (C2), 81.81 (C3),
94.50 (C1), 97.60 (C10), 101.02 (PhCH), 117.14
(CH2CH), 126.22–138.62 (Ph), 134.98 (CH2CH),
170.82 (C@O); FAB-MS m/z 795.3374 (C46H51O12;
[MꢀH]+ requires 795.3380). A significant quantity
(300 mg) of alkene 8 was also isolated, with 1H
(300 MHz) and 13C (75.5 MHz) NMR spectral data con-
sistent with those reported.19
0
0
4.1.8. 6-O-(6-O-Acetyl-2,3,4-tri-O-benzyl-a-D-glucopyr-
anosyl)-1,2:3,4-di-O-isopropylidene-a-D-galactopyranose
(7). Using general procedure (A), acceptor alcohol 5
(112 mg) gave disaccharide 7 (280 mg, 90%) as a colour-

P. J. Meloncelli et al. / Carbohydrate Research 342 (2007) 1793–1804
1799
less oil and as predominantly the a-anomer (a:b, 97:3).
[a]D +16.1; H NMR (600 MHz): dH 1.32, 1.33, 1.45,
(125.8 MHz): dC 55.60 (CH3O), 62.26 (C5), 69.01,
69.55 (C6, C60), 70.59 (CH2O), 73.41, 74.29, 74.92,
75.40 (4C, PhCH2), 74.44 (C50), 75.60, 76.58 (C2, C3,
C4), 77.62, 82.02, 84.65 (C20, C30, C40), 100.42 (C10),
101.08 (PhCH), 104.95 (C1), 117.09 (CH2CH), 126.32–
138.90 (Ph), 135.23 (CH2CH); FAB-MS m/z 844.3812.
1
1.55 (4 · s, 12H, CH3C), 2.03 (s, CH3CO), 3.51 (dd,
J4 ,5 ꢁ J3 ,4 = 9.8, H40), 3.56 (dd, J2 ,3 = 9.6,
0
0
0
0
0
0
J1 ,2 = 3.6, H20), 3.74 (dd, 1H, J6,6 = 10.3, J5,6 = 7.3,
H6), 3.79 (dd, 1H, J5,6 = 6.3, H6), 3.93–3.96 (m, H50),
0
0
4.01–4.06 (m, H30, H5), 4.24 (dd, 1H, J6 ,6 = 11.9,
0
0
J5 ,6 = 1.9, H60), 4.31–4.37 (m, H2, H3, H4), 4.57,
4.1.10. Methyl 2,4,6-tri-O-benzyl-3-O-(2,3,4,6-tetra-O-
benzyl-a-D-glucopyranosyl)-a-D-galactopyranoside (11).
Using general procedure (B), acceptor alcohol 332
(110 mg) gave an inseparable mixture of a-linked 11
and b-linked disaccharides (75:25) (233 mg, 72%) as a
colourless oil.
0
0
0
0
4.88 (AB, J = 10.8, PhCH2), 4.62 (dd, 1H, J5 ,6 = 1.3,
H60), 4.71, 4.76 (AB, J = 11.9, PhCH2), 4.82, 5.02
(AB, J = 10.8, PhCH2), 4.96 (d, H10), 5.53 (d,
J1,2 = 5.0, H1), 7.25–7.40 (m, Ph); 13C NMR
(150.9 MHz): dC 21.00 (CH3CO), 24.75, 25.04, 26.18,
26.25 (4C, CH3C), 63.20, 66.76 (C6, C60), 66.00, 68.76
(C5, C50), 70.70, 70.77, 70.99 (C2, C3, C4), 72.52,
74.99, 75.80 (3C, PhCH2), 77.30 (C40), 79.92 (C20),
81.97 (C30), 96.42, 97.12 (C1, C10), 108.75, 109.37 (2C,
CH3C), 127.76-138.82 (Ph), 170.91 (C@O); FAB-MS
m/z 734.3325 (C41H50O12; [M]+Å requires 734.3302). A
significant quantity (500 mg) of alkene 8 was again
isolated.
4.1.11. Methyl 2,3,6-tri-O-benzyl-4-O-(2,3,4,6-tetra-O-
benzyl-a-D-glucopyranosyl)-a-D-galactopyranoside (12).
Using general procedure (B), acceptor alcohol 437
(105 mg) gave an inseparable mixture of a-linked 12
and b-linked disaccharides (65:35) (223 mg, 72%) as a
colourless oil.
4.1.12. 1,2:3,4-Di-O-isopropylidene-6-O-(2,3,4,6-tetra-O-
benzyl-a-D-glucopyranosyl)-a-D-galactopyranose
Using general procedure (B), acceptor alcohol
(70 mg) gave an inseparable mixture of a-linked 13
and b-linked disaccharides (80:20) (210 mg, 71%) as a
colourless oil. Partial 1H NMR (600 MHz); dH 1.32,
4.1.9. Methyl 3-O-allyl-4,6-O-benzylidene-2-O-(2,3,4,
6-tetra-O-benzyl-a-D-glucopyranosyl)-a-D-galactopyran-
oside (10). Using general procedure (B), acceptor alco-
hol 2 (65 mg) gave a-linked disaccharide 10 (105 mg,
63%) as a colourless oil, [a]D +91.7. 1H NMR
(500 MHz): dH 3.46 (s, CH3O), 3.56–3.60 (m, 2H, H20,
H60), 3.65–3.72 (m, 3H, H40, H5, H60), 3.94 (dd,
(13).
5
0
0
1.33, 1.34, 1.46, 1.54 (5 · s, CH3), 3.60 (dd, J2 ,3 = 9.6,
J1 ,2 = 3.6, H20a), 4.00 (dd, J3 ,4 = 9.6, H30a), 4.44 (d,
0
0
0
0
J2,3 = 10.3, J3,4 = 3.4, H3), 4.04 (dd, J3 ,4 ꢁ J2 ,3 = 9.4,
H30), 4.08 (dd, J6,6 = 12.6, J5,6 = 1.3, H6), 4.15–4.24 (m,
CH2O, H50), 4.25–4.31 (m, 2H, H4, H6), 4.32 (dd,
J1,2 = 3.4, H2), 4.39, 4.57 (AB, J = 12.1, PhCH2), 4.49,
4.79 (AB, J = 11.3, PhCH2), 4.69, 4.81 (AB, J = 12.0,
PhCH2), 4.82, 4.96 (AB, J = 10.8, PhCH2), 4.96 (d,
J1 ,2 = 7.5, H10b), 5.01 (d, H10a), 5.54 (d, J1,2 = 5.0,
H1a), 5.58 (d, J1,2 = 5.0, H1b); Partial 13C NMR
(150.9 MHz): dC 24.35, 24.54, 24.82, 24.93, 25.90,
25.94, 25.96, 26.06 (4C, CH3), 65.63 (C5a), 66.11,
68.28, 68.64, 69.60 (C6a, C60a, C6b, C60b), 79.70
(C20a), 81.53 (C20b), 81.84 (C30a), 84.43 (C30b), 96.20
(C1a), 96.28 (C1b), 96.88 (C10a), 104.26 (C10b),
108.52, 109.15, 109.30 (CH3C).
0
0
0
0
0
0
J1 ,2 = 2.9, H10), 5.01 (d, H1), 5.07–5.13 (m, CH2CH),
5.55 (s, PhCH), 5.88–5.97 (m, CH2CH), 7.10–7.50 (m,
Ph); 13C NMR (125.8 MHz): dC 55.41 (CH3O), 62.73
(C5), 68.32, 69.47 (C6, C60), 70.01, 71.18, 74.37, 74.83,
77.82, 79.57 (C2, C20, C3, C4, C40, C50), 71.79
(CH2O), 73.18, 73.35, 74.64, 75.72 (4C, PhCH2), 82.17
(C30), 94.77 (C10), 97.84 (C1), 101.19 (PhCH), 117.43
(CH2CH), 126.52–138.55 (Ph), 135.17 (CH2CH); FAB-
MS m/z 844.3731 (C51H56O11 [M]+Å requires 844.3823).
Also obtained was the b-linked disaccharide (15 mg,
0
0
4.1.13. 2-O-(a-D-Glucopyranosyl)-D-galactopyranose. (i)
Disaccharide 6 (246 mg) was stirred in AcOH/H2O (4:1,
3 mL) (1 h, 50 ꢁC). The solution was then concentrated
and subjected to flash chromatography (EtOAc/
petrol, 1:1) to give methyl 2-O-(6-O-acetyl-2,3,4-tri-O-
benzyl-a-D-glucopyranosyl)-3-O-allyl-a-D-galactopyran-
1
oside (168 mg, 78%) as a colourless oil. [a]D +80.4; H
7%) as
a
colourless oil, [a]D +98.2; 1H NMR
NMR (600 MHz): dH 2.00 (s, CH3CO), 3.43 (s, CH3O),
3.49 (dd, J = 9.5, 9.3, H30), 3.55 (dd, J2,3 = 9.6,
J1,2 = 3.5, H2), 3.80–3.85 (m, 3H, H4, H5, H6), 3.95
(dd, J6,6 = 10.9, J5,6 = 5.2, H6), 4.05–4.08 (m, H20,
H3), 4.12–4.25 (m, 6H, CH2O, H40, H50, H60), 4.58,
4.80 (AB, J = 11.1, PhCH2), 4.68, 4.78 (AB, J = 12.6,
PhCH2), 4.86, 5.01 (AB, J = 10.6, PhCH2), 4.87 (d,
(500 MHz): dH 3.44 (s, CH3O), 3.55–3.75 (m, 7H, H20,
H30, H40, H5, H50, H60), 3.98 (dd, J3,4 = 3.5,
J2,3 = 10.3, H3), 4.08–4.15 (m, 3H, CH2O, H6), 4.25
(dd, J1,2 = 3.5, H2), 4.31 (dd, J6,6 = 12.4, J5,6 = 1.4,
H6), 4.34 (dd, J4,5 = 0.3, H4), 4.52, 4.60 (AB, J = 12.0,
PhCH2), 4.56, 4.82 (AB, J = 10.8, PhCH2), 4.66 (d,
J1 ,2 = 7.7, H10), 4.74, 5.15 (AB, J = 11.3, PhCH2),
4.77, 4.91 (AB, J = 11.0, PhCH2), 5.04–5.09, 5.19–5.23
(2 · m, CH2CH), 5.17 (d, H1), 5.58 (s, PhCH), 5.81–
5.89 (m, CH2CH), 7.15–7.58 (m, Ph); 13C NMR
H1), 4.89 (d, J1 ,2 = 3.4, H10), 5.13–5.17, 5.57–5.31
(2 · m, CH2CH), 5.89–5.96 (m, CH2CH), 7.26–7.37
(m, Ph); 13C NMR (150.9 MHz): dC 20.95 (CH3CO),
55.20 (CH3O), 63.07, 63.17 (C6, C60), 68.61, 68.63
0
0
0
0

1800
P. J. Meloncelli et al. / Carbohydrate Research 342 (2007) 1793–1804
(C40, C50), 69.10, 75.76 (C4, C5), 71.15 (C20), 71.76
(CH2O), 73.13, 74.73, 75.79 (3C, PhCH2), 77.38 (C30),
79.46 (C2), 82.09 (C3), 94.54 (C1), 96.95 (C10), 118.34
(CH2CH), 127.77–138.55 (Ph), 134.22 (CH2CH),
170.88 (C@O); FAB-MS m/z 708.3177 (C39H48O12
[M]+Å requires 708.3146).
(ii) The diol from (i) (170 mg) in EtOH (20 mL) was
treated with Wilkinson’s catalyst (27 mg) and the mix-
ture refluxed (12 h); the mixture was then treated with
hydrochloric acid (3 M, 1 mL) and refluxed (1 h). The
mixture was neutralized with Et3N (1 mL) and concen-
trated to give a pale coloured oil that was dissolved in
CH2Cl2 (3 mL) and treated with pyridine (8 mL),
Ac2O (3 mL) and DMAP (10 mg) and the solution stir-
red (rt, 10 h). The solution was then treated with MeOH
(4 mL) (1 h, rt); concentration of the mixture followed
by flash chromatography (EtOAc/petrol, 1:1) gave
(C5), 67.65 (C50), 68.28, 68.37 (C4, C40), 68.86 (C3),
70.02 (C30), 71.34, 71.60 (C2, C20), 94.02 (C1), 97.15
(C10), 169.67–170.65 (7C, C@O); FAB-MS m/z
651.2145 (C27H39O18 [M+H]+ requires 651.2136).
(iv) The hepta-acetate from (iii) (103 mg) was stirred
with Ac2O (3 mL) and H2SO4 (50 lL) (6 h, 0 ꢁC). The
solution was then poured onto ice and kept (rt, 12 h).
The mixture was extracted with EtOAc, the extracts
were washed with water, saturated NaHCO3 and dried.
Concentration of the organic extract gave 1,3,4,6-tetra-
O-acetyl-2-O-(2,3,4,6-tetra-O-acetyl-a-D-glucopyranos-
yl)-D-galactopyranose (102 mg) as a colourless oil
and as a mixture of anomers (a:b, 4:1); 1H NMR
(600 MHz): dH 1.97–2.19 (CH3CO), 4.02–4.20 (m,
H2b, H50, H5b, H6, H60), 4.19 (dd, J2,3 = 10.6,
J1,2 = 3.6, H2a), 4.24 (dd, J6,6 = 12.2, J5,6 = 3.5, H6a),
4.25–4.28 (m, H6b), 4.32 (dd, J5,6 = 7.3, H5a), 4.75
(dd, J2,3 = 10.3, J1,2 = 3.9, H20b), 4.92 (dd, J2 ,3
=
0
0
methyl
3,4,6-tri-O-acetyl-2-O-(6-O-acetyl-2,3,4-tri-O-
10.4, J1 ,2 = 3.5, H20a), 5.03–5.05 (m, H3b, H40b),
5.07–5.09 (m, H40a), 5.10 (d, H10a), 5.31 (dd,
J2,3 = 10.6, J3,4 = 2.5, H3a), 5.33–5.35 (m, H30b), 5.35
0
0
benzyl-a-D-glucopyranosyl)-a-D-galactopyranoside (160
mg, 84%) as a colourless oil. [a]D +81.3; 1H NMR
(600 MHz): dH 2.01, 2.05, 2.06, 2.11 (4 · s, 12H,
CH3CO), 3.42 (s, CH3O), 3.48 (dd, J = 9.8, 9.3, H40),
(dd, J3 ,4 = 10.3, H30a), 5.40–5.45 (m, H10b, H4b),
5.52 (d, J3,4 = 2.5, H4a), 5.65 (d, J1,2 = 8.1, H1b), 6.29
(d, H1a); 13C NMR (150.9 MHz): dC 20.65–21.10
(CH3), 61.11, 61.27, 61.61 (C6, C60), 66.99–72.12 (C2,
C20, C3, C30, C4, C40, C5, C50), 89.21 (C1a), 93.75
(C1b), 95.17 (C10b), 95.72 (C10a), 169.08–170.73
(C@O); FAB-MS m/z 619.1857 (C26H35O17 [MꢀOAc]+
requires 619.1874).
0
0
3.52 (dd, J2 ,3 = 9.6, J1 ,2 = 3.5, H20), 3.95–4.00 (m,
H30, H5), 4.07 (dd, J2,3 = 10.7, J1,2 = 3.5, H2), 4.09–
4.10 (m, 2H, H6), 4.17–4.20 (m, 2H, H50, H60), 4.24
0
0
0
0
(dd, J6 ,6 = 12.0, J5 ,6 = 3.8, H60), 4.56, 4.86 (AB,
J = 11.1, PhCH2), 4.66, 4.78 (AB, J = 11.9, PhCH2),
4.80, 4.98 (AB, J = 10.7, PhCH2), 4.84 (d, H10), 4.91
(d, H1), 5.36 (dd, J3,4 = 2.7, H3), 5.46 (d, H4), 7.22–
7.36 (m, Ph); 13C NMR (150.9 MHz): dC 20.78, 20.86,
20.96 (CH3CO), 55.56 (CH3O), 62.03, 62.89 (C6, C60),
66.37 (C50), 68.50 (C4), 68.80 (C3), 69.19 (C5), 70.69
(C2), 73.32, 74.94, 75.80 (3C, PhCH2), 77.07 (C40),
79.60 (C20), 81.78 (C30), 95.50 (C10), 97.32 (C1),
127.85–138.67 (Ph), 169.87, 170.21, 170.61, 170.79 (4C,
C@O). Anal. Calcd for C42H50O15: C, 63.5; H, 6.3.
Found: C, 63.3; H, 6.6.
0
0
0
0
(v) A small piece of Na in MeOH (1 mL) was added to
the octa-acetate from (iv) (90 mg) in MeOH (3 mL) and
the solution stirred (rt, 4 h). The solution was neutral-
ized with resin (Amberlite IR-120, H+), filtered and
the filtrate concentrated to give 2-O-(a-D-glucopyran-
osyl)-D-galactopyranose (41 mg) as a colourless oil and
as a mixture of anomers (a:b, 1:1.4). [a]D +129.0
(H2O); 1H NMR (600 MHz, D2O): dH 3.41–3.45 (m,
H5), 3.52 (m, H2, H20b), 3.67–3.92, 4.01–4.04, 4.07–
4.11 (3 · m, H20a, H3, H30, H4, H40, H50b, H6, H60),
3.96 (dd, J = 10.2, 3.3, H50a), 4.69–4.71 (m, H1b),
(iii) The tetra-acetate from (ii) (140 mg) in MeOH
(10 mL) was stirred with Pd/C (10%, 10 mg) and H2
(1 atm, 12 h, 35 ꢁC). The mixture was filtered, the filtrate
concentrated and treated with pyridine (3 mL), Ac2O
(1 mL) and DMAP (10 mg) (rt, 5 h). Treatment with
MeOH (5 mL) (rt, 1 h), followed by concentration of
the mixture and flash chromatography (EtOAc/petrol,
1:1), gave methyl 3,4,6-tri-O-acetyl-2-O-(tetra-O-acetyl-
5.09 (d, J1,2 = 3.7, H1a), 5.37 (d, J1 ,2 = 3.8, H10b),
0
0
5.46 (d, J1 ,2 = 3.5, H10a); 13C NMR (150.9 MHz,
D2O): dC 63.12, 63.66 (C6b, C60b), 63.15, 63.82 (C6a,
C60a), 70.34–79.71 (C2, C20, C3, C30, C4, C40, C5,
C50), 92.35 (C10a), 98.91 (C1a), 99.33 (C1b), 100.64
(C10b); FAB-MS m/z 343.1253 (C12H23O11 [M+H]+
requires 343.1240).
0
0
a-D-glucopyranosyl)-a-D-galactopyranoside
(103 mg,
90%) as a colourless oil. [a]D +108; 1H NMR
(600 MHz): dH 1.98, 1.99, 1.99, 2.02, 2.06, 2.11 (6 · s,
21H, CH3CO), 3.38 (s, CH3O), 4.01 (dd, J2,3 = 10.6,
J1,2 = 3.6, H2), 4.04–4.11 (m, 4H, H50, H6, H60), 4.13
4.1.14. 3-O-(a-D-Glucopyranosyl)-D-galactopyranose. (i)
Disaccharide 11 (156 mg), containing the b-anomer, in
MeOH (20 mL) was stirred with Pd/C (10%, 15 mg)
and H2 (1 atm, 12 h). The mixture was filtered, the fil-
trate concentrated and treated with pyridine (2 mL),
Ac2O (1 mL) and DMAP (5 mg) and stirred (rt, 8 h).
Treatment with MeOH (2 mL), followed by concentra-
tion of the mixture and flash chromatography (EtOAc/
0
0
(dd, J5,6 = 6.8, 6.4, H5), 4.22 (dd, J6 ,6 = 12.3,
J5 ,6 = 3.9, H60), 4.71 (dd, J2 ,3 = 10.1, J1 ,2 = 3.7,
H20), 4.83 (d, H1), 5.02 (dd, J = 9.9, 9.8, H40), 5.26
(dd, J3,4 = 3.5, H3), 5.27 (d, H10), 5.40 (dd, H30), 5.42
(d, H4); 13C NMR (150.9 MHz): dC 20.68, 20.72, 20.77
(CH3CO), 55.49 (CH3O), 61.67, 61.84 (C6, C60), 66.26
0
0
0
0
0
0

P. J. Meloncelli et al. / Carbohydrate Research 342 (2007) 1793–1804
1801
petrol, 1:2), gave first methyl 2,4,6-tri-O-acetyl-3-O-
(2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl)-a-D-galacto-
pyranoside (65 mg, 64%) as a colourless oil. [a]D +154.2;
1H NMR (600 MHz): dH 1.98, 2.01, 2.05, 2.05,
2.12, 2.14, 2.14 (7 · s, 21H, CH3CO), 3.36 (s, CH3O),
4.06–4.10 (m, 4H, H5, H6, H60), 4.27–4.29 (m, H50),
4.30 (dd, J3,4 = 3.0, J2,3 = 10.5, H3), 4.34 (dd,
H3, H30, H40, H5, H50, H6, H60), 4.07 (dd, J1,2 = 7.0,
J2,3 = 6.1, H2b), 4.15 (d, J = 2.5 Hz, H4b), 4.21 (s,
0
0
0
H4a), 4.62 (d, H1b), 5.10 (d, J1 ,2 = 3.5, H1 b), 5.12
(d, J1 ,2 = 3.6, H10a), 5.28 (d, J1,2 = 1.4, H1a); 13C
NMR (150.9 MHz, D2O): dC 63.03, 63.08 (C6b, C60b),
63.14, 63.87 (C6a, C60a), 67.74 (C4b), 68.31 (C4a),
69.42–76.82 (C2, C20, C3a, C30, C40, C5a, C50), 77.57
(C5b), 80.20 (C3b), 95.04 (C1a), 97.69 (C10a), 97.98
(C10b), 99.10 (C1b); FAB-MS m/z 343.1230
(C12H23O11 [M+H]+ requires 343.1240).
0
0
J6 ,6 = 12.2, J5 ,6 = 1.5, H60), 5.00–5.04 (m, H20, H40),
5.05 (d, J1,2 = 3.5, H1), 5.08 (dd, H2), 5.19 (d,
0
0
0
0
J1 ,2 = 3.4, H10), 5.36 (dd, J3 ,4 ꢁ J2 ,3 = 9.8, H30),
5.40 (d, J = 2.8, H4); 13C NMR (150.9 MHz): dC
20.44, 20.49, 20.53, 20.58, 20.61 (CH3CO), 55.25
(CH3O), 61.68, 61.81 (C6, C60), 65.68 (C4), 66.19 (C5),
67.74, 68.07 (C3, C50), 68.66, 69.18 (C20, C40), 69.31
(C2), 69.69 (C30), 91.88 (C10), 96.98 (C1), 169.39,
169.76, 169.91, 170.06, 170.32 (C@O); FAB-MS m/z
651.2179 (C27H39O18 [M+H]+ requires 651.2136).
0
0
0
0
0
0
4.1.15. 4-O-(a-D-Glucopyranosyl)-D-galactopyranose. (i)
Disaccharide 12 (150 mg), containing the b-anomer, in
MeOH (20 mL) was stirred with Pd/C (10%, 15 mg)
and H2 (12 h, 1 atm). The mixture was filtered, the fil-
trate concentrated and treated with pyridine (2 mL),
Ac2O (1 mL) and DMAP (5 mg) and the solution stirred
(rt, 8 h). Treatment with MeOH (2 mL), followed by
concentration of the mixture and flash chromatography
(EtOAc/petrol, 1:2), gave methyl 2,3,6-tri-O-acetyl-4-O-
(2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl)-a-D-galacto-
pyranoside (57 mg, 55%) as a colourless oil, [a]D +32.2
Further elution gave methyl 2,4,6-tri-O-acetyl-3-
O-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-a-D-galac-
topyranoside (25 mg, 24%) as a colourless oil, [a]D +69.0
1
(lit.38 +75.0). The H (600 MHz) and 13C (150.9 MHz)
NMR spectral data were in good agreement with those
reported.39
1
(ii) The a-hepta-acetate (46 mg) was stirred with Ac2O
(2 mL) and H2SO4 (100 lL) (0 ꢁC, 6 h). The solution
was then poured onto ice and stirred (12 h), the mixture
extracted with EtOAc, the extract washed with saturated
NaHCO3 and brine and then dried. Concentration of
the organic extract followed by flash chromato-
graphy (EtOAc/petrol, 1:3) gave 1,2,4,6-tetra-O-acetyl-3-
O-(2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl)-a-D-galac-
topyranose (45 mg, 94%) as a colourless oil, containing
some 5% of the b-D-galactose anomer. (a-D-anomer)
1H NMR (600 MHz): dH 1.98, 2.00, 2.04, 2.06, 2.07,
2.11, 2.14, 2.27 (8 · s, 24H, CH3CO), 4.05–4.25 (m,
(lit.36 +36.0). The H (600 MHz) and 13C (150.9 MHz)
NMR spectral data were in good agreement with those
reported.36
Further elution gave methyl 2,3,6-tri-O-acetyl-4-O-
(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-a-D-galacto-
pyranoside (20 mg, 20%) as a colourless oil, [a]D +8.5
1
(lit.39 +7.0). The H (600 MHz) and 13C (150.9 MHz)
NMR spectral data were in good agreement with those
reported.39
(ii) The a-hepta-acetate from (i) (40 mg) was stirred
with Ac2O (2 mL) and H2SO4 (100 lL) (rt, 6 h). The
solution was poured onto ice and stirred (6 h); the mix-
ture was then extracted with EtOAc, the extract washed
with saturated NaHCO3, brine and then dried. Concen-
tration of the organic extract gave 1,2,3,6-tetra-O-
acetyl-4-O-(2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl)-
D-galactopyranose (40 mg) as a colourless oil and as a
mixture of anomers (a:b, 5:1). 1H NMR (600 MHz):
dH 1.98–2.15 (24H, CH3), 3.93 (dd, J5,6 = 6.6, 6.4,
H5b), 4.09–4.29 (m, H4, H5a, H50, H6, H60), 4.36 (dd,
J6,6 = 11.1, J5,6 = 6.7, H6a), 4.41 (dd, J6,6 = 11.4,
H6b), 4.84 (dd, J3,4 = 2.6, J2,3 = 10.7, H3b), 4.93–4.96
(m, H10, H3a), 5.14–5.17 (m, H20, H40), 5.30 (dd,
J1,2 = 8.1, H2b), 5.44 (dd, J2,3 = 11.2, J1,2 = 3.7, H2a),
6H, H5, H50, H6, H60), 4.27 (dd, J3,4 = 3.0, J2,3
=
10.8, H3), 5.02 (dd, J2 ,3 = 10.0, J1 ,2 = 3.3, H20), 5.09
0
0
0
0
(dd,J4 ,5 ꢁ J3 ,4 = 9.4, H40), 5.22 (d, H10), 5.30 (dd,
J1,2 = 3.6, H2), 5.35 (dd, H30), 5.45 (d, J = 2.1, H4),
6.42 (d, H1); 13C NMR (150.9 MHz): dC 20.56, 20.71,
20.76, 20.79, 20.83, 20.89, 21.01, 21.05 (8C, CH3),
61.19, 61.58 (C6, C60), 65.28 (C4), 67.51, 68.52, 68.75,
68.83, 69.17, 69.52 (C2, C20, C3, C40, C5, C50), 67.69
(C30), 89.64 (C1), 92.57 (C10), 168.94, 169.51, 169.54,
170.01, 170.13, 170.15, 170.52, 170.71 (8C, C@O);
FAB-MS m/z 619.1835 (C26H35O17 [MꢀOAc]+ requires
619.1874).
0
0
0
0
5.47 (dd, J3 ,4 ꢁ J2 ,3 = 9.6, H30a), 5.48 (dd, J3 ,4
ꢁ
0
0
0
0
0
0
(iii) The (mainly) a-D-anomer from (ii) (39 mg) in
MeOH (3 mL) was treated with NaOMe in MeOH
(1 mL) and the solution stirred (rt, 4 h). The solution
was neutralized with resin (Amberlite IR-120, H+),
filtered and the filtrate concentrated to give 3-O-(a-D-
glucopyranosyl)-D-galactopyranose (20 mg) as a colour-
less oil and as a mixture of anomers (a:b, 1:1.4). [a]D
+146.2 (H2O); 1H NMR (600 MHz, D2O): dH 3.42–
3.46, 3.56–3.62, 3.67–3.86, 3.92–3.96 (4 · m, H2a, H20,
J2 ,3 = 9.7, H30b), 5.71 (d, H1b), 6.36 (d, H1a); 13C
NMR (150.9 MHz): dC 20.65–21.19 (CH3), 61.17,
62.04 (C6b, C60b), 61.30, 61.96 (C6a, C60a), 66.07
(C2a), 67.74 (C2b), 68.12, 68.74, 69.64, 70.09, 70.48,
71.12 (C20a, C3a, C30a, C40a, C5a, C50a), 68.18,
68.47, 70.26, 71.18, 72.83, 73.07 (C20b, C3b, C30b,
C40b, C5b, C50b), 77.94 (C4b), 78.43 (C4a), 89.99
(C10a), 92.05 (C10b), 99.43 (C1a), 99.54 (C1b),
0
0

1802
P. J. Meloncelli et al. / Carbohydrate Research 342 (2007) 1793–1804
168.98–170.81 (C@O); FAB-MS m/z 619.1847
(C26H35O17 [MꢀOAc]+ requires 619.1874).
3.67–3.77, 3.82–3.88 (2 · m, H3a, H40, H5b, H50, H6,
H60), 3.79 (dd, J2,3 = 10.3, J1,2 = 3.9, H2a), 3.97 (d,
H4b), 4.02 (d, J3,4 = 3.0, H4a), 4.26 (dd, J5,6 = 6.4,
5.9, H5a), 4.58 (d, H1b), 4.93–4.95 (m, H10), 5.25 (d,
H1a); 13C NMR (150.9 MHz, D2O): dC 63.22 (C60),
69.25, 69.55 (C6), 71.04 (C2a), 71.25 (C5a), 71.50,
71.79, 72.05, 72.26 (C4, C40, C5b, C50), 74.00, 74.03
(C20), 74.52, 74.55, 75.44, 75.70, 75.75 (C2b, C3, C30),
95.07 (C1b), 99.17 (C1a), 100.95, 100.98 (C10); FAB-
MS m/z 343.1239 (C12H23O11 [M+H]+ requires
343.1240).
(iii) The octa-acetate from (ii) (37 mg) in MeOH
(4 mL) was stirred with NaOMe in MeOH (1 mL) (rt,
1 h). The solution was neutralized with resin (Amberlite
IR-120, H+), filtered and the filtrate concentrated to give
4-O-(a-D-glucopyranosyl)-D-galactopyranose (18 mg) as
a colourless oil and as a mixture of anomers (a:b, 0.4:1).
1
[a]D +116.5 (H2O); H NMR (600 MHz, D2O): dH 3.45
(dd, J = 9.7, 9.7, H30b), 3.50–3.55, 3.70–3.93, 4.09–4.15
(3 · m, H2, H20, H3, H30a, H40, H5, H50, H6, H60), 4.00
(d, J = 2.9, H4b), 4.07 (d, J = 2.7, H4a), 4.64 (d,
J1,2 = 7.8, H1b), 4.92–4.93 (m, H10), 5.29 (d, J1,2 = 3.8,
H1a); 13C NMR (150.9 MHz, D2O): dC 62.79, 62.82,
62.87, 63.08 (C6, C60), 71.17–77.81 (C2, C20, C3, C30,
C40a, C5, C50), 80.09 (C4b), 81.39 (C4a), 95.08 (C1a),
99.35 (C1b), 102.70 (C10b), 102.90 (C10a); FAB-MS
m/z 342.1235 (C12H23O11 [M+H]+ requires 343.1240).
4.2. Evaluation of the four disaccharides
4.2.1. General methods. a-Amylase (human saliva), a-
galactosidase (green coffee beans) and a-glucosidase
and isomaltase (baker’s yeast) were purchased from Sig-
ma Chemicals. The ‘glucose oxidase reagent 1’ consisted
of glucose oxidase (10.4 U/mL), peroxidase (3.1 U/mL)
and ABTS (300 lg/mL); ‘glucose oxidase reagent 2’ con-
sisted of glucose oxidase (20.8 U/mL), peroxidase
(12.2 U/mL), tris buffer (pH 7.0, 0.5 M) and ABTS
(600 lg/mL).
4.1.16. 6-O-(a-D-Glucopyranosyl)-D-galactopyranose. (i)
Disaccharide 7 (139 mg) in MeOH (25 mL) was stirred
with Pd/C (10%, 20 mg) and H2 (2 d, 1 atm). The mix-
ture was filtered, the filtrate concentrated and subjected
to flash chromatography (EtOAc/petrol, 2:1) to give
6-O-(a-D-glucopyranosyl)-1,2:3,4-di-O-isopropylidene-a-
4.2.2. Method for glucose measurement. The enzyme in
phosphate buffer (Table 5; 50 lL, 4–8 U/mL, pH 5.0–
6.9) was added to samples and standards in doubly-
deionized (DDI) water (5 lL, 0–3 mM) in the wells of
a microtiter plate, and the plate mixed on a plate shaker.
The plate was incubated at 37 ꢁC for 60 min. After
this time the ‘glucose oxidase reagent 1’ in phosphate
buffer (150 lL) was added to each well and the plate
mixed. Absorbance of the ABTS was monitored at
405 nm every 5 min until peak absorbance reached a
maximum.
1
D-galactopyranose (60 mg, 89%) as a colourless oil. H
NMR (600 MHz): dH 1.30, 1.32, 1.41, 1.52 (4 · s, 12H,
CH3), 3.53–3.58, 3.62–3.63, 3.73–3.76 (3 · m, 5H, H20,
H30, H40, H5, H6), 3.69 (dd, J6 ,6 = 10.8, J5 ,6 = 5.2,
0
0
0
0
H60), 3.80 (dd, J5 ,6 = 7.2, H60), 3.84–3.86 (m, 1H,
H6), 3.96 (m, H50), 4.25 (dd, J4,5 = 1.5, J3,4 = 8.0, H4),
4.29 (dd, J2,3 = 2.4, J1,2 = 4.9, H2), 4.59 (dd, H3), 4.86
0
0
(d, J1 ,2 = 3.5, H10), 5.51 (d, H1); 13C NMR
(150.9 MHz): dC 24.64, 25.02, 26.09, 26.13 (4C,
CH3C), 61.35, 67.25 (C6, C60), 66.65, 69.62 (C5, C50),
70.56 (C2), 70.71 (C3), 71.24 (C4), 71.99, 72.11, 74.24
(C20, C30, C40), 96.34 (C10), 99.38 (C1), 108.96, 109.58
(2C, CH3C); FAB-MS m/z 423.1873 (C18H31O11
[M+H]+ requires 423.1866).
0
0
4.2.3. Processing of pig intestinal samples. Intestinal
mucosa samples were taken from a freshly euthanased
19.5 kg pig. The pig was restricted from feed for 12 h
prior to death to minimize the quantity of undigested
material. Sampling commenced 20 cm into the small
intestine, with an interval of 50 cm between samples.
At each interval a 20 cm portion of small intestine was
removed, the sample was washed with isosmotic saline,
cut open, patted dry with filter paper and the mucosa
scraped using a glass microscope slide. Approximately
1 g of the mucosa was suspended in DDI water
(20 mL) and the sample homogenized by sonication.
(ii) The tetrol from (i) (26 mg) was stirred in
CF3COOH/H2O (4:1, 2 mL) (0 ꢁC, 1 h). The solution
was then concentrated, applied to a Sephadex (IR 120)
column and eluted with H2O. Concentration of the elu-
ant gave 6-O-(a-D-glucopyranosyl)-D-galactopyranose
(18.5 mg) as a colourless oil and as a mixture of anomers
1
(a:b, 0.6:1); [a]D +119.0 (H2O); H NMR (600 MHz,
D2O): dH 3.39 (m, H30), 3.48 (dd, J2,3 = 9.8, J1,2 = 7.9,
H2b), 3.54–3.56 (m, H20), 3.65 (dd, J3,4 = 3.4, H3b),
Table 5. The enzymes used in glucose measurement
Enzyme
Enzyme concentration (U/mL)
Buffer system (concn)
pH
Additional reagent
NaCl (67 mM)
a-Amylase
8
4
8
8
Potassium phosphate (20 mM)
Potassium phosphate (100 mM)
Potassium phosphate (100 mM)
Potassium phosphate (50 mM)
6.9
5.0
5.0
6.7
a-Galactosidase
a-Glucosidase
Isomaltase

P. J. Meloncelli et al. / Carbohydrate Research 342 (2007) 1793–1804
Table 6. The disaccharide reagents used in disaccharidase assays
1803
Maltose reagent
Sucrose reagent
Lactose reagent
Isomaltose reagent
a-Glcp-(1!6)-Gal reagent
Maleate buffer,
pH 6.9, 0.1 M
Maltose, 0.1 M
Maleate buffer,
pH 6.9, 0.1 M
Sucrose, 0.1 M
Maleate buffer,
pH 6.9, 0.1 M
Lactose, 0.1 M
Maleate buffer,
pH 6.9, 0.1 M
Isomaltose, 0.1 M
Maleate buffer,
pH 6.9, 0.1 M
a-Glcp-(1!6)-Gal, 0.1 M
Table 7. The pig intestinal homogenates used in disaccharidase assays
Enzyme
Reagent
volume (lL)
Homogenate
volume (lL)
Incubation
time (min)
Blank incubation
time (min)
Maltase
Sucrase
Lactase
Isomaltase
90
50
50
50
50
10
50
50
50
50
120
120
120
120
360
60
60
60
60
180
Disaccharidase acting on a-Glcp-(1!6)-Gal
The volume was made up to 50 mL with DDI water and
the sample centrifuged to remove cellular debris
(2000 rpm, 10 min). The supernatant was collected and
stored (ꢀ20 ꢁC) until required for testing.
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