Efficient synthesis and activity of beneficial intestinal flora of two lactulose-derived oligosaccharides

Article abstract of DOI:10.1016/j.ejmech.2016.03.007

Lactulose is considered as a prebiotic because it promotes the intestinal proliferation of Lactobacillus acidophilus which is added to various milk products. Moreover, lactulose is used in pharmaceuticals as a gentle laxative and to treat hyperammonemia. This study was aimed at the total synthesis of two Lactulose-derived oligosaccharides: one is 3-O-β-d-galactopyranosyl-d-fructose, d-fructose and β-d-galactose bounded together with β-1,3-glycosidic bound, the other is 1-O-β-d-galactopyranosyl-d-fructose, d-fructose and β-d-galactose bounded together with β-1,1-glycosidic bound, which were accomplished in seven steps from d-fructose and β-d-galactose and every step of yield above 75%. This synthetic route provided a practical and effective synthetic strategy for galactooligosaccharides, starting from commercially available monosaccharides. Then we evaluated on their prebiotic properties in the search for potential agents of regulating and improving the intestinal flora of human. The result showed that the prebiotic properties of Lactulose-derived oligosaccharides was much better than Lactulose. Among them, 3-O-β-d-galactopyranosyl-d-fructose displayed the most potent activity of proliferation of L. acidophilus.

Full text of DOI:10.1016/j.ejmech.2016.03.007

European Journal of Medicinal Chemistry 114 (2016) 8e13
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Efficient synthesis and activity of beneficial intestinal flora of two
lactulose-derived oligosaccharides
Zhen-Yuan Zhu a, b, , Di Cui , Hui Gao , Feng-Ying Dong , Xiao-cui Liu , Fei Liu ,
*
a
a
a
a
a
a
c
Lu Chen , Yong-min Zhang
Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Science and Biotechnology, Tianjin University of Science and
Technology, Tianjin, 300457, PR China
Tianjin Food Safety & Low Carbon Manufacturing Collaborative Innovation Center, 300457, Tianjin, PR China
Sorbonne Universit ꢀe s, UPMC Univ Paris 06, Insititut Parisien de Chimie Mol ꢀe culaire, CNRS UMR 8232, 4 Place Jussieu, 75005, Paris, France
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 September 2015
Received in revised form
Lactulose is considered as a prebiotic because it promotes the intestinal proliferation of Lactobacillus
acidophilus which is added to various milk products. Moreover, lactulose is used in pharmaceuticals as a
gentle laxative and to treat hyperammonemia. This study was aimed at the total synthesis of two
1
March 2016
Lactulose-derived oligosaccharides: one is 3-O-
galactose bounded together with -1,3-glycosidic bound, the other is 1-O-
tose, -fructose and -galactose bounded together with -1,1-glycosidic bound, which were accom-
plished in seven steps from -fructose and -galactose and every step of yield above 75%. This synthetic
b
-D-galactopyranosyl-D-fructose,
D
-fructose and
b-D-
Accepted 2 March 2016
Available online 3 March 2016
b
b
-D
-galactopyranosyl-D-fruc-
D
b
-D
b
D
b-D
Keywords:
Lactulose
Lactulose-derived oligosaccharides
Prebiotics
Lactobacillus acidophilus
route provided a practical and effective synthetic strategy for galactooligosaccharides, starting from
commercially available monosaccharides. Then we evaluated on their prebiotic properties in the search
for potential agents of regulating and improving the intestinal flora of human. The result showed that the
prebiotic properties of Lactulose-derived oligosaccharides was much better than Lactulose. Among them,
3-O-
b
-D
-galactopyranosyl-D-fructose displayed the most potent activity of proliferation of L. acidophilus.
©
2016 Elsevier Masson SAS. All rights reserved.
1
. Introduction
stool, hence the lactulose can be used as a laxative [2]. Lactulose has
prebiotic property, because it stimulates the growth of health-
promoting bacteria in the gastrointestinal tract, such as lactoba-
cilli and bifidobacteria and at the same time inhibits pathogenic
bacteria such as Salmonella [3]. In addition, lactulose is used in the
pharmaceutical field for treating constipation, hepatic encepha-
lopathy and complications of liver disease, and it maintains blood
glucose and insulin levels [4].
Lactulose synthetic methods usually can be divided into
chemical and enzymetic. The chemical methods are essentially
based on the isomerization of lactose by using many catalysts, such
as sodium hydroxide, potassium hydroxide, sodium carbonate,
magnesium oxide [5e7]. Montgomery and Hudson synthesized
lactulose with calcium hydroxide for the first time [8]. These pro-
cesses generally produce a high level of lactulose degradation,
which leads to the formation of a considerable percentage of
difficult to separate, colored by-products which lower the lactulose
yield. Another group of processes uses complexing reagents such as
aluminates and borates, which facilitate the reaction with a mini-
mum of secondary reactions and result in a high yield of lactulose
Lactulose (4-O-
disaccharide composed of two sugar molecules
galactose bounded together with -1,4-glycosidic bound. Lactulose
is 1.5 times sweeter than Lactose and can be crystallized from
alcohol solution. The -glycosidic linkage of the lactulose is not
hydrolyzed by mammalian digestive enzymes and ingested lactu-
lose passes the stomach and small intestine without degradation. It
is characteristically utilized by all the species of Lactobacillus aci-
dophilus, which resides in the human intestine tract [1]. In the
colon, large number of bacteria metabolizes lactulose and con-
sumes it as their own food. In doing so, these bacteria produce
lactic, acetic, and formic acid as well as carbon dioxide gas. These
acids biochemically draw fluid into the bowel which softens the
b-
D
-galactopyranosyl-
D
-fructose),
a synthetic
D
-fructose and
b-D-
b
b
*
Corresponding author. Key Laboratory of Food Nutrition and Safety, Ministry of
Education, College of Food Science and Biotechnology, Tianjin University of Science
and Technology, Tianjin, 300457, PR China.
E-mail address: zhyuanzhu@tust.edu.cn (Z.-Y. Zhu).
http://dx.doi.org/10.1016/j.ejmech.2016.03.007
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

Z.-Y. Zhu et al. / European Journal of Medicinal Chemistry 114 (2016) 8e13
9
by eliminating lactulose from the reaction equilibrium mixture in
the form of a complex [9,10]. However, they are unsatisfactory from
the industrial aspect because of the difficulty of eliminating the
aluminate and borate. Also in these processes a large excess of
borate and aluminate is necessary for optimal yield. The chemical
methods have several drawbacks since reaction is poorly specific,
side reactions occurring so that low product yields are obtained,
colored by-products are formed and intense purification is required
compounds after deprotection with acetic acid conditions to give
compound 10,13. The structures of all the synthetic compounds
were fully characterized by spectroscopic data (NMR, MS).
3. Results and discussion
For many methods, including the indican enzyme synthesis and
chemical synthesis, enzymatic synthesis is of advantages, such as
mild reaction, good region, and stereoselectivety. However, it re-
quires glycosyltransferase and glycosidase-catalysed, and the
enzyme is expensive. The enzyme on the substrate specificity is
strong. Relatively, the chemical synthesis is indispensable. The core
of the formation of glycosidic bond is an anomeric acetal synthesis,
because the dehydration directly generating glycosidic bond is a
violent reaction, so the usual method is to use a leaving groups at
anomeric position (donor), which reacts with an alcohol (accepter)
to give, in the presence of a promoter, obtained the desired
glycoside.
Knorr-Koenigs reaction is a classic glycosylation, using a bro-
mide as donor and heavy metals as promoters, and the problem of
this reaction is that the promoting agents are expensive and envi-
ronment unfriendly. The Schmidt method in 1980, so-called imi-
date method, is the most valuable glycosidic bond synthesis
method, which has advantage of good stability, easy operation, high
yield, good selectivity, etc. In the reaction, we chose tri-
[11,12]. For these reasons any study to develop a feasible process to
produce lactulose is very important.
The enzymatic transgalactosylation reaction from lactose by b-
galactosidases obtaining complex mixtures of oligosaccharides
with different glycosidic linkages and degree of polymerization
depending on the source of the enzymes and experimental condi-
tions [13e16]. In addition, these products contain glucose, galac-
tose and lactose, without prebiotic properties, which increase the
calorific value of the product. Until recently, lactulose have been
obtained only from lactose, but currently there is a great interest in
obtaining new prebiotic carbohydrates with improved properties,
addressed to reach the distal regions of the colon unaltered to
promote the growth of specific bacteria. Due to the prebiotic
properties that lactulose show, Martínez-Villaluenga, C. and
Cardelle-Cobas, A. carried out enzymatic and chemical approaches
for the synthesis of new lactulose-derived oligosaccharides, which
may be also bioactive compounds and whose beneficial properties
should be investigated [17,18]. Makras et al. reported Gal-
chloroacetimidate as b-D-galactose 1-OH activation group, Trime-
actooligosaccharides synthesized by
bacilli and bifidobacteria contain mainly
di- and trisaccharides, which may have prebiotic effects specifically
targeting those strains better than -(1e4) linkages [19].
In this work we report the chemical synthesis of two Lactulose-
derived oligosaccharides: 3-O- -galactopyranosyl- -fructose and
-O- -galactopyranosyl- -fructose from commercially available
-fructose and -galactose (Fig. 1). It is valuable to develop an
b
-galactosidases from lacto-
thylsilyl trifluoromethanesulfonate (TMSOTf) as promoter, Acetyl
groups have been used as hydroxyl protection. As shown in
Schemes 1 and 2, were readily prepared in good yields from the
corresponding 1-OH sugars. The reaction of glycosylation achieved
b
-(1e1) or -(1e3) linked
b
b
in the conditions described above, gave the compound 3-O-
Galactopyranosyl- -fructose in 86% yield and 1-O- -Galactopyr-
anosyl- -fructose in 85% yield. The stereochemistry of the newly
introduced linkage was determined to be on the basis of the Glc
b-D-
b
-D
D
D
b-D
1
D
b-
D
D
D
b-
D
b
efficient way to prepare this kind of oligosaccharides to promote
further studies, such as thorough pharmacological research and
further structure-activity relationship investigation. The pre-
liminary activity of regulating and improving the intestinal flora of
these synthetic oligosaccharides was then investigated.
H-1, H-2 coupling constant (J1,2 ¼ 7.6 Hz) and Gal H-1, H-2 Coupling
constant (J1,2 ¼ 8.4 Hz) [20]. L. acidophilus was grown in simulated
intestinal fluid demonstrated that 3-O-b-D-galactopyranosyl-D-
fructose is the best carbon source for promoting the growth of L.
acidophilus, which proliferation activity is better than lactulose.
2
. Chemistry
4. Conclusion
The synthetic procedures and reaction conditions are shown in
Schemes 1 and 2. Reaction of b-D-galactose (4) in acetic anhydride
with sodium acetate gave compound 5. Reaction of compound 5 in
diethyl ether with benzylamine provided compound 6, which, after
protection with trichloroacetimidate, gave compound 7. Reaction of
Lactulose chemical structures show a great variability, this af-
fects prebiotic properties. Nevertheless, Studies including synthetic
lactulose are scarce, and little information is available about the
specific preferences of bacteria with respect to lactulose linkage or
composition to compare with. In this work the influence of factors
have been investigated both qualitatively and quantitatively. In-
testinal flora (L. acidophilus) were shown to be able to utilize lac-
tulose and different lactulose-derived as carbon sources. It was
observed that glycosidic linkage affected the individual strains
growth. Our data showed a general preference of the strains
D
-fructose (1) in acetone with sulfuric acid gave compound 2,3.
Compound 7 reacted with compound 2,3 respectively in the pres-
ence of trimethylsily trifluoromethanesulfonate (TMSOTf) to pro-
vide compound 8,11, which were deprotected in methanol with
sodium methylate to give compound 9,12. Then these two
HO
HO
OH
HO
OH
O
OH
O
OH
O
O
O
HO
O
HO
HO
OH
OH
OH
OH
HO
OH
OH
O
HO
O
HO
OH
Lactulose
O
OH
OH
HO
OH
Compound 13
Compound 10
Fig. 1. Structures of compounds Lactulose (4-O-b-D-galactopyranosyl-D-fructose), Compound 13 (1-O-b-D-galactopyranosyl-D-fructose) and Compound 10 (3-O-b-D-galactopyr-
anosyl-D-fructose).

10
Z.-Y. Zhu et al. / European Journal of Medicinal Chemistry 114 (2016) 8e13
ꢁ
3 2
CO) O, 120 C,
Scheme 1. The synthetic route of Lactulose-derived oligosaccharides(Compound 10). Reagents and conditions: (a) H
2
SO
4
, acetone, r.t, 2 h, 88%; (b) CH
3
COONa, (CH
ONa, CH OH, r.t, 2 h, 85%;
ꢁ
ꢁ
ꢁ
1
.5 h, 96%; (c) benzylamine, diethyl ether, 0 C, 2.5 h, 86%; (d) trichloroacetonitrile, DBU, CH
2
Cl
2
, 0 C, 4 h, 88%; (e) TMSOTf, CH
2
Cl2, -20 C, 2 h, 75%; (f) CH
3
3
ꢁ
(
g) CH
3
COOH, 60 C, 8 h, 86%.
towards
over those of
b
-galactosyl residues having
b
(1e3) and
b
(1e1) linkages
aperture 0.20 microns of microporous membrane filter, made
simulated intestinal fluid.
b
(1e4). Besides, results indicate that oligosaccharides
derived from lactulose emerge as candidates to have prebiotic
properties, although further research on their functionality and
safety is required.
L. acidophilus was provided by the Research Center of Food
Biotechnology, Tianjin University of Science and Technology. The
Prefabricated MRS(Man-Rogosa-Sharpe) medium contained 10 g
proteose peptone, 10 g beef extract, 5 g sodium acetate, 0.1 g
magnesium sulfate, 0.05 g manganese sulfate and 2 g dipotassium
phosphate per liter at pH ¼ 6.5. For long-time storage, glycerol-
stocks (33% v/v) of a stationary phase culture were prepared and
5
. The activity of promoting Lactobacillus acidophilus
proliferation
ꢁ
maintained at ꢀ80 C, MRS broth medium was used for activation
A drug's solubility and dissolution behavior within the gastro-
intestinal tract is a key property for successful administration by
the oral route and one of the key factors in the biopharmaceutics
classification system. A compound's pKa for example can induce
extreme effects due to the variation of pH in the intestine about 6.8,
This can greatly affect solubility of for example weakly basic com-
pounds, which could rapidly dissolve in the intestine [21]. This
property can be determined by investigating drug solubility in
human intestinal fluid but this is difficult to obtain and highly
variable, which has led to the development of multiple simulated
intestinal fluid recipes. In this work stock solution of the various
components of the simulated intestinal media were freshly pre-
of strains.
1% inoculum of L. acidophilus was grown in simulated intestinal
fluid with added to different cabon sources (4-O- -galactopyr-
b-D
anosyl-
D
-fructose, 3-O-
b
-
D
-galactopyranosyl-
D
-fructose and 1-O-
b
-
ꢁ
D-galactopyranosyl-
D
-fructose) and incubated together at 36 C in a
shaker. The cultures were halted after 1 h, 2 h, 3 h and their optical
densities were measured at 600 nm (OD600), the results are illus-
trated in (Fig. 2).
6. Experimental section
pared: 3.4 g KH
2
PO
4
dissolved in 250 mL distilled water, with 0.4 g/
6.1. Chemistry
100 mL NaOH solution to adjust pH value to 6.8, then diluted to
5
00 mL, and added to trypsin in the solution, made its mass con-
All chemicals (reagent grade) used were purchased from Sig-
centration of 1 g/100 mL, blending dissolves adequately, used
maeAldrich (U.S.A) and Aladdin-Reagent Co.,Ltd (China). TLC (thin

Z.-Y. Zhu et al. / European Journal of Medicinal Chemistry 114 (2016) 8e13
11
Scheme 2. The synthetic route of Lactulose-derived oligosaccharides(Compound 13). Reagents and conditions as same as Scheme 1.
Fig. 2. Comparison of Lactobacillus acidophilus using different carbon source in simulated intestinal fluid growth of OD600
.
layer chromatography) was run on the silica gel coated aluminum
6.1.1. The acetyl-b-D-galactopyranose 5
1
13
sheets (Silica Gel 60 GF254, E, Merck, Germany). H NMR and
C
Sodium acetate (1.0 g, 12 mmol) was added in acetic anhydride
ꢁ
NMR were recorded on a Bruker AVANCE instrument (400 MHz)
with tetramethylsilane as internal standard, and chemical shifts
values were recorded. ESI (Electrospray Ionization) mass spectra
were obtained on an LCQ-Advantayc-MAX (LAM10188, Finnigan,
Co., Ltd, USA). Specific rotation were obtained on an Automatic
Polarimeter (WZZ-2B, Shanghai precision scientific instrument,
Co.,Ltd, China). Flash column chromatography was carried out us-
ing silica gel (200e300 mesh, Qingdao Marine Chemical Group Co.,
Qingdao, china).
20 mL was heated to 120 C for 30 min, then
b
-D-galactopyranose
(2.0 g, 11 mmol) was added dropwise in the solution under stirring.
ꢁ
After 1 h of the reaction at 120 C, the mixture was cooled and
3
poured into cold water, then washed with saturated NaHCO so-
lution to pH ¼ 7. The precipitate was separated and the mixture was
extracted with dichloromethane. The combined organic phases
were washed with saturated brine and water, successively. Then,
the organic layer was dried with sodium sulfate and concentrated.
The residue was purified by silica gel column chromatography

12
Z.-Y. Zhu et al. / European Journal of Medicinal Chemistry 114 (2016) 8e13
(
5
petroleum ether-ethyl acetate 3:1, Rf ¼ 0.35) to obtain compound
4.112 (m, J ¼ 10.8 Hz, 2H); 4.218 (m, J ¼ 6.2 Hz, 1H); 4.140 (s, 1H);
(4.2 g, 96%).
4.123 (m, J ¼ 10.2 Hz, 2H); 1.984 (s,1H).
6.1.2. 2,3,4,6-Tetra-O-acetyl-b-D-galactopyranose 6
To a mixture of compound 5 (4 g, 10.3 mmol) and 4 Å molecular
6.1.6. 1,2:4,5-di-O-(1-methyl)ethylidene-3-O-(2,3,4,6-tetra-O-
sieves (1.8 g) in diethyl ether 20 mL, then benzylamine 40 mL was
added dropwise in the solution under stirring. The reaction mixture
was continuously stirred for 2.5 h at 0 C, the solvent was removed
acetyl-b-D-Galactopyranosyl)-D-f-ructose 8
To a mixture of compound 7(108.0 mg, 0.22 mmol), compound 2
(29 mg, 0.11 mmol) and 4 Å molecular sieves (300 mg) in anhy-
ꢁ
ꢁ
by rotary evaporator. The residue was dissolved in dichloro-
methane, washed by 1 M HCl solution to pH ¼ 7, The organic phases
were washed with saturated brine. Then, the organic layer was
dried with sodium sulfate and concentrated in vacuo. The residue
was purified by silica gel column chromatography (petroleum
drous dichloromethane 5 mL. It was stirred for 0.5 h at 0 C, and
then TMSOTf (4.0
m
L, 0.022 mmol) was added under Ar protection
ꢁ
and the mixture was stirred at ꢀ20 C for 2 h, TLC showed no
3
remaining material, then neutralized with Et N. The reaction
mixture was then diluted with dichloromethane, and filtered to
remove the molecular sieves, which was washed with water and
saturated brine, dried with sodium sulfate, filtered and concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (petroleum ether-ethyl acetate 3:2) to give com-
1
ether-ethyl acetate 2:1, Rf ¼ 0.5) to give compound 6 (3.1 g, 86%). H
3
NMR (400 MHz, CDCl , dppm): 2.051, 2.067, 2.097, 2.106 (s, 12H),
4
.440 (m, J ¼ 8.2 Hz,1H), 5.176 (d, J ¼ 3.6 Hz,1H), 5.414 (s,1H), 5.433
(
s, 1H), 5.441 (s, 1H), 5.474 (s, 1H), 5.802 (s, 1H), 7.324 (m, 1H).
2
0
1
pound 8 (65 mg, 75%, Rf ¼ 0.55). [
a]
D
ꢀ 28 (c ¼ 0.01, CH
2 2
Cl ). H
6.1.3. 2,3,4,6-Tetra-O-acetyl-
b
-D-galactopyranosyl
NMR (400 MHz, CDCl ppm): 1.259, 1.350, 1.458, 1.526 (s, 12H);
3
, d
trichloroacetimidate 7
To a mixture of compound 6 (2.65 g, 7.6 mmol) and 4 Å mo-
lecular sieves (1.2 g) in anhydrous dichloromethane 10 mL, after
1.977 (s, 3H); 2.035 (m, J ¼ 8.4 Hz, 8H); 2.170 (s, 3H); 3.711 (m, 2H);
3.933 (d, J ¼ 9.4 Hz, 3H); 4.152 (m, J ¼ 8.6 Hz, 4H); 4.240 (d,
J ¼ 7.2 Hz, 1H); 4.426 (s, 1H); 4.590 (d, J ¼ 5.6 Hz, 2H); 5.014 (dd,
13
that DBU (0.23 g) was added in the solution under stirring for
J ¼ 12.6 Hz, 1H); 5.226 (m, J ¼ 10.4 Hz, 1H); 5.382 (s, 1H). C NMR
ꢁ
1
0 min at 0 C. Then trichloroacetonitrile (2.7 mL) was added
3
(400 MHz, CDCl , dppm): 20.56, 20.64, 20.72, 20.81, 24.02, 25.45,
dropwise in the solution for 3.5 h at room temperature. The
mixture was poured into cold water, then washed with saturated
brine. Then, the organic layer was dried with sodium sulfate and
concentrated in vacuo. The residue was purified by silica gel column
25.83, 26.59, 61.07, 61.32, 67.15, 68.86, 70.12, 70.83, 70.98, 71.20,
76.72, 77.04, 77.36, 100.74, 101.88, 108.69, 108.98, 169.31, 170.16,
170.33, 170.42. HR-ESI-MS m/z: 590.5711, calculated for C26
H
38
O
15
þ
[M þ NH
4
]
608.5211.
chromatography (petroleum ether-ethyl acetate 1:1, Rf ¼ 0.55) to
1
give compound 7 (3.25 g, 88%). H NMR (400 MHz, CDCl
3
,
dppm):
2
1
1
.017, 2.021, 2.030, 2.169 (s, 12H), 4.109 (m, J ¼ 8.2 Hz, 1H), 4.176 (m,
6.1.7. 2,3:4,5-di-O-(1-methyl)ethylidene-1-O-(2,3,4,6-tetra-O-
acetyl- -Galactopyranosyl)-D-f-ructose 11
H), 4.443 (m, J ¼ 13.6 Hz, 1H), 5.385 (m, 2H), 5.449 (s, 1H), 6.661 (s,
b-D
H), 8.667 (s, 1H).
The procedure is the same as that for compound 8, silica gel
column chromatography (petroleum ether-ethyl acetate 3:2) to
2
0
6
.1.4. 1,2:4,5-di-O-(1-methyl) ethylidene-
Sulfuric acid (0.3 mL) was added in acetone (60 mL) under
stirring for 10 min, then -fructopyranose (3.2 g) was added, and
D-fructose 2
give compound 11 (65 mg, 75%, Rf ¼ 0.55). [
a
]
D
ꢀ 32 (c ¼ 0.01,
1
CH Cl ). H NMR (400 MHz, CDCl , dppm): 1.344, 1.351, 1.458, 1.526
2 2 3
D
(s, 12H); 1.977, 2.026, 2.036, 2.171 (s, 12H); 3.711 (m, J ¼ 11.4 Hz,
2H); 3.932 (d, J ¼ 10.4 Hz, 3H); 4.153 (d, J ¼ 7.4 Hz, 2H); 4.241 (d,
J ¼ 8.0 Hz, 1H), 4.425 (d, J ¼ 8.4 Hz, 1H); 4.612 (d, J ¼ 8.0 Hz, 2H);
5.015 (dd, J ¼ 14.2 Hz, 1H); 5.231 (m, J ¼ 10.4 Hz, 1H); 5.380 (d,
the mixture was stirred at room temperature. After 2 h, TLC showed
no remaining material, and 1 mol/L sodium hydroxide solution
ꢁ
(
8.5 mL) was added dropwise in the solution at 0 C. The precipitate
1
3
was separated and the mixture was concentrated in vacuo, then
dissolved in the dichloromethane 100 mL, and washed with water
and saturated brine, successively. The organic layer was dried with
sodium sulfate and concentrated in vacuo. Obtain light yellow solid
and recrystallization with petroleum ether, after filtration and
concentrated in vacuo to give the white solid 2(3.9 g, 88%,
J ¼ 3.2 Hz, 1H). C NMR (400 MHz, CDCl3,
dppm): 24.01, 25.42,
25.81, 26.56, 61.05, 61.30, 67.14, 68.85, 69.60, 69.78, 70.10, 70.81,
70.95, 71.17, 76.72, 77.04, 77.36, 100.72, 101.86, 108.65, 108.94,
169.26, 170.10, 170.28, 170.36. HR-ESI-MS m/z: 590.5782, calculated
þ
for C26H
38
O15 [M þ NH
4
]
608.6204.
2
0
1
Rf ¼ 0.45). [
a]
D
ꢀ 98 (c ¼ 0.01, CH
2 2 3
Cl ). H NMR (400 MHz, CDCl ,
dppm): 1.357, 1.404, 1.488, 1.554 (s, 12H); 3.682 (m, 2H); 3.942 (d,
6.1.8. 1,2:4,5-di-O-(1-methyl)ethylidene-3-O-(
b-D-
J ¼ 2.0 Hz, 1H); 3.910 (d, J ¼ 1.6 Hz, 1H); 4.238 (d, J ¼ 1.2 Hz, 1H);
Galactopyranosyl)- -fructose 9
D
4
.257 (s, 1H); 1.625 (d, 1H); 1.918 (s, 1H).
To a mixture of compound 8(100 mg, 0.17 mmol) and sodium
methylate (10.8 mg) in 10 mL of methanol was stirred at room
temperature for 2 h. The solvent was removed by rotary evaporator.
The residue was dissolved in dichloromethane, washed by water
and saturated brine, successively. The organic layer was dried with
sodium sulfate and concentrated in vacuo. The residue was purified
by silica gel column chromatography (dichloromethane-
methanol ¼ 10:1, Rf ¼ 0.45) to give compound 9 as white powder
6
.1.5. 2,3:4,5-di-O-(1-methyl) ethylidene-
sulfuric acid (2.0 mL)was added in acetone (46 mL) under stir-
ring for 10 min, then -fructose (4.7 g) was added, and the mixture
D
-fructose 3
D
was stirred at room temperature. After 2 h, TLC showed no
remaining material, and 1 mol/L sodium hydroxide solution
ꢁ
(
7.5 mL) was added dropwise in the solution at 0 C. The precipitate
2
0
1
was separated and the mixture was concentrated in vacuo, then
dissolved in the dichloromethane 100 mL, and washed with water
and saturated brine, successively. The organic layer was dried with
sodium sulfate and concentrated in vacuo. Obtain light yellow solid
and recrystallization with petroleum ether, after filtration and
concentrated in vacuo to give the white solid 3 (5.6 g, 83%,
(60 mg, 85%). [
a]
D
ꢀ 65 (c ¼ 0.01, CH
2 2
Cl ). H NMR (400 MHz,
CDCl ppm): 1.422, 1.446, 1.472, 1.523 (s, 12H); 3.460 (s, 1H); 3.561
3
, d
(d, J ¼ 4.8 Hz, 1H); 3.781 (m, 5H); 3.932 (m, 3H); 4.117 (s, 1H); 4.247
(d, J ¼ 8.2 Hz, 1H); 4.349 (m, J ¼ 10.8 Hz, 2H); 4.491 (s, 1H); 4.616 (d,
13
J ¼ 10.2 Hz, 1H). C NMR (400 MHz, CDCl3,
dppm): 23.92, 25.40,
25.74, 26.12, 26.47, 27.97, 29.69, 70.16, 70.81, 71.26, 71.41, 76.73,
2
0
1
Rf ¼ 0.45). [
a
]
D
ꢀ 49 (c ¼ 0.01, CH
2
Cl
2
). H NMR (400 MHz, CDCl
3
,
77.05, 77.37, 102.21, 103.56, 108.77, 109.02. HR-ESI-MS m/
þ
d
ppm): 1.372, 1.444, 1.516, 1.536 (s, 12H); 3.674 (d, J ¼ 7.2 Hz, 1H);
z:422.4162, calculated for C18
H
30
O
11 [M þ NH
4
]
440.4056.

Z.-Y. Zhu et al. / European Journal of Medicinal Chemistry 114 (2016) 8e13
13
6
.1.9. 2,3:4,5-di-O-(1-methyl)ethylidene-1-O-(
Galactopyranosyl)- -fructose 12
The procedure is the same as that for compound 9, silica gel
column chromatography dichloromethane-methanol 10:1,
Rf ¼ 0.45) to give compound 12 as white powder (65 mg, 75%,
b
-
D
-
Appendix A. Supplementary data
D
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.03.007.
¼
2
0
1
Rf ¼ 0.55). [
a
]
D
ꢀ 68 (c ¼ 0.01, CH
2 2 3
Cl ). H NMR (400 MHz, CDCl ,
References
d
3
1
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.747 (m, 2H); 3.769 (s, 1H); 3.778 (s, 1H); 3.807 (s, 1H); 3.834 (s,
H); 3.897 (d, J ¼ 1.6 Hz, 1H); 3.933 (m, 2H); 4.009 (m, 1H); 4.131
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2
7
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1.53, 71.77, 73.36, 75.23, 76.70, 77.02, 77.34, 102.23, 103.67, 108.61,
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18 30 11
C H O
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[M þ NH
[
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2
0
1
[
(
(
a
]
D
ꢀ 44 (c ¼ 0.01, H
2 3
O). H NMR (400 MHz, CDCl , dppm): 3.374
m, J ¼ 12.4 Hz, 1H); 3.498 (m, J ¼ 10.6 Hz, 2H); 3.581 (s, 2H); 3.724
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1
3
C NMR (400 MHz, CDCl3, dppm): 60.23, 61.49, 62.42, 69.32, 71.14,
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þ
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22
H O
11 [Mþ2NH
4
]
377.0762.
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[
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.1.11. 1-O-
b-D-Galactopyranosyl-D-fructose 13
2
0
The procedure is the same as that for compound 10, [
a
]
D
ꢀ 42
1
(
c ¼ 0.01, H
2
O). H NMR (400 MHz, CDCl , dppm): 3.497 (m,
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3
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(
7
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d
[
[
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H
22
O11 [Mþ2NH
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Acknowledgments
This work was financially supported by the National Spark Key
Program of China (2015GA610001), the Foundation of Tianjin
University of Science and Technology (Nos. 20120106), the Inter-
national Science and Technology Cooperation Program of China
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(
2013DFA31160), and the Foundation of Tianjin Educational Com-
757.
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