Synthesis of an allergy inducing tetrasaccharide “4P-X”

Article abstract of DOI:10.1016/j.carres.2016.11.013

4P-X (β-D-galactopyranosyl-(1?→?4)-β-D-galactopyranosyl-(1?→?6)-[β-D-galactopyranosyl-(1?→?4)]-β-D-glucopyranose) is included in galacto-oligosaccharides (GOSs) produced by β-galactosidase derived from Bacillus circulans. 4P-X has been known to induce particularly strong allergies. High purity 4P-X is essential for use as a standard to quantify the amount of 4P-X in GOSs; however, the isolation of high purity 4P-X has never been reported. In this study, we achieved the synthesis of 4P-X by a combination of organic and enzymatic chemical syntheses in a short time. This is the first report of isolated, high purity 4P-X.

Full text of DOI:10.1016/j.carres.2016.11.013

Accepted Manuscript
Synthesis of an allergy inducing tetrasaccharide “4P-X”
Takashi Moriya, Naoki Nagahata, Rei Odaka, Hirohide Nakamura, Jun Yoshikawa,
Katsumi Kurashima, Tadao Saito
PII:
S0008-6215(16)30341-X
10.1016/j.carres.2016.11.013
CAR 7296
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 31 August 2016
Revised Date: 23 November 2016
Accepted Date: 23 November 2016
Please cite this article as: T. Moriya, N. Nagahata, R. Odaka, H. Nakamura, J. Yoshikawa, K.
Kurashima, T. Saito, Synthesis of an allergy inducing tetrasaccharide “4P-X”, Carbohydrate Research
(2016), doi: 10.1016/j.carres.2016.11.013.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
OH OH
O
OAc OAc
O
OH
enzymatic synthesis
chemical synthesis
HO
O
OAc
O
AcO
OH
OAc
AcO
OH
OH
O
OH OH
O
OH
AcO
4
steps
O
CCl
NH
3
HO
O
O
OH
O
O
OH
chemical synthesis
OH
HO
HO
HO
OH
OH
O
OH OH
O
OH
2
steps
O
OH
O
OAc OAc
OH
HO
HO
OH
O
chemical synthesis
OH
OH
HO
HO
O
O
OH
OAc
O
AcO
AcO
4P-X
OH
OAc
OAc
5
steps

ACCEPTED MANUSCRIPT
Synthesis of an allergy inducing tetrasaccharide “4P-X”
a,
†
a,b,
†
a
a
Takashi Moriya , Naoki Nagahata , Rei Odaka , Hirohide Nakamura ,
a
a
b
Jun Yoshikawa , Katsumi Kurashima , Tadao Saito
a
Enzymes and Pharmaceuticals Laboratory, GODO SHUSEI Co., Ltd.
50 Nakahara, Kamihongo, Matsudo City, Chiba, 271-0064, Japan
Graduate School of Agricultural Science, Tohoku University.
-1 Amamiya-machi Tsutsumidori, Aoba-ku, Sendai City, Miyagi, 981-8555, Japan
These authors contributed equally to this work.
2
b
1
†
Abstract
P-X
β-D-galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→6)-[β-D-galactopyranosyl-(1
4)]-β-D-glucopyranose) is included in galacto-oligosaccharides (GOSs) produced by
4
(
→
β-galactosidase derived from Bacillus circulans. 4P-X has been known to induce
particularly strong allergies. High purity 4P-X is essential for use as a standard to
quantify the amount of 4P-X in GOSs; however, the isolation of high purity 4P-X has
never been reported. In this study, we achieved the synthesis of 4P-X by a combination
of organic and enzymatic chemical syntheses in a short time. This is the first report of
isolated, high purity 4P-X.
Keywords: Galacto-oligosaccharides, 4P-X, allergy, oligosaccharide synthesis
Galacto-oligosaccharides (GOSs) is a general term for oligosaccharides that consist
of a certain number of galactose units combined with lactose. GOSs exist naturally in
1
human breast milk and bovine colostrum. Various physiological functions of GOSs
have been reported including regulating intestinal functions, promoting mineral
absorption by improving the balance of enterobacterial flora, regulating the immune
2
,3
system, and preventing and improving inflammatory bowel disease. GOSs have
received particular attention for their prebiotic effects that promote the growth of
4
,5
Bifidobacterium, Lactobacillus, and other enteric bacteria. Therefore, GOSs are
commonly used in infant formula, beverages fermented by Lactobacillus, and yogurts.
Some of these foods containing GOSs are certified as Food for Specified Health Uses
by the Consumer Affairs Agency in Japan, and GOSs are certified as generally
1

ACCEPTED MANUSCRIPT
recognized as safe (GRAS) substances by the U.S. Food and Drug Administration
(
GRAS Notices: GRN 233, 236, 285, 286, 334, 484, 489, 495, 518, and 569). In general,
GOSs are produced by a transgalactosylating reaction with β-galactosidase
EC.3.2.1.23). β-Galactosidase is produced in many microorganisms such as Bacillus
circulans, Aspergillus oryzae, Kluyveromyces marxianus, Kluyveromyces fragilis,
(
6
–11
Sporobolomyces singularis, and Lactobacillus fermentum.
As far as we know,
β-galactosidases differ in their three-dimensional structures, resulting in stereo- and
regioselectivity of glycosidic bonds. For example, fungal species such as Aspergillus
regioselectively produce β1-6 bond (6′-GOS), while bacteria such as Bacillus
1
2
regioselectively produce β1-4 bond (4′-GOS). Moreover, β-galactosidase produced by
B. circulans possesses particularly strong transglycosidation activity, and thus, GOS
®
prepared by B. circulans are sold worldwide as Vivinal GOS by FrieslandCampina
(Netherlands).
Recently, several reports of allergy symptoms caused by GOSs have been
1
3–15
published.
The first was a 1996 report from Japan that determined allergy symptoms
among workers at an oyster farm in Hiroshima Prefecture after they consumed a
1
6
lactobacillus beverage containing 6′-GOS produced by fungal β-galactosidase. This
report indicated a correlation between the allergies with sea-squirt asthma, and the
authors suspected there may be some commonalities between 6′-GOS and sea-squirt
antigens. Allergy symptoms have also been observed with 4′-GOS produced by bacterial
β-galactosidase. In 2014, Kaneko et al. observed that GOS produced by β-galactosidase
derived from B. circulans induced particularly strong allergic reactions compared with
1
7
GOSs produced by other microorganisms and revealed that the allergies were caused
by two tetrasaccharides [Gal β1-4 (Gal β1-4 Gal β1-6) Glc, Gal β1-4 Gal β1-4 Gal β1-3
Glc]. Most notably, the former branched tetrasaccharide, commonly referred to as 4P-X,
is seen as the most problematic. In that report, 4P-X was separated and quantitated by
pyridylamination of a GOS sample followed by the use of three types of HPLC columns
(Shodex KS-802, Shimadzu STR-ODS-II, Shimadzu ODS); however, this analysis
system was extremely complex. Alternatively, Van Leeuwen et al. reported that it was
®
generally possible to isolate structural isomers of Vivinal GOS by ion chromatography
1
8,19
®
(Dionex ICS-3000)
and over 40 structures (over 99%) of Vivinal GOS have been
characterized, including release of their NMR spectra; however, 4P-X was mixed with
another isomer, and high purity 4P-X is not yet available. Since 4P-X is a high-risk
allergen and important oligosaccharide, it is essential to obtain high purity 4P-X for the
sake of future investigations. The goal of this research is to establish a fast and accurate
quantitative analysis system for 4P-X. This system will enable us to evaluate the allergy
2

ACCEPTED MANUSCRIPT
risk of GOS-containing foods. To accomplish this, high purity 4P-X is needed for use as
a reference standard. By combining organic and enzymatic chemical syntheses, we
successfully prepared high purity 4P-X in a short time.
Synthetic strategy for 4P-X (15). In order to efficiently prepare 4P-X, we used
retrosynthetic analysis to reveal the convergent synthetic route shown in Figure 1. The
tetrasaccharide is formed by coupling lactose 7 and galactobiose 13 derivatives.
The synthesis of the lactose derivative 7 has been reported. S. Tejima synthesized the
lactose derivative 7 in short steps, starting from commercially available lactose
2
0
monohydrate. However, the use of highly toxic mercuric acetate was problematic.
Therefore, we planned to synthesize the lactose derivative 7 by glycosylation of the
glucose derivative 4 and the commercially available galactose derivative 5 without the
use of highly toxic reagents.
The synthesis of the galactobiose derivative 13 has not been reported. We deduced
that the synthesis of the galactobiose derivative 13 by the organic chemical synthesis
requires multi-step processes. Therefore, in order to reduce the synthetic processes, we
planned to adopt a combination of enzymatic and organic chemical methods. In
particular, Galactobiose 10 is prepared by the enzymatic synthesis, and converted to the
galactobiose derivative 13 by the organic chemical reactions.
Galactobiose 10 is not commercilally available, so we planned to prepare by
transglycosylation of lactose and galactose using β-galactosidase. We selected Biolacta
FN5 derived from B. circulans. It was known that β-galactosidase derived from B.
2
1
circulans regioselectively produces β1-4 bond (4’-GOS), we inferred that this is
suitable for the preparation of Galactobiose 10. We expected that this combination of
enzymatic and chemical synthesis will result in the efficient synthesis of this
galactobiose derivative.
Through the all processes, in order to simplify the deprotection process, we chose
acetyl group as protecting groups as much as possible.
3

ACCEPTED MANUSCRIPT
OH OH
O
HO
O
OH
O
OH
HO
O
OH OH
O
OH
O
OH
O
HO
HO
OH
OH
4
P-X (15)
OAc
OAc
O
O
OAc
O
OAc
AcO
OAc
O
OH
O
OAc
AcO
O
OAc
AcO
AcO
AcO
OAc
OAc
O
CCl3
NH
7
13
OAc
OAc
O
OH OH
O
OBn
O
AcO
HO
O
OH
O
HO
OAc
AcO
AcO
OH
HO
10
O
CCl3
NH
OH
OAc
5
4
OH
OH OH
O
OH
OH
O
OH
O
OH
O
HO
HO
OH
OH
OH
O
HO
HO
HO
OH
OH
OH
OH
glucose (1)
galactose (8)
lactose (9)
Figure 1. Retrosynthetic analysis of 4P-X (15).
Synthesis of lactose acceptor 7. The lactose acceptor 7 was synthesized as shown in
Scheme 1. Glucose (1) was treated with benzaldehyde dimethyl acetal in the presence of
a catalytic p-toluenesulfonic acid monohydrate (p-TsOH–H O) to give the
2
2
2
benzyliden-protected glucose 2 (53.7%). The protected glucose 2 was treated with
acetic anhydride and 4-dimethylaminopyridine (DMAP) to afford the further acetylated
glycoside 3 (93.8%). The benzylidene acetal in 3 was regioselectively cleaved by
triethylsilane in the presence of trifluoroacetic acid (TFA) to afford the protected
2
3
glycoside 4 (61.0%). Glycosylation of glucose donor 4 and the commercially available
galactose acceptor 5 in the presence of trimethylsilyl trifluoromethanesulfonate
(TMSOTf) and 4 Å molecular sieves afforded disaccharide 6 (53.1%). Deprotection of
the benzyl group from disaccharide 6 was performed by palladium-catalyzed
hydrogenation delivering disaccharide 7 (93.3%).
4

ACCEPTED MANUSCRIPT
OH
O
OBn
O
a
O
b
O
c
O
O
HO
HO
O
O
OAc
HO
AcO
OAc
OH
OH
HO
AcO
OH
OH
OAc
OAc
1
2
3
4
OAc
OAc
O
OAcOAc
O
OBn
O
OAcOAc
O
OH
O
d
e
AcO
4
+
AcO
O
OAc
O
OAc
AcO
AcO
O
CCl
NH
3
AcO
AcO
OAc
OAc
OAc
OAc
5
6
7
Scheme 1. Reagents and conditions: (a) PhCH(OMe) , p-TsOH–H O, DMF, 60 °C, 6 h,
2
2
5
4%; (b) Ac O, DMAP, pyr., rt, 1 h, 94%; (c) Et SiH, TFA, MS4Å, CH Cl , 0 °C to rt,
2 3 2 2
4
.5 h, 61%; (d) TMSOTf, MS4Å, CH Cl , −40 °C, 1 h; −20 °C, 2 h, 53%; (e) H gas,
2 2 2
Pd/C, MeOH, rt, 22 h, 93%.
Synthesis of galactobiose donor 13. The galactobiose donor 13 was synthesized as
shown in Scheme 2. Transglycosylation of galactose acceptor 8 and lactose donor 9 by
®
Biolacta FN5 afforded GOSs containing galactobiose 10; a mixture of galactobiose 10
and lactose (9) (3.3:1) was obtained by activated carbon purification. The mixture was
acetylated to acetylated galactobiose 11 and acetylated lactose. This mixture was
purified by silica gel column chromatography to afford pure acetylated galactobiose 11
(
67.9%). The anomeric acetyl group was selectively deprotected from acetylated
2
4
galactobiose 11 by piperidine, resulting in disaccharide 12 (76.0%). Disaccharide 12
was treated with trichloroacetonitrile and 1,8-diazabicyclo[5,4,0]-7-undecane (DBU) to
afford the trichloroimidate derivative 13 (86.1%).
OH OH
OH OH
O
OH
OH
O
OH
O
O
a
O
OH
O
b
HO
OH
OH
O
9
HO
HO
HO
OH
OH
OH
OH
OH
HO
OH
8
9
10
OAc
OAc
O
OAc
OAc
OAc
O
OAc
O
O
OAc
O
AcO
O
OAc
O
c
O
OAc
O
d
AcO
AcO
OAc
AcO
OAc
AcO
OAc
AcO
OAc
OH
AcO
O
CCl
NH
3
OAc
OAc
13
11
12
®
Scheme 2. Reagents and conditions: (a) Biolacta FN5 , 50 °C, 24 h, 45% (10:9 = 3.3:1);
b) Ac O, DMAP, pyr., rt, 21.5 h, 68%; (c) piperidine, THF, rt, 30 h, 76%; (d) CCl CN,
(
2
3
DBU, CH Cl , rt, 4 h, 86%.
2
2
5

ACCEPTED MANUSCRIPT
Synthesis of 4P-X (15). 4P-X was synthesized as shown in Scheme 3. Glycosylation
of galactobiose donor 13 and lactose acceptor 7 in the presence of TMSOTf and 4 Å
molecular sieves afforded tetrasaccharide 14 (43.4%). The acetyl groups were
deprotected from tetrasaccharide 14 by NaOMe, resulting in disaccharide 4P-X (15)
1
(
81.9%). The structure of 15 was confirmed by H-NMR (δ = 4.49 ppm, d, J = 7.8 Hz,
1
,2
1
3
0
.6H, H-1″ and δ = 4.48 ppm, d, J = 7.8 Hz, 0.4H, H-1″) and C-NMR spectroscopy.
1,2
OAc
OH OH
O
OAc
O
O
O
HO
AcO
OH
O
OAc
O
OAc
AcO
OAc
OH
a
O
O
b
HO
7
+
13
OH
OAc
OH OH
O
HO
OAc
O
O
O
OAc
O
OH
O
HO
AcO
AcO
OAc
OH
OH
OAc
14
15
Scheme 3. Reagents and conditions: (a) TMSOTf, MS4Å, CH Cl , −40 °C, 2 h, 43%;
2
2
(
b) NaOMe, MeOH–H O, rt, 6 h, 82%.
2
In summary, we have succeeded in the synthesis of high purity 4P-X. Isolation and
purification of 4P-X from a GOS prepared from lactose by incubation with
β-galactosidase is difficult and, as a result, this is the first time high purity 4P-X has
been reported. In future, we plan to use the synthesized 4P-X as a standard material to
establish a fast and accurate analysis method for its quantification in the
GOS-containing foods. This analysis method will enable us to evaluate the allergy risk
of GOS-containing foods.
1
1
. Experimental
.1. General methods
All reagents and solvents were obtained from commercial suppliers and used without
further purification. The production of galactobiose was accomplished with Biolacta
®
FN5 (Amano Enzyme Inc.). Reactions were monitored by thin-layer chromatography
(TLC) carried out on silica gel plates 60 F254 (Merck). Silica gel column
chromatography was performed on silica gel 60N (63–210 µm) (Kanto Chemical Co.,
Inc.) or silica gel 60 (40–100 µm) (Kanto Chemical Co., Inc.). Carbon column
chromatography was performed on activated carbon (Wako Pure Chemical Industries,
Ltd.). Optical rotations were measured with a JASCO DIP-1000 Digital Polarimeter. IR
6

ACCEPTED MANUSCRIPT
spectra were recorded on a JASCO FT/IR-4200 Fourier transform infrared
1
13
spectrophotometer. H- (400 and 600 MHz) and C-NMR spectra (100 and 150 MHz)
were recorded on JEOL JNM-ECX-400P and Varian NMR System 600 spectrometers,
1
respectively. For H-NMR spectra, chemical shifts (δ) are referenced using TMS (0.00
1
3
ppm) in CDCl and acetone (2.23 ppm) in D O as internal standards. For C-NMR
3
2
spectra, chemical shifts (δ) are referenced using CDCl (77.0 ppm) and acetone (31.1
3
ppm) in D O as internal standards. High-resolution mass spectra were recorded on
2
JEOL JMS-T100LP AccuTOF LC-plus4G.
1
.2. 4,6-O-Benzyliden-D-glucopyranose (2)
A suspension of D-glucose (1) (1.00 g, 5.55 mmol) in DMF (11 mL) was heated to
6
0 °C to dissolve the solid. Benzaldehyde dimethyl acetal (1.24 mL, 8.33 mmol) and
p-toluenesulfonic acid monohydrate (10 mg) were added. The resulting solution was
stirred at 60 °C for 6 h, depressurizing 10 min for each hour in order to remove MeOH.
The mixture was concentrated in vacuo. The residue was purified by silica gel column
chromatography (MeOH/AcOEt = 1:10, v/v) to give glucopyranose 2 (799.5 mg, 2.98
mmol, 53.7%) as a white solid.
2
0
1
2
: [α] +12.9 (c 0.20, MeOH); H-NMR (400 MHz, CD
3
OD): δ 7.46–7.42 (m, 2H),
.38–7.35 (m, 3H), 6.31 (d, J = 4.1 Hz, 0.5H), 5.79 (d, J = 7.8 Hz, 0.5H), 5.60 (t, J = 9.9
Hz, 0.5H), 5.52 (s, 0.5H), 5.51 (s, 0.5H), 5.37 (t, J = 9.2 Hz, 0.5H), 5.17–5.10 (m, 1H),
D
7
4
3
.39 (dd, J = 10.3, 4.6 Hz, 0.5H), 4.32 (dd, J = 10.5, 5.0 Hz, 0.5H), 4.08–4.00 (m, 0.5H),
.80–3.63 (m, 2.5H), 2.19 (s, 1.5H), 2.11 (s, 1.5H), 2.08 (s, 1.5H), 2.06 (s, 1.5H), 2.05
1
3
(s, 1.5H), 2.04 (s, 1.5H); C-NMR (100 MHz, CD
3
OD): δ 139.2, 139.1, 129.9, 129.0,
1
27.5, 103.0, 102.9, 99.0, 94.7, 83.1, 82.4, 77.2, 74.7, 74.4, 71.8, 70.3, 69.8, 67.7, 63.5;
+
16 6
HRMS (ESI): calcd for C13H O Na ([M+Na] ): 291.0845, found: 291.0842.
1
.3. Acetyl 2,3-di-O-acetyl-4,6-O-benzyliden-D-glucopyranoside (3)
To a solution of glucopyranose 2 (1.42 g, 5.28 mmol) in pyridine (26 mL) was added
acetic anhydride (7.5 mL, 79.2 mmol) and 4-dimethylaminopyridine (193 mg, 1.58
mmol). The resulting solution was stirred at rt for 1 h. The mixture was concentrated in
vacuo. The residue was purified by silica gel column chromatography (AcOEt/Hexane =
1
:1, v/v) to give glucopyranoside 3 (1.95 g, 4.96 mmol, 93.8%) as a white solid.
20
−1 1
3
2 2 2 2
: [α] +16.8 (c 0.20, CH Cl ); IR (CH Cl ): 1756, 1370, 1261, 1071 cm ; H-NMR
D
(400 MHz, CDCl
3
): δ 7.46–7.42 (m, 2H), 7.38–7.35 (m, 3H), 6.31 (d, J = 4.1 Hz, 0.5H),
7

ACCEPTED MANUSCRIPT
5
.79 (d, J = 7.8 Hz, 0.5H), 5.60 (t, J = 9.9 Hz, 0.5H), 5.52 (s, 0.5H), 5.51 (s, 0.5H), 5.37
(t, J = 9.2 Hz, 0.5H), 5.17–5.10 (m, 1H), 4.39 (dd, J = 10.3, 4.6 Hz, 0.5H), 4.32 (dd, J =
1
0.5, 5.0 Hz, 0.5H), 4.08–4.00 (m, 0.5H), 3.80–3.63 (m, 2.5H), 2.19 (s, 1.5H), 2.11 (s,
1
3
1
.5H), 2.08 (s, 1.5H), 2.06 (s, 1.5H), 2.05 (s, 1.5H), 2.04 (s, 1.5H); C-NMR (100 MHz,
CDCl
3
): δ 169.93, 169.90, 169.86, 169.5, 169.1, 168.8, 136.64, 136.56, 129.2, 129.1,
1
2
28.2, 126.10, 126.06, 78.6, 77.9, 71.7, 71.2, 69.8, 68.7, 68.5, 68.2, 67.0, 64.9, 20.9,
+
22 9
0.8, 20.7, 20.6, 20.5; HRMS (ESI): calcd for C19H O Na ([M+Na] ): 417.1162,
found: 417.1165.
1
.4. Acetyl 2,3-di-O-acetyl-6-O-benzyl-D-glucopyranoside (4)
To a solution of glucopyranoside 3 (1.01 g, 2.57 mmol) in dry CH Cl (26 mL) was
2
2
added activated 4 Å molecular sieves, the resulting mixture was stirred at rt for 10 min,
then triethylsilane (4.09 mL, 25.7 mmol) was added. The resulting mixture was cooled
to 0 °C then TFA (1.97 mL, 25.7 mmol) was added dropwise over 5 min. The resulting
mixture was stirred at rt for 4.5 h, then cooled to 0 °C and quenched with
trimethylamine (4 mL) and H O (40 mL). The solution was separated into CH Cl and
2
2
2
aqueous layers. The aqueous layer was extracted with AcOEt (50 mL × 2). The
combined organic layers were dried over sodium sulfate, filtered, and concentrated in
vacuo. The residue was purified by silica gel column chromatography (AcOEt/Hexane =
1
:1, v/v) to give glucopyranoside 4 (622 mg, 1.57 mmol, 61.0%) as a colorless oil.
20
4
: [α] +50.2 (c 0.30, CH
2
Cl
2
2 2
); IR (CH Cl ): 2873, 1754, 1371, 1221, 1083, 1044
D
−
1 1
cm ; H-NMR (400 MHz, CDCl
3
): δ 7.38–7.27 (m, 5H), 6.30 (d, J = 3.7 Hz, 0.6H),
5
2
.69 (d, J = 7.8 Hz, 0.4H), 5.33 (t, J = 9.9 Hz, 0.6H), 5.13–5.00 (m, 1.4H), 4.63–4.52 (m,
H), 3.97–3.91 (m, 0.6H), 3.86–3.76 (m, 2H), 3.76–3.62 (m, 1.4H), 2.15 (s, 1.8H), 2.10
1
3
(s, 1.8H), 2.088 (s, 1.2H), 2.086 (s, 1.2H), 2.03 (s, 1.2H), 2.01 (s, 1.8H); C-NMR (100
MHz, CDCl
3
): δ 171.3, 171.1, 169.9, 169.5, 169.1, 137.40, 137.35, 128.4, 127.9, 127.8,
1
2
4
27.7, 91.8, 89.3, 75.4, 74.9, 73.8, 73.7, 72.5, 72.4, 70.3, 70.0, 69.9, 69.3, 69.2, 20.88,
+
0.85, 20.78, 20.77, 20.55, 20.46; HRMS (ESI): calcd for C19
24 9
H O Na ([M+Na] ):
19.1318, found: 419.1320.
1
.5. Acetyl ,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3-di-O-acetyl-6-O-
benzyl-D-glucopyranoside (6)
A mixture of 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl 2,2,2-trichloroacetimidate
(5) (374 mg, 0.759 mmol) (Tokyo Chemical Industry Co., Ltd.) and glycosyl donor 4
8

ACCEPTED MANUSCRIPT
(201 mg, 0.506 mmol) was co-evaporated with toluene (2 mL) and dissolved in dry
CH Cl (2 mL). Activated 4 Å molecular sieves were added and the resulting mixture
2
2
was stirred at rt for 10 min. TMSOTf in CH Cl (51 mM, 1.0 mL, 51 µmol) was added
2
2
dropwise and the mixture was stirred at −40 °C for 30 min after which TMSOTf in
CH Cl (51 mM, 2.0 mL, 102 µmol) was added dropwise and the mixture was stirred at
2
2
−40 °C for 30 min. The mixture was then heated to −20 °C and stirred for 2 h. Then, the
mixture was diluted with CH Cl (10 mL) and stirred for 30 min. The mixture was
2
2
poured into sat. NaHCO aq. (20 mL) at 0 °C and was separated into CH Cl and
3
2
2
aqueous layers. The aqueous layer was extracted with AcOEt (50 mL × 2). The
combined organic layers were dried over sodium sulfate, filtered, and concentrated in
vacuo. The residue was purified by silica gel column chromatography (AcOEt/Hexane =
1
:1 to 2:1, v/v) to give disaccharide 6 (195 mg, 0.269 mmol, 53.1%) as a white solid.
20
−1 1
6
: [α] +26.9 (c 0.25, CH Cl ); IR (CH Cl ): 1754, 1370, 1223, 1049 cm ; H-NMR
2 2 2 2
D
(
400 MHz, CDCl ): δ 7.43–7.34 (m, 5H), 6.30 (d, J = 3.6 Hz, 0.7H), 5.63 (d, J = 8.2 Hz,
3
0.3H), 5.40 (t, J = 9.9 Hz, 0.7H), 5.29 (d, J = 2.7 Hz, 0.7H), 5.26 (d, J = 2.8 Hz, 0.3H),
5.17 (t, J = 9.4 Hz, 0.3H), 5.10–4.95 (m, 2H), 4.81–4.73 (m, 2H), 4.47 (d, J = 11.9 Hz,
0.7H), 4.40–4.33 (m, 1H), 4.12–3.97 (m, 3.3H), 3.85 (d, J = 10.1 Hz, 0.7H), 3.77 (d, J =
2.8 Hz, 0.3H), 3.74 (d, J = 2.3 Hz, 1.0H), 3.68–3.56 (m, 2H), 2.17 (s, 2.1H), 2.14 (s,
2.1H), 2.13 (s, 0.9H), 2.10 (s, 0.9H), 2.08 (s, 3H), 2.03 (s, 2.1H), 2.023 (s, 0.9H), 2.017
(
s, 0.9H), 2.00 (s, 2.1H), 1.963 (s, 2.1H), 1.957 (s, 0.9H), 1.94 (s, 2.1H), 1.93 (s, 0.9H);
1
3
C-NMR (100 MHz, CDCl ): δ 170.4, 170.2, 170.0, 169.9, 169.5, 169.1, 168.8, 168.7,
3
1
7
2
7
37.4, 128.7, 128.3, 128.2, 100.3, 100.2, 91.9, 89.3, 75.2, 74.2, 74.0, 73.8, 73.7, 72.7,
2.5, 71.0, 70.9, 70.5, 70.4, 69.51, 69.48, 69.1, 69.0, 66.8, 66.7, 66.6, 21.0, 20.8, 20.74,
+
0.66, 20.63, 20.5; HRMS (ESI): calcd for C H O Na ([M+Na] ): 749.2269, found:
3
3
42 18
49.2257.
1
.6. Acetyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3-di-O-acetyl-D-
glucopyranoside (7)
To a solution of a disaccharide 6 (151 mg, 0.207 mmol) in MeOH (4.1 mL) was
added 5% Pd/C (151 mg). The suspension was stirred at rt for 20 h under a hydrogen
atmosphere. The mixture was filtered over Celite by elution with MeOH (10 mL × 4)
and AcOEt (10 mL × 4). The combined filtrates were concentrated in vacuo. The residue
was purified by silica gel column chromatography (AcOEt/Hexane = 2:1 to 4:1 to 1:0,
v/v) to give disaccharide 7 (123 mg, 0.193 mmol, 93.3%) as a colorless oil.
2
0
7
: [α] +26.6 (c 0.10, CH Cl ); IR (CH Cl ): 2993, 1754, 1605, 1370, 1223, 1059
2 2 2 2
D
9

ACCEPTED MANUSCRIPT
−
1 1
cm ; H-NMR (400 MHz, CDCl ): δ 6.26 (d, J = 4.1 Hz, 0.7H), 5.68 (d, J = 8.7 Hz,
3
0
5
0
9
4
1
1
7
2
6
.3H), 5.48 (t, J = 9.8 Hz, 0.7H), 5.36 (d, J = 3.7 Hz, 1H), 5.24 (t, J = 9.4 Hz, 0.3H),
.16–5.09 (m, 1H), 5.05–4.97 (m, 1.7H), 4.96 (d, J = 3.7 Hz, 0.3H), 4.63 (d, J = 8.2 Hz,
.7H), 4.62 (d, J = 8.2 Hz, 0.3H), 4.17–4.05 (m, 2H), 4.03–3.72 (m, 4.7H), 3.54 (d, J =
.6 Hz, 0.3H), 2.17 (s, 2.1H), 2.16 (s, 2.1H), 2.15 (s, 0.9H), 2.10 (s, 0.9H), 2.07 (s,
.2H), 2.06 (s, 3H), 2.052 (s, 0.9H), 2.047 (s, 0.9H), 2.03 (s, 0.9H), 2.02 (s, 2.1H),
1
3
.972 (s, 2.1H), 1.969 (s, 0.9H); C-NMR (100 MHz, CDCl ): δ 170.5, 170.24, 170.15,
3
70.05, 169.8, 169.6, 169.2, 169.11, 169.06, 101.2, 101.0, 91.8, 89.3, 77.8, 75.7, 74.6,
4.4, 73.1, 71.1, 71.0, 70.8, 70.7, 69.8, 69.7, 69.4, 69.3, 66.9, 61.0, 60.1, 60.0, 21.0,
+
0.94, 20.87, 20.8, 20.7, 20.6; HRMS (ESI): calcd for C H O Na ([M+Na] ):
2
6
36 18
59.1799, found: 659.1795.
1
.7. Acetyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-D-
glactopyranoside (11)
Galactobiose 10 was synthesized by the transglycosylation reaction of
β-galactosidase [accepter:galactose (8), donor:lactose (9)]. β-Galactosidase was
®
obtained from Biolacta FN5 (Amano Enzymes) and derived from B. circulans. The
transglycosylation reaction was performed at 50 °C. The buffer employed was 100 mM
sodium phosphate (10 mL), pH 6.5. 0.01% of enzyme was added to the substrate
solution containing 25% (w/v) lactose (2.50 g, 7.30 mmol) and 25% (w/v) galactose
(2.50 g, 13.9 mmol), and was incubated for 24 h with shaking. The reaction was stopped
by boiling (5 min at 100 °C). After boiling, the supernatant was collected by
centrifugation (14,000 rpm, 5 min). The supernatant was loaded onto a charcoal column
(
φ 4.6 cm × 20 cm; solvent, H O), and monosaccharides were eluted with H O. After
2
2
that, disaccharides that adhered to the charcoal were eluted stepwise with EtOH. The
7
.5 % (v/v) of EtOH fraction gave a mixture of galactobiose 10 and lactose (9) (1.13 g,
.29 mol, 45.0%) as a yellowish white solid. The ratio of galactobiose 10 and lactose (9)
3
was 3.3:1, confirmed by HPLC analysis (CARBOSep CHO-620CA column,
®
Transgenomic ; φ 6.5 mm × 300 mm).
To a solution of the mixture of galactobiose 10 and lactose (9) (555 mg, 1.62 mmol,
1
0:9 = 3.3:1) in pyridine (16 mL) was added acetic anhydride (2.3 mL, 24.3 mmol) and
4
-dimethylaminopyridine (19.5 mg, 0.16 mmol). The resulting solution was stirred at rt
for 3 h. Then, acetic anhydride (2.3 mL, 24 mmol) and DMAP (19.5 mg, 0.16 mmol)
were added and the solution was stirred for 18.5 h. The mixture was concentrated in
vacuo. The residue was purified by silica gel column chromatography (AcOEt/Hexane =
1
0

ACCEPTED MANUSCRIPT
1
:1.5 to 1:1 to 1:0 to MeOH/AcOEt/ = 1:10, v/v) to give disaccharide 11 (748 mg, 1.10
mmol, 67.9%) as a white solid.
20
−1
1
1: [α] +48.0 (c 0.10, CH Cl ); IR (CH Cl ): 1752, 1371, 1225, 1068 cm ;
D
2 2 2 2
1
H-NMR (400 MHz, CDCl ): δ 6.29 (d, J = 3.7 Hz, 0.67H), 5.65 (d, J = 8.2 Hz, 0.33H),
3
5
.38–5.36 (m, 1H), 5.35 (d, J = 4.1 Hz, 0.33H), 5.32 (d, J = 3.7 Hz, 0.33H), 5.29–5.18
m, 1.67H), 5.00 (dd, J = 10.6, 3.7 Hz, 1H), 4.94 (dd, J = 10.1, 3.2 Hz, 0.33H), 4.44 (d,
J = 7.8 Hz, 0.67H), 4.43 (d, J = 7.8 Hz, 0.33H), 4.41–4.33 (m, 1H), 4.24 (d, J = 2.8 Hz,
(
0
2
3
.67H), 4.21–4.05 (m, 4.33H), 3.85 (t, J = 6.2 Hz, 1.33H), 2.19 (s, 1H), 2.17 (s, 2H),
.15 (s, 1H), 2.14 (s, 2H), 2.13 (s, 2H), 2.12 (s, 1H), 2.113 (s, 2H), 2.110 (s, 1H), 2.07 (s,
1
3
H), 2.050 (s, 2H), 2.047 (s, 1H), 2.02 (s, 1H), 2.002 (s, 3H), 1.998 (s, 2H); C-NMR
(
100 MHz, CDCl ): δ 170.6, 170.5, 170.28, 170.25, 170.19, 170.1, 169.5, 169.4, 169.14,
3
1
7
2
7
69.11, 168.9, 101.9, 101.8, 91.8, 89.9, 74.5, 74.2, 73.1, 72.9, 70.72, 70.67, 70.63, 70.3,
0.0, 68.6, 68.5, 68.2, 66.8, 66.1, 63.4, 63.2, 61.3, 61.2, 20.89, 20.85, 20.82, 20.7, 20.63,
+
0.58, 20.50; HRMS (ESI): calcd for C H O Na ([M+Na] ): 701.1905, found:
2
8
38 19
01.1895.
1
.8. 2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-D-
galactopyranose (12)
To a solution of disaccharide 11 (86.7 mg, 0.128 mmol) in THF (2.4 mL) was added
piperidine in THF (2.0 M, 0.25 mL, 0.50 mmol). The resulting solution was stirred at rt
for 30 h, then cooled to 0 °C and quenched with HCl aq. (1 N, 1 mL), H O (10 mL).
2
The solution was extracted with AcOEt (2 × 30 mL). The combined organic layers were
dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was purified
by silica gel column chromatography (AcOEt/Hexane = 2:1 to 4:1, v/v) to give
disaccharide 12 (61.9 mg, 97.3 µmol, 76.0%) as a white solid.
2
0
−1
1
2: [α] +43.1 (c 0.20, CH Cl ); IR (CH Cl ): 2941, 1750, 1370, 1227, 1057 cm ;
D
2 2 2 2
1
H-NMR (400 MHz, CDCl ): δ 5.40 (d, J = 3.6 Hz, 0.66H), 5.37 (d, J = 3.2 Hz, 1H),
3
5
.31–5.17 (m, 2H), 5.03–4.89 (m, 2H), 4.61 (d, J = 7.3 Hz, 0.33H), 4.43–4.29 (m,
.66H), 4.24–4.13 (m, 2H), 4.10 (d, J = 6.4 Hz, 2H), 3.85 (t, J = 6.6 Hz, 1H), 3.76 (dd, J
2
=
7.1, 4.6 Hz, 0.33H), 2.17 (s, 1H), 2.16 (s, 2H), 2.15 (s, 1H), 2.12 (s, 5H), 2.085 (s, 4H),
1
3
2
.081 (s, 2H), 2.05 (s, 3H), 2.004 (s, 1H), 1.997 (s, 2H); C-NMR (100 MHz, CDCl ):
3
δ 170.8, 170.6, 170.5, 170.4, 170.30, 170.26, 170.22, 170.15, 170.06, 169.7, 169.4,
1
6
2
01.9, 101.8, 95.6, 90.9, 75.3, 74.4, 72.7, 72.2, 71.2, 70.7, 70.5, 69.8, 68.7, 68.6, 67.8,
8.6, 67.85, 67.79, 66.9, 66.8, 63.9, 63.6, 61.4, 61.3, 20.9, 20.84, 20.77, 20.69, 20.64,
+
0.61, 20.58; HRMS (ESI): calcd for C H O Na ([M+Na] ): 659.1799, found:
2
6
36 18
11

ACCEPTED MANUSCRIPT
6
59.1790.
.9. 2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-α-D-
1
galactopyranosyl 2,2,2-trichloroacetimidate (13)
To a solution of disaccharide 12 (270 mg, 0.425 mmol) in CH Cl (8.4 mL) was
2
2
added Cl CCN (1.7 mL, 17.0 mmol) and DBU in THF (0.85 mM, 0.3 mL, 0.26 mmol).
3
The resulting solution was stirred at rt for 4 h. The mixture was concentrated in vacuo.
The residue was purified by silica gel column chromatography (0.5% Et N in
3
AcOEt/Hexane = 2:1, v/v) to give disaccharide 13 (286 mg, 0.366 mmol, 86.1%) as a
white solid.
20
−1
1
3: [α] +75.1 (c 0.20, CH Cl ); IR (CH Cl ): 1751, 1370, 1226, 1070 cm ;
D
2 2 2 2
1
H-NMR (400 MHz, CDCl ): δ 8.64 (s, 1H), 6.51 (d, J = 3.7 Hz, 1H), 5.40–5.35 (m,
3
2
H), 5.28 (dd, J = 11.0, 2.8 Hz, 1H), 5.20 (dd, J = 10.5, 7.8 Hz, 1H), 5.00 (dd, J = 10.5,
.2 Hz, 1H), 4.43 (d, J = 8.2 Hz, 1H), 4.38 (dd, J = 11.9, 4.1 Hz, 1H), 4.31 (d, J = 3.2
3
Hz, 1H), 4.29 (d, J = 4.6 Hz, 1H), 4.15 (dd, J = 11.5, 7.3 Hz, 1 H), 4.10 (d, J = 6.4 Hz,
2
H), 3.86 (t, J = 6.6 Hz, 1H), 2.17 (s, 3H), 2.14 (s, 3H), 2.12 (s, 3H), 2.05 (s, 3H), 2.02
1
3
(s, 3H), 2.01 (s, 3H), 2.00 (s, 3H); C-NMR (100 MHz, CDCl ): δ ; 170.54, 170.49,
3
1
6
70.3, 170.2, 170.1, 169.5, 169.3, 160.8, 101.9, 93.8, 90.9, 74.5, 70.8, 70.6, 70.5, 70.2,
8.7, 66.8, 66.5, 63.5, 61.4, 20.9, 20.8, 20.7, 20.62, 20.57, 20.5; HRMS (ESI): calcd for
+
C H NO Cl Na ([M+Na] ): 802.0896, found: 802.0876.
28
36
18
3
1
.10. Acetyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-β
-D-galactopyranosyl-(1→6)-[2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)]-2,
3
-di-O-acetyl-β-D-glucopyranoside (14)
A mixture of glycosyl acceptor 13 (47.9 mg, 61.4 µmol) and glycosyl donor 7 (26.0
mg, 40.9 µmol) was co-evaporated with toluene (2 mL) and dissolved in dry CH Cl (1
2
2
mL). Activated 4 Å molecular sieves were added and the resulting mixture was stirred at
rt for 10 min. TMSOTf in CH Cl (4.1 mM, 1.0 mL, 4.1 µmol) was added dropwise and
2
2
the mixture was stirred at −40 °C for 2 h. Then, the mixture was diluted with CH Cl
2
2
(
10 mL) and stirred for 10 min. The mixture was poured to sat. NaHCO aq. (30 mL) at
3
0
°C, and the solution was separated to CH Cl layer and the aqueous layer. The
2
2
aqueous layer was extracted with AcOEt (50 mL × 2). The combined organic layers
were dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (AcOEt/Hexane = 4:1 to 1:0, v/v) to give
1
2

ACCEPTED MANUSCRIPT
tetrasaccharide 14 (22.3 mg, 17.8 µmol, 43.4%) as a colorless oil.
20
−1
1
4: [α] +22.5 (c 0.41, CH Cl ); IR (CH Cl ): 1752, 1370, 1225, 1055 cm ;
D
2 2 2 2
1
H-NMR (400 MHz, CDCl ): δ 6.25 (d, J = 3.7 Hz, 0.8H), 5.62 (d, J = 8.2 Hz, 0.2H),
3
5
.42 (dd J = 10.1, 8.2 Hz, 0.8H), 5.37 (t, J = 3.6 Hz, 2H), 5.20–5.05 (m, 4H), 5.03–4.97
(m, 2H), 4.89 (dd, J = 10.3, 3.2 Hz, 1H), 4.66 (d, J = 7.3 Hz, 0.8H), 4.60–4.56 (m, 1H),
4
6
3
2
.50 (d, J = 12.1, 4.1 Hz, 0.8H), 4.42–4.38 (m, 1H), 4.21–4.05 (m, 6.4H), 4.00 (t, J =
.9 Hz, 1H), 3.93–3.85 (m, 4.8H), 3.73 (dd, J = 6.4, 4.6 Hz, 1H), 2.19 (s, 3H), 2.16 (s,
H), 2.149 (s, 3H), 2.146 (s, 3H), 2.13 (s, 3H), 2.11 (s, 3H), 2.09 (s, 3H), 2.06 (s, 6H),
1
3
.054 (s, 3H), 2.046 (s, 3H), 2.00 (s, 6H), 1.96 (s, 3H); C-NMR (100 MHz, CDCl ):
3
δ 170.7, 170.6, 170.4, 170.33, 170.27, 170.2, 170.1, 170.0, 169.95, 169.89, 169.7, 169.6,
1
8
6
2
69.51, 169.46, 169.39, 169.36, 169.2, 169.0, 102.2, 102.1, 100.8, 100.6, 100.4, 91.7,
9.2, 77.7, 75.5, 75.1, 74.8, 74.4, 73.1, 72.7, 72.4, 71.1, 71.0, 70.8, 70.7, 70.6, 70.5,
9.8, 69.6, 69.3, 68.5, 68.4, 68.1, 67.0, 65.6, 65.2, 63.7, 63.5, 61.3, 61.1, 61.0, 32.0,
9.8, 29.4, 22.8, 21.1, 21.0, 20.94, 20.89, 20.83, 20.71, 20.69, 20.66, 20.58; HRMS
+
(ESI): calcd for C H O Na ([M+Na] ): 1277. 3595, found: 1277.3582.
5
2
70 35
1
.11. β-D-Galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→6)-[β-D-
galactopyranosyl-(1→4)]-β-D-glucopyranose (15)
To a solution of tetrasaccharide 14 (21.9 mg, 17.4 µmol) in 45% MeOH aq. (1 mL)
was added NaOMe in 45% MeOH aq. (2.9 mM, 1.0 mL, 2.9 µmol). The resulting
solution was stirred at rt for 9 h. The solution was concentrated in vacuo. The residue
was dissolved in MQ (2 mL) and poured to Cellulose Ester Membrane (Spectrum,
MWCO: 0.1–0.5 kDa, FW: 31 mm, Dia: 20 mm, vol/L: 3.1 mL/cm) and dialyzed
against MQ (1L) at rt for 6 h. The dialyzed solution was lyophilized to give
tetrasaccharide 15 (9.50 mg, 14.3 µmol, 81.9%) as a white solid.
2
0
1
1
5: [α] +23.3 (c 0.19, D O); H-NMR (600 MHz, D O): δ 5.23 (d, J = 4.2 Hz,
D
2 2
0.4H), 4.68 (d, J = 8.4 Hz, 0.6H), 4.59 (d, J = 7.8 Hz, 1H), 4.50 (d, J = 7.8 Hz, 1H),
4.49 (d, J = 7.8 Hz, 0.6H), 4.48 (d, J = 7.8 Hz, 0.4H), 4.28 (dd, J = 11.1, 1.8 Hz, 0.6H),
4.20 (dd, J = 11.1, 2.4 Hz, 0.4H), 4.18 (d, J = 3.6 Hz, 1H), 4.10–4.07 (m, 0.4H), 3.96
(
dd, J = 11.4, 3.6 Hz, 0.4H), 3.93–3.52 (m, 20.6H), 3.30 (dd, J = 6.2, 5.2 Hz, 0.6H);
1
3
C-NMR (150 MHz, D O): δ 106.9, 105.8, 105.7, 105.52, 105.49, 98.6, 94.6, 80.5,
2
7
7
9.80, 79.76, 78.0, 77.8, 77.0, 76.9, 76.4, 76.3, 75.8, 75.4, 75.2, 74.1, 73.8, 73.6, 71.7,
1.30, 71.26, 70.3, 63.79, 63.76, 63.7, 63.2; HRMS (ESI): calcd for C H O Na
2
4
42 21
+
([M+Na] ): 689.2116, found: 689.2104.
1
3

ACCEPTED MANUSCRIPT
Acknowledgment
The authors are grateful to Ms. M. Shioya (Graduate School of Agricultural Science,
Tohoku University) for providing NMR spectrum data of 4P-X (15).
Supplementary data
Supplementary data associated with this article can be found, in the online
version, at http://xxxxxxxx.
References
1
. Sumiyoshi, W.; Urashima, T.; Nakamura, T.; Arai, I.; Nagasawa, T.; Saito, T.; Tsumura, N.; Wang,
B.; Brand-Miller, J.; Watanabe, Y.; Kimura, K. J. Appl. Glycosci., 2004, 51, 341-344.
2
3
4
5
6
2
7
. Macfarlane, G. T.; Steed, H.; Macfarlane, S. J. Appl. Microbiol., 2008, 104, 305-344.
. Chonan, O.; Takahashi, R.; Watanabe, M. Biosci. Biotechnol. Biochem., 2001, 65, 1872-1875.
. Davis, L. M. G.; Martinez, I.; Walter, J.; Hutkins, R. Int. J. Food Microbiol., 2010, 144, 285-292.
. Veereman-Wauters, G.; Br. J. Nutr., 2005, 93, S57-60.
. Warmerdam, A.; Paudel, E.; Jia, W.; Boom, R. M.; Janssen, A. E. M. Appl. Biochem. Biotechnol.,
013, 170, 340-358.
. Urrutia. P.; Rodriguez-Colinas, B.; Fernandez-Arrojo, L.; Ballesteros, A. O.; Wilson, L.; Illanes,
A.; Plou, F. J. J. Agric. Food. Chem., 2013, 61, 1081-1087.
8
2
9
1
1
. Cheng, C. C.; Yu, M. C.; Cheng, T. C.; Sheu, D. C.; Duan, K. J.; Tai, W. L. Biotechnol. Lett., 2006,
8, 793-797.
. Liu, H.; Liu, J.; Tan, B.; Zhou, F.; Qin, Y.; Yang, R. Bioprocess. Biosyst. Eng., 2012, 35,
287-1295.
0. Kaneko, K.; Watanabe, Y.; Kimura, K.; Matsumoto, K.; Mizobuchi, T.; Onoue, M. Biosci.
Biotechnol. Biochem., 2014, 78, 100-108.
1
2
1
1
1. Liu, G. X.; Kong, J.; Lu, W. W.; Kong, W. T.; Tian, H.; Tian, X. Y.; Huo, G. C. J. Dairy. Sci.,
011, 94, 5811-5820.
2. Sako, T.; Matsumoto, K.; Tanaka, R. Int. Dairy. J., 1999, 9, 69-80.
3. Chiang, W. C.; Huang, C. H.; Llanora, G. V.; Gerez, I.; Goh, S. H.; Shek, L. P.; Nauta, A. J.;
Van Doorn, W. A.; Bindels, J.; Ulfman, L. H.; Knipping, K.; Delsing, D. J.; Knol, E. F.; Lee, B. W.
J. Allergy Clin. Immunol., 2012, 130, 1361-1367.
1
9
1
4. Vo, T. H.; Le, N. H.; Patel, M. S.; Phan, L. T.; Tran Minh, N. N. Foodborne Pathog. Dis., 2012,
, 156-159.
5. Soh, J. Y.; Huang, C. H.; Chiang, W. C.; Llanora, G. V.; Lee, A. J.; Loh, W.; Chin, Y. L.; Tay, V.
1
4

ACCEPTED MANUSCRIPT
Y.; Chan, Y. H.; Delsing, D.; Lee, B. W. Allergy, 2015, 70, 1020-1023.
1
6. Jyo, T.; Katsutani, T.; Otshuka, T.; Tsuboi, S.. Occup. Environ. Allergy., 1996, 3, 12-20.
7. Kaneko, K.; Watanabe, Y.; Kimura, K.; Matsumoto, K.; Mizobuchi, T.; Onoue, M. Biosci.
1
Biotechnol. Biochem., 2014, 78, 100-108.
1
2
1
2
2
2
8. Van Leeuwen, S. S.; Kuipers, B. J. H.; Dijkhuizen, L.; Kamerling, J. P. Carbohydrates. Res.,
014, 400, 59-73.
9. Van Leeuwen, S. S.; Kuipers, B. J. H.; Dijkhuizen, L.; Kamerling, J. P. Carbohydrates. Res.,
016, 425, 48-58.
0. Tejima, S. Carbohydrates. Res., 1971, 20, 123-132.
1. Torres, D. P. M.; Goncalves, M. do P. F.; Teixeira, J. A.; Rodrigues, L. R. Comprehensive Rev.
Food Science and Food Safety, 2010, 9, 438-454.
2
8
2
2
2. Fürstner, A.; Radkowski, K.; Grabowski, J.; Wirtz, C.; Mynott, R. J. Org. Chem., 2000, 65,
758-8762.
3. DeNinno, M. P.; Etienne, J. B.; Duplantier, K. C. Tetrahedron Lett., 1995, 36, 669-672.
4. Van Boeckel, C. A. A.; Beetz, T.; Vos, J. N.; de Jong, A. J. M.; van Aelst, S. F.; van den Bosch,
R. H.; Mertens, J. M. R.; van der Vlugt, F. A. J. Carbohydr. Chem., 1985, 4, 293-321.
1
5

ACCEPTED MANUSCRIPT
・
・
・
The total synthesis of 4P-X is achieved for the first time.
A combination of enzymatic and organic chemical syntheses result in efficient synthesis of 4P-X.
High purity 4P-X is obtained for the first time.