A novel two-step synthesis of α-linked mannobioses based on an acid-assisted reverse hydrolysis reaction

Article abstract of DOI:10.1016/j.carres.2011.10.037

Instead of an enzyme-assisted reverse hydrolysis reaction for the synthesis of manno-oligosaccharides, we propose here a versatile new approach. By Fischer type glycosylation, a d-mannose solution of extremely high concentration (approximately 83% (w/w)) was incubated at 60 °C for 65 h in 0.5 M HCl. After dilution and neutralization, the small amount of formed β-linked oligosaccharides was hydrolyzed by β-mannosidase. The yields of α-d-Manp-(1→2)-d-Manp (7.9%), α-d-Manp-(1→3)-d-Manp (7.9%), and α-d-Manp-(1→6)-d-Manp (29.1%) isolated by an activated carbon column chromatography were almost identical to those of the enzymatic reaction, but the yield of α-d-Manp-(1→3)-d-Manp increased enormously by the present method.

Full text of DOI:10.1016/j.carres.2011.10.037

Carbohydrate Research 347 (2012) 147–150
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
A novel two-step synthesis of a-linked mannobioses based on an
acid-assisted reverse hydrolysis reaction
⇑
Katsumi Ajisaka , Misato Yagura, Tatsuo Miyazaki
Department of Food Science, Niigata University of Pharmacy and Applied Life Sciences, 265-1, Higashijima, Akiha-ku, Niigata 956-8603, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 August 2011
Received in revised form 24 October 2011
Accepted 25 October 2011
Available online 4 November 2011
Instead of an enzyme-assisted reverse hydrolysis reaction for the synthesis of manno-oligosaccharides,
we propose here a versatile new approach. By Fischer type glycosylation, a D-mannose solution of extre-
mely high concentration (approximately 83% (w/w)) was incubated at 60 °C for 65 h in 0.5 M HCl. After
dilution and neutralization, the small amount of formed b-linked oligosaccharides was hydrolyzed by b-
mannosidase. The yields of
a-D-Manp-(1?2)-D-Manp (7.9%), a-D-Manp-(1?3)-D-Manp (7.9%), and a-D-
Manp-(1?6)- -Manp (29.1%) isolated by an activated carbon column chromatography were almost iden-
D
Keywords:
Mannobiose
Acid-assisted reverse hydrolysis reaction
Fischer glycosylation
Acid catalyst
tical to those of the enzymatic reaction, but the yield of
by the present method.
a
-
D-Manp-(1?3)-
D-Manp increased enormously
Ó 2011 Elsevier Ltd. All rights reserved.
Among the approaches for the synthesis of oligosaccharides
In our current study, we focused on the practical synthesis of
linked manno-oligosaccharides due to the following reasons. Sev-
eral -linked manno-oligosaccharides have been reported to bind
to various bacteria, and the
derivatives are anticipated to show potential as new agents in
the prevention of bacterial infection. In addition, -Manp-
(1?2)- -Manp and -Manp-(1?2)- -Manp- -(1?2)- -Manp
have been used as components in the chemical synthesis of high-
a-
1,2
including chemical approaches or enzymatic approaches to use
3
,4
5,6
transferases or glycosidases, the reverse hydrolysis reaction
with the aid of glycosidases seems to be the most practical option
for a large scale oligosaccharide synthesis. The reverse hydrolysis
reaction using glycosidases gives oligosaccharides of various link-
a
1
6
a
-linked manno-oligosaccharide
a
-D
D
7
ages simultaneously in one step, although the yield may not be
D
a
-
D
D
a
-D
so high as the reaction using glycosyltransferases.
1
7,18
Previously, we have reported the synthesis of
a-linked manno-
mannose type sugar chains.
In the previous paper, we reported a one step synthetic method
-Manp-(1?2)- -Manp, -Manp-(1?3)- -Manp,
Manp-(1?6)- -Manp, and -Manp-(1?2)- -Manp- -(1?2)-
-Manp using -mannose as the starting material in a reverse
hydrolysis reaction catalyzed by
the -mannosidase-assisted reaction, b-linked oligosaccharides
should also be formed together with -linked oligosaccharides in
the acid-assisted reaction. However, -linked manno-oligosaccha-
oligosaccharides by a reverse hydrolysis reaction with the aid of
a
-
8
,9
mannosidase.
Subsequent improvements to the yield and
for
a
-D
D
a-
D
D
a-D-
regioselective production of the oligosaccharides have since been
D
a
-D
D
a-D
1
0,11
reported.
However, the reaction using enzyme remains costly
D
D
8
as large quantities of enzyme are needed for the reverse hydrolysis
reaction. As the reverse hydrolysis reaction is based on equilibrium
between monosaccharides and oligosaccharides, the catalytic role
of the enzyme can intrinsically be replaced by an acid catalyst,
a-mannosidase. In contrast to
a
a
a
although a mixture of
a
- and b-glycosidic linkages would be pro-
rides would be produced at higher levels than the corresponding b-
linked manno-oligosaccharides since this reaction is controlled by
thermodynamical stability. Actually, the Fischer glycosylation
duced in this reaction. We thus aimed to develop a versatile meth-
od for the preparation of oligosaccharides using an acid catalyst
instead of enzyme. This type of acid assisted glycosylation is based
reaction in alcohol also predominantly affords
a
-linked alkyl man-
The purpose of our present study was to con-
firm the above hypothesis and to establish an efficient and
12
13–15
on the Fischer glycosylation reaction, which is usually performed
nopyranosides.
in an anhydrous alcoholic solution with an acid catalyst.1
3–15
How-
ever, our aim was to react unprotected sugars in aqueous solution
so that the equilibrium might shift toward hydrolysis. Therefore
our strategy to overcome this was to raise the concentration of
monosaccharide to the maximum possible level.
practical process for the synthesis of
oligosaccharides.
a-linked manno-
We prepared a
centration for the test reactions (1 g of
in 200 L of H O). This produced a molar ratio of
O of approximately 1:2.5, as 5.5 mmol of -mannose and
13.9 mmol of H O are present in this reaction mixture. This
D
-mannose solution of an extremely high con-
-mannose was dissolved
-mannose to
D
l
2
D
⇑
Corresponding author. Tel.: +81 250 25 5168; fax: +81 250 25 5021.
E-mail address: ajisaka@nupals.ac.jp (K. Ajisaka).
H
2
D
2
0
008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.10.037

1
48
K. Ajisaka et al. / Carbohydrate Research 347 (2012) 147–150
concentration of
products with practical yield.
As a preliminary assessment of our approach, we compared the
use of two different HCl concentrations, 0.1 M and 0.5 M, and three
different temperatures, 4, 37, and 60 °C. The reactions performed at
D
-mannose is sufficient to produce condensation
800
7
6
5
00
00
00
4
0
°C did not yield any product even after 210 h. The reaction with
.1 M HCl at 37 °C showed only a small disaccharide peak by HPLC
after 210 h. The reactions with 0.1 M HCl at 60 °C, and with 0.5 M
HCl at both 37 °C and 60 °C showed measurable oligosaccharide
peaks by HPLC. A representative HPLC chart is shown in Figure 1.
The time course of these reactions could be demonstrated by plot-
ting the peak area of the disaccharide products against time
400
3
2
00
00
r
e
l
a
t
i
v
e
p
e
a
k
a
r
e
a
(
Fig. 2). The reaction with 0.5 M HCl at 60 °C was thereby con-
100
0
cluded to be optimal among the tested conditions and we next per-
formed a preparative scale synthesis using these conditions.
0
30
60
90
120
150
180
210
In the experiment, 5 g of
water to give 5.5 mL solution. After the addition of 240
D
-mannose was dissolved in 1 mL of
L concen-
reaction time (h)
l
trated HCl to make 0.5 M final HCl concentration, this solution was
incubated at 60 °C. The reaction was stopped after 65 h when the
peak area of mannobiose reached to plateau. The mixture was then
diluted and neutralized immediately with a 0.1 M NaOH solution.
To observe the existence of b-linked oligosaccharides qualita-
tively in the reaction mixture, NMR spectra were measured before
and after b-mannosidase treatment (Fig. 3). The broad peaks at
Figure 2. Time course of the production of mannobioses obtained from a HPLC
chart of each reaction. Triangle, reaction in 0.5 M HCl at 37 °C; circle, reaction in
0.1 M HCl at 60 °C; square, reaction in 0.5 M HCl at 60 °C.
the isolated products (Scheme 1) are summarized in Table 1. The
yields of
were almost similar to those of our previous enzymatic reaction.
The yield of -Manp-(1?3)- -Manp was also 7.9%, although it
was very low in the previous enzymatic reaction. The difference
would be ascribed to the regioselectivity of the -mannosidase
used in the enzymatic reaction. Namely, -Manp-(1?2)- -Manp
and -Manp-(1?6)- -Manp reached equilibrium in the early
stages of the enzymatic reaction, but -Manp-(1?3)- -Manp
a-D-Manp-(1?2)-D-Manp and a-D-Manp-(1?6)-D-Manp
9
1
00.3–100.6 ppm (arrow, Fig. 3a) disappeared and the intensity
a
-D
D
of two peaks at 93.4 ppm and 93.9 ppm increased (arrows,
Fig. 3b) following b-mannosidase treatment. The former broad
peaks are assumed to be signals from manno-oligosaccharides hav-
ing a b-linkage at the non-reducing end, and the latter signals to be
anomeric carbons of a- and b-D-mannoses, which are generated by
the hydrolysis of b-linked manno-oligosaccharides. Consequently,
a
a-
D
D
a-
D
D
a-
D
D
has not yet reached equilibrium when the reaction was stopped.
Of course, as both the acid and the enzyme-assisted reverse hydro-
lysis reactions are thermodynamically controlled, the composition
and the yield might be equal in principle after more extended reac-
the remaining signals at 99–102 ppm in the spectrum after b-man-
nosidase treatment (Fig. 3b) must be those of the a-linked manno-
oligosaccharides. These results thus indicated qualitatively that a-
linkages were much larger than b-linkages in this reaction.
1
3
tion time.
Present results thus indicate that acid catalysts can successfully
replace -mannosidase in the synthesis of -linked mannobioses.
After the separation of mannobioses by an activated carbon col-
umn chromatography, the structures of mannobioses 2–4 were
identified by comparing their 1 C NMR spectra with those of the
3
a
a
13
As a result of the experiments to examine the unnatural com-
pounds generated from the hydrolyzed products under an acid
hydrolysis condition of yeast mannan, Jones and Nicholson re-
corresponding authentic standards. From the C NMR and DEPT
spectra, the structure of the by-product was determined to be
1
,6-anhydro-b-D-mannopyranoside 5. The amounts and yields of
ported in 1958 that
-Manp, b- -Manp-(1?4)-
were isolated when a solution of
mately 37% (w/w)) was incubated at room temperature for
a-
D
-Manp-(1?6)-
-Manp, and 1,6-ahydro-b-
-mannose in 6 M HCl (approxi-
D
-Manp, b-
D
-Manp-(1?6)-
D
D
D
D-mannose
D
1
9
1
68 h. However, the amounts and yields of the products were
not described in this report. We speculate however that the yields
were not preparative ones, since the concentration of -mannose
D
was low and the reaction was performed at room temperature.
In conclusion, three kinds of mannobioses were obtained simul-
taneously at a moderate yield (45% in total) in an acid-assisted re-
verse hydrolysis reaction. Our present process is therefore the most
practical method yet reported for the synthesis of a-linked mann-
obioses. Moreover, the merit is that multiple oligosaccharides of
different linkages can be obtained simultaneously in a two-step
reaction. Although our present method is based on the classical
Fischer type reaction, we used an extremely highly concentrated
aqueous solution of
general Fischer type reaction that uses an anhydrous alcoholic
solution. Moreover, as expected, -linked mannobioses were ob-
D-mannose, in contrast to the conditions of
a
tained in practical yield due to the relative thermodynamic stabil-
ities of these molecules.
It is noteworthy that as our novel reaction is based on equilib-
rium between mono-, di-, and higher oligosaccharides, it can be
Figure 1. HPLC chart of the reaction mixture containing 0.5 M HCl incubated at
0 °C for 65 h. Column: Asahi-pak NH2-P50 (Shoko Co., Tokyo, Japan) was eluted
with 80% acetonitrile at a flow rate of 1 mL/min. Peaks were detected using a
6
performed repeatedly by the simple addition of
D-mannose of the
same weight as the isolated target products. The present process
Refractive Index monitor.

K. Ajisaka et al. / Carbohydrate Research 347 (2012) 147–150
149
Figure 3. 13C NMR spectra of anomeric regions (a) before and (b) after the addition of b-mannosidase isolated from Helix pomatia. The arrow in (a) indicates the non-reducing
end anomeric carbon of b-linked manno-oligosaccharides. The arrows in (b) indicate anomeric carbon signals of
D
-mannose.
OH
1
2
) 0.5 M HCl / 60 °C / 65 h
O
1
3
OR
) β-Mannosidase from
R O
OH
O
OH
HO
HO
O
Helix pomatia
HO
O
2
+
OH
R O
OH
OH
HO
5
1
2
3
1
2: R = Manα-, R = H, R = H
: R = H, R = Manα-, R = H
: R = H, R = H, R = Manα-
1
2
3
3
4
1
2
3
Scheme 1.
Table 1
Amounts and yields of the isolated products in an acid-catalyzed reverse hydrolysis
Co., Tokyo, Japan). H and 13C NMR spectra were recorded with a
1
reaction for the synthesis of
a-mannobioses
Bruker AVANCE DPX-250 spectrometer.
Products
Isolated amounts (mg) Yielda (%)
2900
1
.3. General procedure for the synthesis of mannobioses by the
1
D-Mannose (recovered)
acid catalyzed reverse hydrolysis reaction
2
3
4
5
a
a
a
-
-
-
D
D
D
-Manp-(1?2)-
-Manp-(1?3)-
-Manp-(1?6)-
D
D
D
-Manp
-Manp
-Manp
166
167
614
14
7.9
7.9
29.1
0.7
D-Mannose (5.0 g) was dissolved in 1 mL of water. The final vol-
1,6-Anhydro-b-
mannopyranoside
Higher oligosaccharides
D
-
ume of the solution became 5.5 mL, to which 240 L of concen-
l
trated HCl (12.5 M) was added. The resulting viscous solution
was mixed magnetically at 60 °C. At appropriate time intervals,
HPLC was taken to confirm the progress of the reaction. After
6
158
a
Calculated on the basis of the consumed D-mannose 1.
6
5 h, the reaction was stopped by dilution with 600 mL water
and immediately neutralized with a 0.1 M NaOH solution. b-Man-
nosidase from Helix pomatia (500 L, 19.7 unit/mL, Sigma) was
can therefore construct the basis of an efficient reaction system for
the production of -linked mannobioses.
a
l
then added to hydrolyze the b-linkages, and the solution was incu-
bated at 37 °C for 24 h. The b-mannosidase-treated solution was
next applied to an activated carbon column (7 cm / Â 40 cm
length), which was eluted with a gradient of water (5 L) and 12%
aqueous ethanol (5 L). Sequential 25 mL fractions were collected,
and the reducing sugar contents of each fraction were assayed
using a phenol–sulfuric acid method which afforded four broad
1
1
. Experimental section
.1. Materials
-Mannose was purchased from Sigma–Aldrich, and the other
D
chemicals including an activated carbon for column chromatogra-
phy were from Wako Chemicals (Tokyo, Japan).
peaks.
lowed by
(1?3)- -Manp 3 (167 mg, 7.9%), and
614 mg, 29.1%). After the elution of
D
-Mannose 1 (2.9 g) was eluted at first, subsequently fol-
-Manp-(1?2)- -Manp (150 mg), -Manp-
-Manp-(1?6)- -Manp 4
-Manp-(1?6)- -Manp 4,
a-
D
D
2
a-D
D
1
.2. General methods
D
a
-D
(
a
-
D
D
Shimadzu LC-10ATvp system was used by connecting Shodex
all of the remaining higher oligosaccharides were eluted with
RI-201H refractive index monitor and NH2-P50 column (Shoko
aqueous 50% ethanol solution to yield a syrup (158 mg). In several

1
50
K. Ajisaka et al. / Carbohydrate Research 347 (2012) 147–150
2. Crich, D.; Banerjee, A.; Yao, Q. J. Am. Chem. Soc. 2004, 126, 14930–14934.
of the latter fractions of
a
-D
-Manp-(1?2)-
D-Manp 2, an impurity
3
4
.
.
Wong, C.-H. Chimia 2009, 63, 318–326.
Miyazaki, T.; Sato, T.; Furukawa, K.; Ajisaka, K. Methods Enzymol. 2010, 480,
was detected. This impurity and mannobiose 2 were separated
by HPLC using NH2-P50 column which was subsequently eluted
with 80% acetonitrile to isolate mannobiose 2 (16 mg, 166 mg in
total, 7.9%) and a by-product 5 (14 mg, 0.7%) .
511–524.
5. Kroger, L.; Thiem, J. Carbohydr. Res. 2007, 342, 467–481.
6.
7.
8.
Miyasato, M.; Ajisaka, K. Biosci. Biotechnol. Biochem. 2004, 68, 2086–2090.
Muthana, S.; Cao, H.; Chen, X. Curr. Opin. Chem. Biol. 2009, 13, 573–581.
Ajisaka, K.; Matsuo, I.; Isomura, M.; Fujimoto, H.; Shirakabe, M.; Okawa, M.
Carbohydr. Res. 1995, 270, 123–130.
Acknowledgement
9.
Ajisaka, K.; Yamamoto, Y. Trends Glycosci. Glycotechnol. 2002, 14, 1–11.
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0. Suwasono, S.; Rastall, R. A. Biotechnol. Lett. 1996, 18, 815–856.
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We thank the Uchida Energy Science Promotion Foundation for
financial assistance.
1
1
1
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2. Fischer, E. Ber. Dtsch. Chem. Ges. 1893, 26, 2400–2412.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2011.10.037.
1
91, 6592–6601.
7. Matsuo, I.; Isomura, M.; Miyazaki, T.; Sakakibara, T.; Ajisaka, K. Carbohydr. Res.
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