A new method of synthesis of alkyl β-glycosides using sucrose as sugar donor

Article abstract of DOI:10.1271/bbb.80097

Cellobiose phosphorylase from Clostridium thermocellum catalyzed the β-anomer-selective synthesis of alkyl glucosides from cellobiose. Synthesis of alkyl β-glucoside from inexpensive sucrose using cellobiose phosphorylase and sucrose phosphorylase from Pseudomonas saccharophilia was investigated. By combined use of these two phosphorylases, alkyl β-glucoside was anomer-selectively synthesized from sucrose and alkyl alcohol.

Full text of DOI:10.1271/bbb.80097

Biosci. Biotechnol. Biochem., 72 (9), 2415–2417, 2008
Note
A New Method of Synthesis of Alkyl ꢀ-Glycosides
Using Sucrose as Sugar Donor
Kuniki KINO,y Ryoko SATAKE, Takayuki MORIMATSU, Shoko KURATSU,
Yu SHIMIZU, Masaru SATO, and Kohtaro KIRIMURA
Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University,
3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan
Received February 13, 2008; Accepted May 25, 2008; Online Publication, September 7, 2008
[doi:10.1271/bbb.80097]
Cellobiose phosphorylase from Clostridium thermo-
cellum catalyzed the ꢀ-anomer-selective synthesis of
alkyl glucosides from cellobiose. Synthesis of alkyl
ꢀ-glucoside from inexpensive sucrose using cellobiose
phosphorylase and sucrose phosphorylase from Pseudo-
monas saccharophilia was investigated. By combined use
of these two phosphorylases, alkyl ꢀ-glucoside was
anomer-selectively synthesized from sucrose and alkyl
alcohol.
the reaction is biased toward transglycosylation, but
there has been no report to the effect that phosphorylase
catalyze the ꢀ-anomer-selective synthesis of alkyl
glycosides. Alkyl ꢀ-glycosides are preferred for food
additives, cosmetics, and drugs because they are
naturally occurring glycosides. Hence, the effective
process for alkyl ꢀ-glycoside production is desirable.
Among the several phosphorylases, cellobiose
phosphorylase (CPase) is known to have ꢀ-anomer
selective glucosylation activity toward saccharides10)
Hence, we expected that CPase can be utilized to alkyl
ꢀ-glucoside synthesis, and found that CPase from
Clostridium thermocellum YM4 (CtCPase, AY072794)
catalyzed the ꢀ-anomer-selective synthesis of several
alkyl glucosides (Table 1). Similarly to the glucosyla-
tion of saccharide, CtCPase utilized both cellobiose and
ꢁ-glucose-1-phosphate (ꢁ-G1P) as glucosyl donor (data
not shown). Hence, it was concluded that ꢁ-G1P
Key words: cellobiose phosphorylase; sucrose phospho-
rylase; ꢀ-glucoside; ꢀ-anomer-selective
transglucosylation
Glycosylated compounds usually become more solu-
ble and stable, and they are exploited for pharmaceut-
icals and biocompatible materials. Among the glyco-
sides, alkyl glycosides are utilized as surfactants because
they show high biodegradability and low toxicity. In
biochemical synthesis, alkyl glucosides were usually
synthesized using glucosidases.1,2) These glucosidases
show both hydrolysis and glucosyltransfer activity.
Hence, the yield of alkyl glucoside is often lowered by
hydrolysis activity.
Phosphorylases catalyze reversible phosphorolysis of
saccharides, such as glycogen, maltose, sucrose, and cel-
lobiose. They have very strict regiospecificity and cata-
lyze anomer-selective phosphorolysis. Taking advantage
of this regiospecificity, they are utilized in the synthesis
of oligosaccharides via their reverse reactions.3–5)
Moreover, some sucrose phosphorylases (EC 2.4.1.7;
SPase) catalyze ꢁ-anomer-selective transglycosylation
of compounds having various alcoholic, phenolic, or
carboxylic OH groups.6–8) On the other hand, some
maltose phosphorylases (EC 2.4.1.8) catalyze only
ꢁ-anomer-selective transglycosylation of compounds
having alcoholic OH groups.9) Phosphorylases are useful
for the effective synthesis of alkyl ꢁ-glycosides because
Table 1. Synthesis of ꢀ-Glucosides with CtCPase
Acceptor
activitya
b
Methanol
Ethanol
+
+
+
1-Propanol
2-Propanol
1-Butanol
c
ꢀ
+
ꢀ
+
+
+
+
+
ꢀ
t-Butanol
n-Pentanol
n-Hexanol
n-Heptanol
1,2-Butandiol
1,3-Butandiol
1,4-Butandiol
aActivity was investigated under the following conditions: the reaction
mixture consisted of CtCPase, 100 mM cellobiose, and 30% alkyl alcohol
(v/v) in 100 mM citrate-phosphate buffer (pH 7.0), incubated at 40 ꢁC with
shaking at 160 rpm for 20 h.
b+, Glucoside was detected in TLC analysis.
c
ꢀ, not detected.
y
To whom correspondence should be addressed. Tel: +81-3-5286-3211; Fax: +81-3-3232-3889; E-mail: kkino@waseda.jp
Abbreviations: CPase, cellobiose phosphorylase; SPase, sucrose phosphorylase; ꢁ-G1P, ꢁ-glucose-1-phosphate; ꢀ-MetGlc, methyl ꢀ-D-
glucopyranoside

2416
K. KINO et al.
HOH2C
HO
O
OH
CH2OH
CH2OH
CH2OH
CH2OH
OH
R
OH
HOH2C
O
O
Pi
Pi
O
O
O
O
R
OH
OH
OH
OH
OH
O
P
OH
OH
CH2OH
OH
OH
OH
OH
Sucrose phosphorylase
Cellobiose phosphorylase
Fig. 1. Alkyl ꢀ-Glucoside Production from Sucrose.
a
b
1
2
3
4
5
6
1
2
3
4
5
Fig. 2. Determination of the Reaction Product.
TLC analysis of the reaction mixture (a). The reaction mixture consisted of purified PsSPase, CtCPase, 100 mM sucrose, and 30% methanol
(v/v), with and without inorganic phosphate. It was incubated at 40 ꢁC with shaking at 160 rpm for 20 h. Lanes: 1, reaction mixture with
inorganic phosphate; 2, without inorganic phosphate; 3, sucrose; 4, fructose; 5, ꢁ-G1P; 6, ꢀ-MetGlc. Treatment with ꢁ- and ꢀ-glucosidase (b).
Lanes: 1, reaction product; 2, ꢁ-glucosidase treated; 3, ꢀ-glucoside treated; 4, glucose; 5, fructose.
released from phosphorolysis of cellobiose was trans-
ferred to alkyl alcohol. Although several alkyl ꢀ-
glucosides can be synthesized in one pot using CPase,
cellobiose is too much expensive for industrial produc-
tion of alkyl ꢀ-glucosides. Hence, it was concluded that
effective synthesis of alkyl ꢀ-glucoside from an inex-
pensive disaccharide, such as sucrose, was necessary,
and we focused on the combined use of CPase and
sucrose phosphorylase (Fig. 1).
It was expected that SPase would phosphorolyze
sucrose into ꢁ-G1P and fructose, and that subsequently
CPase would catalyze the synthesis of alkyl ꢀ-glucoside
from ꢁ-G1P and alkyl alcohol. In this process, SPase
should not have transglycosylation activity. The trans-
glycosilation activities of several SPases were tested,
and it was found that the recombinant SPase from
Pseudomonas saccharophilia IAM 14368 (PsSPase,
AF158367) had no transglycosylation activity (data not
shown). In the reaction with CtCPase and PsSPase, a
spot of glucoside was detected using methanol as sugar
acceptor (Fig. 2a).
phate buffer (pH 7.0) was incubated at 30 ꢁC with
shaking at 160 rpm for 20 h. The reaction product was
separated by solvent extraction and then purified with a
silica column packed with Wakogel C-280 (Wako,
Osaka, Japan).
The structure of the product was determined by
enzymatic treatment and NMR analysis (Avance 600
spectrometer, Bruker, Rheinstetten, Germany) in
DMSO-d6 with tetramethylsilane as an internal standard.
Under glucosidase treatment, the spot corresponding to
glucoside disappeared after treatment with ꢀ-glucosi-
dase, but not with ꢁ-glucosidase (Fig. 2b). Hence, it was
suggested that the product was ꢀ-glucoside of methanol.
The product was confirmed to be ꢀ-glucoside by
13C-NMR (150 MHz) and 1H-NMR (600 MHz). The
data suggested that the product consisted of methanol
and D-glucose (data not shown). A doublet at 4.2 ppm
showed the existence of a ꢀ-anomeric proton of the
glucosyl moiety, since this signal had a larger coupling
constant (J ¼ 7:7 Hz) than that for ꢁ-glucoside (J ¼
3:8 Hz). Based on these results, the product was
identified as methyl ꢀ-D-glucopyranoside (ꢀ-MetGlc).
Thus it was found that ꢀ-glucoside could be anomer-
selectively synthesized from inexpensive sucrose in an
one pot reaction with PsSPase and CtCPase.
Methanol was transglycosylated with phosphorylases
under the following conditions: a reaction mixture
containing PsSPase, CtCPase, 100 mM sucrose, and
30% methanol (v/v) in 100 mM citrate-100 mM phos-

A New Method of ꢀ-Glycoside Biosynthesis by Phosphorylases
2417
glycosides in low aqueous media: influence of glycosyl
donor and water activity. J. Mol. Cal. B: Enzymatic, 14,
69–76 (2001).
Subsequently, the reaction was carried out under
several temperatures and pH levels for high yield
synthesis of ꢀ-MetGlc. The optimal temperature and
pH were 10 ꢁC and pH 6.0. The low reaction temper-
ature was preferred for ꢀ-MetGlc synthesis because the
SPase tended to reconvert ꢁ-G1P and fructose to
sucrose at high reaction temperature (data not shown).
Then the time course of ꢀ-MetGlc production from
sucrose was investigated under the following condi-
tions: the reaction mixture containing PsSPase,
CtCPase, 100 mM sucrose, and 30% methanol (v/v)
in 100 mM citrate-100 mM phosphate buffer (pH 6.0)
was incubated at 10 ꢁC with shaking at 160 rpm for
20 h. In this reaction, ꢀ-MetGlc was selectively
synthesized, and no ꢁ-MetGlc was detected. The total
amount of ꢀ-MetGlc reached a maximum (34%, mol/
mol) at 18 h. In the case of reactions with other SPases
that showed higher phosphorolysis activity than
PsSPase, ꢁ-MetGlc was synthesized as a by-product.
Hence, the fact that PsSPase showed no glucosyltrans-
fer activity was essential to this method.
2) Kobayashi, T., Adachi, S., Nakanishi, K., and Matsuno,
R., Synthesis of alkyl glycosides through ꢀ-glucosidase-
catalyzed condensation in an aqueous-organic biphasic
system and estimation of the equilibrium constants for
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(2000).
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Recombinant sucrose phosphorylase from Leuconostoc
mesenteroides: characterization, kinetic studies of trans-
glucosylation, and application of immobilized enzyme
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phenolic compounds by sucrose phosphorylase from
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Enzymatic synthesis of ꢁ-anomer-selective D-glucosides
using maltose phosphorylase. Biosci. Biotechnol. Bio-
chem., 71, 1598–1600 (2007).
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cellobiose phosphorylase (CBP) of the Clostridium
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J. Biochem., 132, 197–203 (2002).
In this study, we developed a unique method for the
synthesis of alkyl ꢀ-glucosides from sucrose by com-
bined use of SPase and CPase. This is the first report to
describe an effective process for ꢀ-anomer-selective
synthesis of alkyl glucoside using phosphorylases. It is
expected that the method can be applied in effective
synthesis of other ꢀ-glucosides using other CPases with
different acceptor specificities.
Acknowledgments
We are grateful to Dr. Motomitsu Kitaoka of the
National Food Research Institute for providing recombi-
nant CtCPase. This research was done at the ‘‘Center for
Practical Chemical Wisdom’’ supported by the Global
COE program of the Ministry of Education, Culture,
Sports, Science and Technology of Japan.
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