Enzymatic synthesis of alkyl α-2-deoxyglucosides by alkyl alcohol resistant α-glucosidase from Aspergillus niger

Article abstract of DOI:10.1016/j.tetasy.2004.11.046

Aspergillus niger α-glucosidase (ANGase) was used for an efficient syntheses of alkyl α-d-2-deoxyglucosides (A2DGs) and for regioselectivity studies of alkoxy-hydro additions of d-glucal in the presence of alkyl alcohols. ANGase showed a high stability wi

Full text of DOI:10.1016/j.tetasy.2004.11.046

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 403–409
Enzymatic synthesis of alkyl a-2-deoxyglucosides by alkyl
alcohol resistant a-glucosidase from Aspergillus niger
Young-Min Kim, Masayuki Okuyama, Haruhide Mori, Hiroyuki Nakai, Wataru Saburi,
Seiya Chiba and Atsuo Kimura*
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
Received 4 November 2004; accepted 19 November 2004
Abstract—Aspergillus niger a-glucosidase (ANGase) was used for an efficient syntheses of alkyl a-D-2-deoxyglucosides (A2DGs) and
for regioselectivity studies of alkoxy-hydro additions of D-glucal in the presence of alkyl alcohols. ANGase showed a high stability
with respect to the high concentration of alkyl alcohols. The reaction conditions were optimized for pH, temperature, alkyl alcohol
concentration, and D-glucal concentration. On the basis of MS and NMR analyses, A2DGs were confirmed to have only an a-2-
deoxyglucosidic bond and the two-dimensional NMR (HMBC) spectra showed to be made up of 2-deoxyglucosyl and alkyl
moieties.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
substrate specificities are diverse.3 a-Glucosidases are di-
vided into two groups, a-glucosidase families I and II
based on their amino acid sequences.4 The primary
structures of a-glucosidase families I and II are members
of families 13 and 31 of glycosyl hydrolases, respec-
tively.5,6 a-Glucosidase family I, for example, Saccharo-
myces carlsbergensis (Saccharomyces cerevisiae) a-
glucosidase,7 Bacillus cereus a-glucosidase,8 and honey-
bee a-glucosidase I, II, and III,9,10 has the same four
amino acid sequence regions as conserved in a-amylase
and its related enzymes.11 On the other hand, the en-
zymes from origins such as human lysosomal,12 A. niger
(ANGase),13 and Schizosaccharomyces pombe14,15
belong to a-glucosidase family II, which has regions
A and B including critical residues for activity.14
There is no homology in the primary structures between
a-glucosidase families I and II.
Some glycosidases possess the hydrolytic activity for
glycosides as well as the hydration activity toward D-gly-
cals.1,2 The stereochemical studies of the hydration reac-
tions with D-glycals have provided insight into the
specificity and function of glycosidases. The hydrations
of D-glucal performed in D2O by a-glucosidase and b-
glucosidase produce 2-deoxy-[2(a)-2H]-a-D-glucose and
2-deoxy-[2(e)-2H]-b-D-glucose through trans-addition
(Fig. 1). The product-anomeric configuration of each
enzyme in the hydrolytic reaction is also maintained in
the hydration reaction of D-glucal. a-Glucosidase family
I enzymes cannot hydrate D-glucal, but a-glucosidase
family II enzymes, such as Aspergillus niger, pig serum,
rice, buckwheat, and sugar beet, are able to catalyze
the hydration of the double bond in D-glucal and pro-
duce the a-anomer of 2-deoxyglucose.1
Alkyl a- or b-2-D-deoxyglucosides are known to occur in
many antibiotics.16 Some b-glycosidases have been re-
ported to produce b-2-deoxyglycosides from D-glycal
and alcoholic or phenolic compounds.17,18 The anomer-
ically selective enzymatic synthesis of a-2-D-deoxygluco-
sides is an interesting approach because their chemical syn-
thesis gives two stereoisomers of a- and b-configurational
compounds.19 We found that a-glucosidase family II
enzymes catalyze the transfer of 2-deoxyglucosyl groups
from D-glucal to various alcohols as acceptors thus syn-
thesizing various alkyl a-D-2-deoxyglucosyl derivatives
a-Glucosidase [EC 3.2.1.20, a-D-glucoside glucohydro-
lase] is a group of typical exo-type carbohydrases, which
catalyze the split of a-glucosyl linkages to liberate a-glu-
cose from the non-reducing terminal of substrate. Vari-
ous types of a-glucosidases are distributed widely in
microorganisms, plants and animal tissues, and their
*
Corresponding author. Present address: Kita-9 Nishi-9, Kita-ku,
Sapporo, 060-8589, Japan. Tel./fax: +81 11 706 2808; e-mail:
kimura@abs.agr.hokudai.ac.jp
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.11.046

404
Y.-M. Kim et al. / Tetrahedron: Asymmetry 16 (2005) 403–409
CH2OD
OD
O
O
D(a)
OD
OD
OD
OD
D
OD
OD
OD
OD
O
DO
+D2O
DO
CH2OD
O
OD
O
OD
OD
OD
OD
OD
D(e)
D
Figure 1. Reaction scheme for D-glucal by a- and b-glucosidases in D2O.
OH
H
H
O
H
HO
H
HO
H2O
H
OH
OH
O
H
OH
HO
+ E
HO
H
H
ROH
O
HO
H
HO
H
H
OR
H
Figure 2. Synthesis of alkyl a-D-2-deoxyglucoside and a-2-deoxyglucose from D-glucal by ANGase. E: ANGase; R: –CH3, –(CH2)nCH3 [n = 1–7],
–CH(CH3)CH3, –CH2CH(CH3)CH3, CH(CH3)CH2CH3, –CH2CH@CH2,
.
CH2
(Fig. 2). Herein we report (i) the screening of a-glucos-
idase from various origins having high stability in high
concentration of alkyl alcohols, (ii) the conditions suit-
able for the synthetic reaction of alkyl a-2-D-deoxyglu-
cosides using D-glucal, and (iii) the determination of
structures of newly synthesized a-D-2-deoxyglucosides
by HPAEC, MS, and NMR.
100
80
60
40
20
0
2. Results and discussion
2.1. Screening for stable enzymes in alkyl alcohol
0
1
2
3
4
Time (h)
In order to select the most suitable enzyme for synthesiz-
ing the A2DG in alcohols, we compared the stabilities of
a-glucosidases from different origins such as A. niger,
pig serum, buckwheat, and S. pombe in 50% methanol
(Fig. 3). Buckwheat a-glucosidase was rapidly inacti-
vated in 0.5 h. S. pombe and pig serum a-glucosidases
retained 6% and 20% of original activity after 4 h,
respectively, but A. niger a-glucosidase (ANGase) re-
tained 91% of initial activity after 4 h. As ANGase
was the most stable of four enzymes, it was selected as
the enzyme suitable for synthesizing A2DGs in alkyl
alcohols. The residual activity of ANGase in the various
concentrations of methanol (0–70%, v/v) was investi-
Figure 3. Stability of a-glucosidases in 50% (v/v) methanol. Each
reaction mixture (0.1 mL) containing ANGase (m, 0.68 U, pH 4.3), pig
serum a-glucosidase (s, 0.65 U, pH 7.0), S. pombe a-glucosidase (d,
0.65 U, pH 4.5), or buckwheat a-glucosidase (j, 0.66 U, pH 5.0), 50%
(v/v) methanol and 20 mM buffer was incubated at 35 ꢁC. In the
indicated time, aliquots of 10 lL were used for measuring the residual
activities.
gated at 35 ꢁC (Fig. 4). ANGase showed 91% of initial
activity in 50% methanol for 4 h, and ANGase main-
tained 75% of initial activity even in 70% methanol.
ANGase was suitable for synthesis of alkyl a-glucosides

Y.-M. Kim et al. / Tetrahedron: Asymmetry 16 (2005) 403–409
405
100
80
60
40
20
0
20
16
12
8
4
0
0
4
8
12
16
20
24
0
1
2
3
4
Time (h)
Time (h)
Figure 5. Time course of M2DG and 2-deoxyglucose syntheses by
ANGase from D-glucal. Reaction mixture (0.1 mL) containing 20 mM
D-glucal, 20 mM sodium acetate buffer (pH 4.3), and ANGase (0.26 U)
in 50% (v/v) methanol was incubated at 35 ꢁC for 24 h. At the
indicated times, the reaction mixture (10 ll) was pipetted out.
Concentration of each carbohydrate was measured by HPAEC. m,
D-Glucal; s, 2-deoxyglucose; d, M2DG.
Figure 4. Stability of ANGase in methanol. Reaction mixture (0.1 mL)
containing ANGase (0.68 U), 0 (s), 30 (j), 50 (m), or 70 (d)% (v/v)
methanol in 20 mM sodium acetate buffer (pH 4.3) was incubated at
35 ꢁC. In the indicated time, aliquots of 10 lL were taken out from the
reaction mixture for measuring the residual activities.
in alkyl alcohols. Pelenc et al., Monsan et al., and Bous-
quet et al. have reported on industrial process for butyl
a-glucoside production using ANGase and Talaromyces
duponti a-transglucosidases.20–23 They found that resid-
ual activity of ANGase was maintained over 50% of
initial activity in 9% (v/v) of 1-butanol for 20 days.
in Figure 5. At the initial stage up to 5 h, the formation
of M2DG, as well as 2-deoxyglucose (by-product made
by hydration), was observed along with a sharp decrease
in D-glucal on HPAEC. After 24 h incubation, the yield
of M2DG reached 83%, while the yield of 2-deoxyglu-
cose was 16%. Reaction conditions of temperature,
pH, and D-glucal concentration were examined in 50%
(v/v) methanol in order to increase the productivity of
M2DG. The reaction mixture was incubated at temper-
atures of 25–60 ꢁC for 6 h, and M2DG was obtained in
the maximum at 35 ꢁC. The optimal pH was observed
around pH 4.5. The effects of D-glucal concentrations
on the synthesis of M2DG were examined at various
concentrations of D-glucal in the 50% methanol for
24 h. The formation of M2DG was proportional to
the concentration of D-glucal up to 500 mM. D-Glucal
concentration showed no effect on the final yield of
M2DG (about 80%). This result implies that the high
concentration of substrate is better for mass production
of M2DG in the industrial field. Figure 6 shows the
effects of methanol concentrations on the synthesis of
M2DG. The production of M2DG was increased up
to 70% (v/v) methanol, and 93% of D-glucal was con-
verted into M2DG after 24 h. The productivity was
steeply decreased more than 70% (v/v) of methanol.
How can ANGase retain high residual activity in high
concentration alkyl alcohols than other a-glucosidases?
Two possibilities were considered for the resistance in
alkyl alcohols. The first possibility is that ANGase is a
glycoprotein containing 25–27% carbohydrate most of
which is mannose.24 It is known that sugar chains on
the surface of proteins protect the enzyme from dena-
tured conditions. ANGase consisting of P1 and P2 sub-
units has 16 Asn residues putatively N-glycosylated.13,24
Subsequent ANGase was treated with Endo H for the
deglycosylation of the sugar chain, however, the degly-
cosylated ANGase showed the same residual activity
as that of native ANGase in 30%, 50%, and 70% meth-
anol for 4 h, meaning that the sugar chain is not a crit-
ical factor to keep the stability. ANGase has also five
residues linked by putative O-glycosylated sugar chain,9
but its role remains obscure.
The finding also implies that the protein moiety is
important, that is, the second possibility. Even if a-glu-
cosidase family II enzymes have homology in their
sequence, the amino acids exposed to surface of protein
are considered to be different. ANGase is thought to
have hydrophilic residues in the surface area of protein
to keep native protein conformation from denaturing
by alkyl alcohols. Even if we elucidated the properties
and amino acid sequence of crystalline ANGase,24–29
its 3D structure is not available. It was reported that
the modification of chymotrypsin by some hydrophilic
reagents showed the strong stabilization of the enzyme
against denaturation by organic solvents.30
ANGase showed 93% maximum yield production of
M2DG at pH 4.5 and 35 ꢁC in 70% methanol (Table
1). Produced M2DG was purified by a silica gel column
chromatography to be homogenous on TLC and
HPAEC. FD/MS and NMR analyses were preformed
to confirm the structure of isolated compound. The
molecular mass of M2DG was 178 by FD/MS spectro-
metry. The NMR spectra of M2DG indicated the forma-
tion of only a-2-deoxyglucosidic bond and the other
signals were in accord with the proposed structure. Par-
ticularly, 2-deoxy form of M2DG was confirmed by the
signals of Heq and Hax of the C2 methylene observed at
2.13 and 1.71 ppm, respectively, which correspond
into 13C NMR signal of C2 at 39.4 ppm. Conse-
quently, isolated compounds were identified as methyl
2.2. M2DG from D-glucal by ANGase
The time course for synthesis of methyl 2-deoxygluco-
side (M2DG) by ANGase in 50% methanol is shown

406
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Y.-M. Kim et al. / Tetrahedron: Asymmetry 16 (2005) 403–409
ing alcohol-soluble product, because A2DG having
water-immiscible alkyl group remains in the organic
phase. Each A2DG was obtained as follows: methyl
2DG (11.8 mg), ethyl 2DG (13.6 mg), 1-propyl 2DG
(9.5 mg), 1-butyl 2DG (20.6 mg), 1-pentyl 2DG
(20.2 mg), 1-hexyl 2DG (12.1 mg), 1-heptyl 2DG
(10.3 mg), 1-octyl 2DG (5.2 mg), 2-methyl-1-propyl
2DG (20.6 mg), 2-propyl 2DG (20.4 mg), 2-butyl 2DG
(10.4 mg), allyl 2DG (20.4 mg), cyclohexyl 2DG
(6.5 mg), and benzyl 2DG (10.5 mg). The molecular
mass of A2DGs was analyzed by FD/MS as described
100
80
60
40
20
0
1
in the experimental section. H NMR and 13C NMR
0
20
40
60
80
100
analyses of all A2DGs were performed. In particular,
1-butyl a-D-2-deoxyglucoside in D2O and 1-pentyl
a-D-2-deoxyglucoside in (CD3)2SO were analyzed by
secondary NMR (COSY, HMBC, HSQC). NMR study
of A2DGs indicated the formation of only a-2-deoxy-
glucosidic bond and all signals in accord with the
proposed structures. Alkyl group connected at the C1
of 2-deoxyglucosyl residue was confirmed by the two
dimensional NMR (HMBC) spectra.
Methanol (%)
Figure 6. M2DG production from D-glucal by ANGase in methanol.
Reaction mixture (0.1 mL) containing different concentration (10–
90%, v/v) of methanol, 20 mM sodium acetate buffer (pH 4.3), 20 mM
D-glucal and ANGase (0.26 U) was incubated at 35 ꢁC for 24 h. At the
indicated times, the reaction mixture (10 lL) was pipetted out. The
reaction was stopped by heating for 5 min in boiling water. Concen-
tration of each carbohydrate was measured by HPAEC. m, D-Glucal,
s, 2-deoxyglucose; d, M2DG.
It is difficult to synthesize glycosides by carbohydrases in
two solvent phases of water and hydrophobic alcohols.
However, in this experiment, the formation of A2DG
was preceded in the water-insoluble alcohol system.
Furthermore, most of the formed A2DGs remained in
the organic layer, resulting in easy separation of
product.
Table 1. Production of various A2DG
Alcohol
Water solubility
Product (%)
2-Deoxyglucose
A2DG
Methanola
Ethanolb
1-Propanolb
Miscible
93
91
88
6
6
11
1-Pentanolb
1-Heptanolb
Immiscible
83
45
16
54
The addition and hydration of D-glucal occurs at the
same time, meaning the two reactions are competitive
with each other. If hydration is reduced, the addition be-
comes the main reaction. The alcohol itself, which also
acts as substrate, results in shifting the thermodynamic
equilibrium toward synthesis not only by the increase
of alcohol (reactant) concentration but also by the low
concentration of water (reducing hydrolysis). The enzy-
matic synthesis of A2DG from D-glucal was first exam-
ined in 70% methanol, 90% ethanol, 90% 1-propanol by
ANGase, and A2DGs were obtained in the high yields
of 93%, 91%, and 88%, respectively. It was found that
ANGase had four benefits: (i) the simplicity of one step
reaction, (ii) the high stability in alcohols, (iii) its strong
addition activity, (iv) the wide acceptability of various
alcohols.
a The reaction mixture contained 70% alcohol.
b The reaction mixture contained 90% alcohol. Analytical method of
products was described in the experimental section.
a-2-deoxyglucoside. From these results it was concluded
that methyl a-2-deoxyglucoside was synthesized by the
ANGase-catalyzed addition of methanol to D-glucal.
2.3. Synthesis of novel A2DG
The analytical scale synthesis of various A2DGs by
ANGase was investigated by HPAEC (Table 1). The
production of ethyl, 1-propyl, 1-pentyl, and 1-heptyl a-
D-2-deoxyglucosides increased with increasing ethanol,
1-propanol, 1-pentanol, and 1-heptanol concentrations
of up to 90% (v/v) from 20 mM D-glucal in 24 h-reac-
tion, giving yields of 91%, 88%, 83%, and 45%, respec-
tively. Newly synthesized A2DGs were obtained in
high yield.
ANGase shows the hydrolytic activity to M2DG, but its
magnitude is very small, implying that A2DGs are can-
didates for inhibitors of a-glucosidase. Currently, we
analyze the inhibition ability of A2DGs to a-glucosidase
as well as glucoamylase.
Enzymatic preparative-scale syntheses were performed
using 70–90% alkyl alcohols as described in the experi-
mental section. A2DGs were isolated according to two
kinds of methods depending on the properties of alkyl
moieties; (1) from the reaction mixture containing alco-
hols miscible with water, A2DGs were purified by a
silica gel column chromatography and (2) from the
reaction mixture containing alcohols immiscible with
water, A2DGs were separated by liquid/liquid extrac-
tion. The latter method is simple and effective for isolat-
3. Experimental
3.1. Materials
3.1.1. Enzyme and chemicals. The a-glucosidases from
A. niger24 pig serum,31 buckwheat,32 and S. pombe14
were purified as previously reported. Maltose was fur-
ther purified by repeated recrystallization. Fucose and
2-deoxyglucose were purchased from Nacalai Tesque

Y.-M. Kim et al. / Tetrahedron: Asymmetry 16 (2005) 403–409
407
Chemical Inc. (Kyoto, Japan) and tri-O-acetyl-D-glucal
from Sigma Chem. Co., (St. Louis, Mo, USA). All
organic solvents used in this study were of the highest
quality available from Nacalai Tesque Chemical Inc.
ANGase (15 lg) in methanol (50%, v/v) was incubated
at 35 ꢁC for 6 h. The effects of D-glucal concentrations
on the synthesis of M2DG were examined in 50% meth-
anol with various concentrations of D-glucal for 24 h.
Reaction mixture was incubated between 25 and 60 ꢁC
for 6 h for obtaining optimum temperature.
3.1.2. D-Glucal preparation. The 3,4,6-tri-O-acetyl-D-
glucal (5 mmol) was deacetylated by freshly prepared so-
dium methoxide (0.025 mL) in 40 mL of dry methanol.1
Mixture was maintained at 25 ꢁC until reaction was
completed as judged by TLC, then 3 g of silica gel 60
(Kieselgel 60) was added to the mixture. The suspension
was dried under vacuum, and the dried powder was put
on a 3 · 30 cm column of silica gel 60, followed by sub-
jecting to chromatography with ethyl acetate/ethanol
(5:1, v/v) as solvent. Fractions containing D-glucal were
collected, and the solvent was evaporated to syrup. The
syrup was further dried with ethanol and benzene, and
then kept for 1–2 days in the vacuum desiccator over
calcium sulfate (yield, 45–50%). D-Glucal (0.74 mg/mL)
was kept in a dry methanol solution at À20 ꢁC.
3.3. Deglycosylation of ANGase by Endo H
ANGase (200 lg) was treated with 50 munit (1.25 lg)
of Endo H (Roche Diagnostics GmbH, Mannheim,
Germany) in 50 mM sodium acetate buffer (pH 5.5) at
28 ꢁC for overnight. The deglycosylation of ANGase
by Endo H was confirmed by decreasing molecular mass
in SDS-PAGE.33
3.4. Production and purification of A2DG
To synthesize A2DG, ANGase (0.51 U) in 20 mM
sodium acetate buffer (pH 4.3) was incubated with
20 mM D-glucal and 70–90% (v/v) of alcohols at 35 ꢁC
in a final volume of 1 mL. After incubation, 0.02 mL
of the medium was withdrawn, and heated in boiling
water for 10 min to stop the reaction. The products were
analyzed by TLC and HPAEC.
3.2. Biochemical assays
3.2.1. Measurements of enzyme activity. The enzyme
concentration was measured spectrophotometrically
1%
1cm
taking E
at 280 nm to be 17.7, 12.9, 2.46, and 8.1
for a-glucosidase from A. niger24 buckwheat,32 S.
pombe,14 and pig serum,31 respectively. The reaction
mixture containing 0.2 mL of 0.5% maltose, 0.2 mL of
0.1 M buffer and 0.1 mL of enzyme solution was incu-
bated at 35 ꢁC. The sodium acetate buffer was used for
a-glucosidase A. niger (pH 4.3), S. pombe (pH 4.5),
and buckwheat (pH 5.0). For pig serum a-glucosidase,
0.1 M sodium phosphate buffer (pH 7.0) was used. The
glucose liberated was measured by the Tris–glucose oxi-
dase–peroxidase method with ÔGlucose ARII-Test
WakoÕ (Wako Pure Chemical Ind., Ltd, Japan).3 One
unit of a-glucosidase activity was defined as the amount
of enzyme that hydrolyzed 1 lmol of maltose per minute
under the above conditions.
Fourteen kinds of alcohols were checked for preparative
scale-synthesis of A2DG. In the synthetic reactions, for
methanol, 1-butanol, 1-pentanol, ally alcohol, and 1-
octanol, 70% (v/v) of concentration was used, and 90%
(v/v) of concentration for ethanol, 1-propanol, 1-hexa-
nol, 1-heptanol, cyclohexanol, 2-butanol, 2-methyl-1-
propanol, benzyl alcohol, and 2-propanol. Reaction
mixture (4 mL) containing ANGase (2.55 U), D-glucal
(40 mM), in 40 mM sodium acetate buffer (pH 4.3)
and 70% or 90% (v/v) alcohol as described above was
incubated at 35 ꢁC for 2 days on a rotary shaker
(180 rpm).
In the reactions using alcohols highly soluble in water,
the produced A2DGs were purified by a silica gel col-
umn (1.5 · 30 cm) with a mixture of acetonitrile–water
(85:15, v/v) at 0.35 mL/min. In the reactions using
poorly soluble alcohol in water, A2DG was isolated
from the excess of 2-deoxyglucose and D-glucal by
liquid–liquid extraction in the following manner: from
the reaction mixture (4 mL) forming two-layer of aque-
ous phase and alcohol phase, the alcohol phase contain-
ing A2DG as well as small amounts of 2-deoxyglucose
and D-glucal was transferred into new test tube, and
then the equal volume of water was added. From the
layers made again, the aqueous phase containing D-glu-
cal and 2-deoxyglucose was discarded, and the same
procedures were repeated 5–10 times.
3.2.2. Enzyme stability in methanol. Reaction mixture
(0.1 mL), containing 50% of methanol, and enzyme of
ANGase (0.68 U, pH 4.3), S. pombe a-glucosidase
(0.65 U, pH 4.5), buckwheat a-glucosidase (0.66 U,
pH 5.0) in 20 mM sodium acetate buffer, or pig serum
a-glucosidase (0.65 U, pH 7.0) in 20 mM sodium phos-
phate buffer, was incubated at 35 ꢁC for 4 h. At the indi-
cated time, 10 lL were pipetted out and diluted 50–100
times with 20 mM buffer used. Then, 0.1 mL of each
diluted enzyme solution, 0.2 mL of 0.5% maltose, and
0.2 mL of 20 mM sodium acetate buffer or 20 mM so-
dium phosphate buffer were incubated at 35 ꢁC. Glucose
liberated was measured by glucose oxidase–peroxidase
method.2 One hundred percent of the residual activity
was defined as the enzyme activity without the methanol
treatment.
3.5. Analysis of AD2G and carbohydrates
3.5.1. Thin layer chromatography (TLC). The reaction
mixtures were put on TLC plate (Kiesel Gel G, Type
60, Merck Co.), and developed two times using a solvent
system (acetonitrile:water = 85:15, v/v) with 2-deoxy-D-
glucose (Rf: 1.62; Rf for glucose being 1.0) and D-glucal
(Rf: 2.38) as the standards. Among the produced
3.2.3. Reaction conditions for formation of M2DG
(methyl a-2-deoxyglucoside). For analysis of pH
effects, reaction mixture (0.1 mL) containing 20 mM
D-glucal, 20 mM sodium acetate buffer (pH 3.6–5.5) or
20 mM sodium phosphate buffer (pH 5.5–8.0), and

408
Y.-M. Kim et al. / Tetrahedron: Asymmetry 16 (2005) 403–409
A2DGs, methyl, ethyl, and propyl a-D-2-deoxygluco-
sides were migrated in the positions between 2-deoxyglu-
cose and D-glucal, and other A2DGs showed the higher
mobilities than D-glucal. The carbohydrates were visual-
ized on the TLC plate by dipping into 0.03% (w/v) N-(1-
naphthyl)ethylenediamine, 5% (v/v) H2SO4 in methanol,
followed by heating at 120 ꢁC for 5 min.34
13C NMR dC (DMSO-d6): d 96, 73.1, 71.7, 68, 66.1,
61, 38.1, 31.1, 29, 25.5, 22.1, 13.9.
Heptyl a-D-2-deoxyglucopyranoside; FD–MS: m/z 262,
1H NMR dH (DMSO-d6): d 4.76, 3.59, 3.53, 3.45, 3.28,
2.99, 2.44, 1.47, 1.84 (Heq), 1.41 (Hax), 1.26, 0.85; 13C
NMR dC (DMSO-d6): d 96.4, 73.1, 71.7, 67.4, 66.1,
38.1, 31.1, 29.1, 28.8, 28.7, 25.8, 22.1, 13.97.
3.5.2. High performance anion exchange chromatography
(HPAEC). The reaction mixture (0.02 mL) was taken
out at the indicated time, and diluted to 200 times.
The sample (50 lL), filtered by Millexꢂ-HV unit (Milli-
pore, Bedford, MA, USA), was injected onto the
HPAEC (Dionex, Osaka, Japan) equipped with a Carbo-
Pac PA-1 column (4 · 250 mm, Dionex). The carbohy-
drates were eluted in 16 mM NaOH with fucose as
internal standard at a flow rate of 1 mL/min with mon-
itoring by a pulsed amperometric detector (Dionex).
Each carbohydrate and its amount were determined by
the retention time and by the area ratio between a fucose
and carbohydrate (0–500 lM), respectively.
Octyl a-D-2-deoxyglucopyranoside; FD–MS: m/z 276,
1H NMR dH (DMSO-d6): d 4.76, 3.53, 2.99, 3.29, 3.44,
3.62, 3.0, 3.3, 1.48, 1.84 (Heq), 1.4 (Hax), 1.25, 0.86,
0.84; 13C NMR dC (DMSO-d6): d 96.4, 73.1, 71.7,
67.4, 66.1, 61, 38.1, 31.1, 29.1, 28.8, 28.7, 25.8, 22.1,
13.97.
Benzyl a-D-2-deoxyglucopyranoside; FD–MS: m/z 254,
1H NMR dH (DMSO-d6): d 7.35, 7.28, 4.77, 4.37, 3.68,
3.65, 3.48, 3.41, 3.05, 1.93 (Heq), 1.48 (Hax); 13C NMR
dC (DMSO-d6): d 138.2, 128.3, 127.7, 127.5, 95.9, 73.5.
71.8, 68.1, 68, 61.1, 37.9.
3.5.3. MS and NMR analysis. The molecular mass of
each A2DG was determined by FD–MS (JEOL JMS-
SX102A, Tokyo, Japan). Isolated A2DG (approx.
10 mg) was dissolved in D2O (0.5 mL, 99.98% D2O) or
(CD3)2SO (0.5 mL, 99.96% of D). NMR spectra were re-
corded with a Bruker AMX-500 spectrometer operated
at 500 MHz for the 1H and at 125 MHz for 13C nucleus.
Chemical shifts (d) were expressed in ppm relative to the
external standard of TSP.
Cyclohexyl a-D-2-deoxyglucopyranoside; FD–MS: m/z
1
246, H NMR dH (DMSO-d6): d 4.94, 3.59, 3.37, 3.33,
2.99, 1.62, 1.79 (Heq), 1.42 (Hax), 1.26, 1.18; 13C NMR
dC (DMSO-d6): d 9 4.4, 73.2, 71.9, 67.9, 61.1, 34, 33,
31.1, 25.3, 23.7, 23.5.
Allyl a-D-2-deoxyglucopyranoside; FD–MS: m/z 204,
1H NMR dH (D2O): d 5.89, 5.23, 5.12, 4.82, 4.13, 4.06,
3.86, 3.61, 3.44, 3, 1.87 (Heq), 1.44 (Hax); 13C NMR dC
(D2O): d 134.9, 116.2, 95.9, 73.2, 71.6, 67.9, 66.6, 60.9,
37.8.
Methyl a-D-2-deoxyglucopyranoside; FD–MS: m/z 178,
1H NMR dH (D2O): d 4.9, 3.85, 3.84, 3.76, 3.59, 3.35,
2.13 (Heq), 1.89, 1.71 (Hax); 13C NMR dC (D2O): d
101.1, 74.9, 73.8, 70.9, 63.5, 57.2, 39.4.
2-Methyl-1-propyl a-D-deoxyglucopyranoside; FD–MS:
1
m/z 220, H NMR dH (DMSO-d6): d 4.75, 3.61, 3.59,
3.44, 3.32, 3.29, 2.99, 1.77, 1.86 (Heq), 1.42 (Hax),
0.86, 0.84; 13C NMR dC (DMSO-d6): d 96.5, 73.1,
72.7, 71.6, 67.9, 60.9, 38, 27.9, 19.4, 19.3.
Ethyl a-D-2-deoxyglucopyranoside; FD–MS: m/z 192,
1H NMR dH (D2O): d 5.02, 3.86, 3.84, 3.77, 3.63, 3.35,
3.52, 2.12 (Heq), 1.71 (Hax), 1.19; 13C NMR dC (D2O):
d 99.6, 74.9, 73.8, 71.1, 65.9, 63.5, 39.5, 16.8.
2-Propyl a-D-2-deoxyglucopyranoside; FD–MS: m/z
1
206, H NMR dH (D2O): d 5.14, 3.97, 3.84, 3.75, 3.66,
Propyl a-D-2-deoxyglucopyranoside; FD–MS: m/z 206,
1H NMR dH (D2O): d 5.01, 3.88, 3.87, 3.77, 3.74, 3.62,
3.34, 3.46, 2.13 (Heq), 1.71 (Hax), 1.59, 0.9; 13C NMR
dC (D2O): d 99.7, 74.98, 73.8, 72, 71.1, 63.5, 39.5, 24.7,
12.7.
3.33, 1.89, 2.06 (Heq), 1.72 (Hax), 1.17; 13C NMR dC
(D2O): d 97.5, 75, 73.9, 72.1, 71.1, 63.5, 39.8, 25.1, 22.9.
2-Butyl a-D-2-deoxyglucopyranoside; FD–MS: m/z 220,
1H NMR dH (D2O): d 4.87, 3.59, 3.56, 3.43, 3.37, 3.3,
2.99, 1.8 (Heq), 1.35 (Hax), 1.35, 1.06, 0.84; 13C NMR
dC (D2O): d 96.1, 93.2, 73.8, 70.8, 67.9, 61, 38.4, 28.4,
20.7, 19.4.
Butyl a-D-2-deoxyglucopyranoside; FD–MS: m/z 220,
1H NMR dH (D2O): d 4.99, 3.87, 3.84, 3.76, 3.61, 3.68,
3.34, 3.49, 2.12 (Heq), 1.7 (Hax), 1.56, 1.35, 0.89; 13C
NMR dC (D2O): d 99.8, 75, 73.8, 71.1, 70.1, 63.4, 39.5,
33.4, 21.6, 15.9.
Acknowledgements
Pentyl a-D-2-deoxyglucopyranoside; FD–MS: m/z 234,
1H NMR dH (DMSO-d6): d 4.99, 3.86, 3.83, 3.75, 3.66,
3.61, 3.49, 3.34, 2.12 (Heq), 1.7 (Hax), 1.59, 1.32, 1.32,
0.87; 13C NMR dC (DMSO-d6): d 99.7, 74.9, 73.8,
71.1, 70.4, 63.4, 31, 30.5, 24.6, 16.1.
We are grateful to Dr. E. Fukushi and Mr. K. Watanabe
in our Graduate School for measuring the NMR and
MS data.
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