Hydrolytic activity of α-galactosidases against deoxy derivatives of p- nitrophenyl α-D-galactopyranoside

Article abstract of DOI:10.1016/S0008-6215(99)00281-5

The four possible monodeoxy derivatives of p-nitrophenyl (PNP) α-D- galactopyranoside were synthesized, and hydrolytic activities of the α- galactosidase of green coffee bean, Mortierella vinacea and Aspergillus niger against them were elucidated. The 2- and 6-deoxy substrates were hydrolyzed by the enzymes from green coffee bean and M. vinacea, while they scarcely acted on the 3- and 4-deoxy compounds. On the other hand, A. niger α- galactosidase hydrolyzed only the 2-deoxy compound in these deoxy substrates, and the activity was very high. These results indicate that the presence of two hydroxyl groups (OH-3 and -4) is essential for the compounds to act as substrates for the enzymes of green coffee bean and M. vinacea, while the three hydroxyl groups (OH-3, -4, and -6) are necessary for the activity of the A. niger enzyme. The kinetic parameters (K(m) and V(max)) of the enzymes for the hydrolysis of PNP α-D-galactopyranoside and its deoxy derivatives were obtained from kinetic studies. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(99)00281-5

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Carbohydrate Research 324 (2000) 107–115
Hydrolytic activity of a-galactosidases against deoxy
derivatives of p-nitrophenyl a-
D-galactopyranoside
Wataru Hakamata, Toshiyuki Nishio *, Tadatake Oku
Laboratory of Bio-organic Chemistry, College of Bioresource Sciences, Nihon Uni6ersity, 3-34-1 Shimouma,
Setagaya-ku, Tokyo 154-8513, Japan
Received 2 February 1999; accepted 28 September 1999
Abstract
The four possible monodeoxy derivatives of p-nitrophenyl (PNP) a-
D
-galactopyranoside were synthesized, and
hydrolytic activities of the a-galactosidase of green coffee bean, Mortierella 6inacea and Aspergillus niger against
them were elucidated. The 2- and 6-deoxy substrates were hydrolyzed by the enzymes from green coffee bean and M.
6inacea, while they scarcely acted on the 3- and 4-deoxy compounds. On the other hand, A. niger a-galactosidase
hydrolyzed only the 2-deoxy compound in these deoxy substrates, and the activity was very high. These results
indicate that the presence of two hydroxyl groups (OH-3 and -4) is essential for the compounds to act as substrates
for the enzymes of green coffee bean and M. 6inacea, while the three hydroxyl groups (OH-3, -4, and -6) are
necessary for the activity of the A. niger enzyme. The kinetic parameters (Km and Vmax) of the enzymes for the
hydrolysis of PNP a-D-galactopyranoside and its deoxy derivatives were obtained from kinetic studies. © 2000
Elsevier Science Ltd. All rights reserved.
Keywords: a-Galactosidase; Substrate specificity; Deoxy galactoside; Synthesis
1. Introduction
and almond a-mannosidase both hydrolyzed
p-nitrophenyl (PNP) a- -rhamnopyranoside
D
Generally, different types of exo-glycosi-
dases are distinguished on the basis of specific-
ity for the glycon structure of their substrates,
and their substrate specificities are expressed
in their relative activities against substrates
having various aglycons. Indications exist that
the specificity of glycosidases for the glycon
moiety of the substrate is not absolute. Several
studies reported that weak hydrolytic reac-
tions with deoxy analogs of the appropriate
at a rate approaching that for PNP a- -
D
mannopyranoside.
The aim of the present study was to gain
further knowledge about the glycon specificity
of a-galactosidase (a- -galactoside galactohy-
D
drolase, EC 3.2.1.22). a-Galactosidases cata-
lyze the hydrolytic cleavage of terminal
a- -galactopyranosyl residues from oligosac-
D
charides; preparations from some sources also
release -galactose from glycopeptides and
D
glycon were catalyzed by several
D
-glucosi-
glycolipids. Many a-galactosidases have been
purified from various sources [11], and the
amino acid sequences of the enzymes from
Mortierella 6inacea [12], Saccharomyces carls-
bergensis [13], and Streptococcus mutans [14]
have been determined. Complementary and
genomic DNAs encoding a-galactosidases
dases [1–7] and by two of the three a-galac-
tosidases used in the present study [8,9]. In
addition, we have reported [10] that jack bean
* Corresponding author.
E-mail address: t-nishio@nna.so-net.ne.jp (T. Nishio)
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00281-5

108
W. Hakamata et al. / Carbohydrate Research 324 (2000) 107–115
2. Results and discussion
Synthesis of deoxy substrates.—The struc-
-galactopyranoside (1) and
tures of PNP a-
D
of the newly synthesized deoxy derivatives (7,
16, 23, and 27, Scheme 1) used in the present
study are compared in Fig. 1. Each derivative,
namely
PNP
2-deoxy-a-
D
-lyxo-hexopyr-
-xylo-hexopyr-
anoside (7), PNP 3-deoxy-a-
D
anoside (16), PNP 4-deoxy-a-
D
-xylo-hexo-
pyranoside (23), and PNP a-
D
-fucopyranoside
Fig. 1. PNP a-D-galactopyranoside and its deoxy derivatives.
(27), was obtained in crystalline form. The
vicinal diaxial proton coupling constants (J2,3)
1
observed in the H NMR spectra of 1, 7, 16,
23, and 27 are 10.5, 12.0, 13.0, 10.4, and 10.4
were cloned from coffee bean [15], guar [16],
human [17], yeast [13], Esherichia coli [18],
Aspergillus niger [19], S. mutans [14], and M.
6inacea [20]. Finally, the aglycon specificities
of many a-galactosidases have been investi-
gated, but the glycon specificities have not
been systematically checked.
Hz, respectively. These values indicated that
4
these deoxygenated glycosides retained the C1
chair conformation in D2O. In addition, low-
energy conformations of 1, 7, 16, 23, and 27
were calculated1. These computed data sup-
1
ported the results of H NMR (data not
shown).
Substrate specificity of h-galactosidase.—
We investigated the specific activities of a-
galactosidases from green coffee beans and
from the molds M. 6inacea and A. niger on the
hydrolysis of 1 and the foregoing deoxy
derivatives. Since different reaction conditions
Here, we report the synthesis of monodeoxy
derivatives of PNP a- -galactopyranoside (1)
D
(Fig. 1) and the hydrolytic activities of a-
galactosidases from green coffee bean, M.
6inacea, and A. niger against them, and we
discuss differences in the substrate specificities
of these enzymes.
1 MM2 force field by CAChe (Sony Tektronix).
Scheme 1. Synthesis of 2-, 3-, 4-, and 6-deoxy derivatives of PNP a-D-galactopyranoside.

W. Hakamata et al. / Carbohydrate Research 324 (2000) 107–115
109
6inacea enzymes against 2- and 6-deoxy galac-
topyranosides are probably due to the loss of
a hydrogen bond, which affects formation of
the enzyme–substrate complex. However, we
think that the foregoing inference is not appli-
cable to the A. niger enzyme because of the
slight increase of the Km value against 7. This
fact indicates that the C-2 hydroxyl group of 1
is not concerned with the hydrogen-bonding
interaction between the substrate and the A.
niger enzyme. We think that the high activity
of the A. niger enzyme against 7 is due to the
inductive effect of OH-2, which depresses the
formation of the carbonium–oxonium ion.
These results indicate that the three a-galac-
tosidases are separable into two classes based
on the hydrolysis of the 2- and 6-deoxy
substrates.
were used for each enzyme, the findings allow
only a rough comparison to be made between
the activities of one enzyme relative to those
of the other a-galactosidases. Of the four de-
oxy derivatives of 1, the A. niger a-galactosi-
dase hydrolyzed only the 2-deoxy ‘galacto-
pyranoside’ (7); its activity with 7 appeared to
be substantially higher than against 1. On the
other hand, the green coffee bean and M.
6inacea enzymes showed activity not only for
the 2-deoxy compound 7 (substantially lower
than for 1 in each case), but also some still
lower activity for the 6-deoxy derivative PNP
a- -fucopyranoside (27). The failure of all
D
three enzymes to hydrolyze detectably the 3-
and 4-deoxy derivatives suggests that the C-3
and C-4 hydroxyl groups of a galactopyra-
noside may be important for substrate recog-
nition by the a-galactosidases.
Amino acid sequences of a-galactosidases
from green coffee bean [15], M. 6inacea [20],
and A. niger [19] have been reported. The
sequence homologies of the a-galactosidase
from A. niger with the enzymes from green
coffee bean and M. 6inacea are 35 and 32%,
respectively, and the sequence homology be-
tween the green coffee bean and M. 6inacea
enzymes is high (43%), suggesting a close rela-
tionship. Various types of glycosyl hydrolases
have been classified into families on the basis
of amino acid sequence similarities [21–23].
a-Galactosidases of A. niger and green coffee
bean were classified into the same family (27)
by this classification system. A different view
of the relationship among a-galactosidases ap-
pears when specificity for the entire substrate
is used as a basis of comparison. Suzuki et al.
[8] noted that, unlike the coffee bean and A.
niger enzymes, the M. 6inacea enzyme does
To examine further the several apparently
large differences between the activities against
2- and 6-deoxy substrates shown by green
coffee bean and M. 6inacea a-galactosidases
versus the A. niger enzyme, kinetic studies of
the hydrolysis of 1, 7, and 27 were carried out
(Table 1). From reaction velocities measured
at different substrate concentrations,
a
Lineweaver–Burk plot of 1/6 versus 1/S was
used to derive Km and Vmax for each enzyme–
substrate combination. Reaction conditions
(pH and/or incubation temperature) were dif-
ferent for the three enzymes. Values of Km for
the hydrolysis of 1, 7, and 27 by the green
coffee bean enzyme were 0.85, 27.4, and 38.5,
and those with the M. 6inacea enzyme were
0.96, 10.2, and 27.0, respectively. The Km val-
ues of the A. niger enzyme for the hydrolysis
of 1 and 7 were 0.54 and 2.63. The data
presented indicate that the increase of Km for
7 relative to 1 is 4.8-fold with the A. niger,
10.6-fold with M. 6inacea, and 32-fold with
the green coffee bean a-galactosidases. The
closeness of the first two values emphasizes
the need to provide standard errors of the
parameters in Table 1 and indicate that the
data were collected only from early-stage reac-
tions. These results show that the affinity for
the deoxy ‘galactopyranoside’ differentiates
the green coffee bean and M. 6inacea a-galac-
tosidases from the A. niger enzyme. The high
Km values of the green coffee bean and M.
not liberate
D-galactose from legume galac-
tomannan [8]. Classification of a-galactosi-
dases has been carried out by Kaneko et al.
[24]. They classified microbial a-galactosidases
into two groups based on the substrate specifi-
city against galacto-manno-oligosaccharides.
Canellakis et al. reported that mammalian
a-glucosidase hydrolyzes PNP 2-deoxy-a- -
D
‘glucopyranoside’ [25], and Nakano et al. re-
ported that A. niger a-glucosidase has
2-deoxy- -‘glucose’ condensation activity [26].
D
Purified a-glucosidases from various sources,
including the crystalline enzyme from A. niger,

110
W. Hakamata et al. / Carbohydrate Research 324 (2000) 107–115
have been found to catalyze the hydration of
D
-glucal and, thus, do not require an OH
group at C-2 [27]. This is true of the a-galac-
tosidase from green coffee beans, which has
been found to hydrate -galactal [9]. Our data
D
are consistent with these various observations
and suggest that a-galactosidases in general
will show 2-deoxy- -‘galactopyranoside’ hy-
D
drolyzing ability. Recently, Notenboom et al.
reported that the C-2 hydroxyl group of the
substrate acts to stabilize the transition state
in the action of b-glucosidases [28], but the
above data with a-galactosidase and a-glu-
cosidase suggest that the glycon C-2 hydroxyl
group of the substrate is not important with
the a-glycosidase.
Further studies using various substituted
substrates, including deoxy glycosides, should
be done to elucidate the substrate specificities
of various exo-a-glycosidases.
3. Experimental
Materials and methods.—New compounds
were characterized by elemental analysis and
1H NMR spectra. Melting points were deter-
mined with a Yamato model MP-21 capillary
apparatus and are uncorrected. Optical rota-
tions were measured with a Perkin–Elmer 141
1
polarimeter at 20 °C. H NMR spectra were
recorded with a Varian VXR-400 spectrome-
ter. Chemical shifts are expressed in ppm
downfield shift from Me4Si. Mass spectra were
obtained with a Jeol JMX SX-102A instru-
ment under positive FAB conditions. Column
chromatography was performed on Silica Gel
60 (230–400 mesh, E. Merck). The progress of
all reactions was monitored by thin-layer
chromatography (TLC) on Silica Gel 60 F254
(0.25 mm, E. Merck).
To prepare each deoxy derivative of PNP
a- -galactopyranoside (1), the following
D
methods were used.
Method A. To a solution of the sugar
derivative (55 mmol) in pyridine (150 mL) at
room temperature (rt) was added Ac2O (50
mL). The mixture was stirred overnight and
poured into water (200 mL). The product was
extracted with EtOAc (3×150 mL), washed
with water, 1 M HCl, satd NaHCO3, and

W. Hakamata et al. / Carbohydrate Research 324 (2000) 107–115
111
brine, and then dried over Na2SO4. Removal
of the solvent afforded the corresponding
acetate.
the solution had been refluxed for 4.5 h, the
solvent was evaporated. The residue was dis-
solved in 1:9 MeCN–Et2O (200 mL) and KF
(5.0 g) was added. The mixture was filtered
and the solvent was evaporated. The product
was purified by column chromatography on
silica gel (1:1 hexane–EtOAc) to afford 13.3 g
(72.7%) of 5. Boron trifluoride ethyl ether
complex (0.61 mL, 4.82 mmol) was added to a
stirred solution of 5 (0.53 g, 1.61 mmol) fol-
lowed by p-nitrophenol (0.45 g, 3.21 mmol) in
dry toluene (50 mL) under N2, and the mix-
ture was stirred for 1.5 h at rt. The mixture
was washed with satd NaHCO3 and brine,
and then concentrated after drying over
Na2SO4. After evaporation of the solvent, the
product was purified by column chromatogra-
phy on silica gel (3:2 hexane–EtOAc) to af-
ford 0.20 g (29.8%) of 6, which was
recrystallized from hot EtOH; mp 140.0–
Method B. 40:40:1 AcOH–Ac2O–H2SO4
(16.2 mL) was added to a stirred solution of
the sugar derivative (2.5 mmol). The mixture
was stirred overnight and then poured into
water (200 mL). The product was extracted
with EtOAc (200 mL). The organic layer was
washed with water (3×200 mL), satd
NaHCO3, and brine, and then dried over
Na2SO4. Removal of the solvent afforded the
corresponding acetate.
Method C [29]. To a melt of the acetate (1
equiv) and p-nitrophenol (4 equiv) was added
0.33 g anhyd ZnCl2 (1 equiv) dissolved in 1
mL of 19:1 AcOH–Ac2O. The flask was kept
in an oil bath at 125 °C for 5 min and evacu-
ated with a water pump. After dissolving the
resulting brown syrup in 300 mL of EtOAc,
the ZnCl2 and p-nitrophenol were removed by
successive washing with satd NaHCO3 and
brine, dried over Na2SO4, and concentrated.
Method D. 5:1:1 MeOH–Et3N–water (10
mL) was added to a stirred solution of the
sugar derivative (16 mmol). After the mixture
had been stirred for 3 h at rt, the solvent was
evaporated.
1
141.0 °C; [h]D +187° (c 0.98, CHCl3); H
NMR (400 MHz, CDCl3): l 1.93, 2.04, 2.17 (3
s, 9 H, 3×OCOCH3), 2.16 (ddd, 1 H, J1,2eq
1.2, J2ax,2eq 12.8, J2eq,3 5.2 Hz, H-2eq), 2.32
(ddd, 1 H, J1,2ax 3.14, J2ax,3 12.8 Hz, H-2ax),
4.07 (m, 1 H, H-6a), 4.08 (m, 1 H, H-6b), 4.18
(m, 1 H, H-5), 5.41 (dd, 1 H, J3,4 3.0, J4,5 2.4
Hz, H-4), 5.48 (ddd, 1 H, H-3), 5.85 (d, 1 H,
H-1), 7.17 (d, 2 H, J 9.2 Hz, aromatic H), 8.22
(d, 2 H, aromatic H); MS: 412 [MH+].
Method E. Sodium methoxide (28%) in
MeOH (1.0 mL) was added to a stirred solu-
tion of the sugar derivative (10 mmol) in dry
MeOH (50 mL). After the mixture had been
stirred for 5 h at rt, Amberlite IR-120 (H+
form) was added to neutrality.
p-Nitrophenyl 2-deoxy-h- -xylo-hexopyra-
D
noside (7).—Compound 7 was prepared from
6 (64.4 mg, 0.16 mmol) according to Method
C, and recrystallized from EtOAc–hexane
(40.6 mg, 90.9%): mp 146.5–148.0 °C; [h]D
p-Nitrophenyl 2-deoxy-3,4,6-tri-O-acetyl-h-
1
D
-xylo-hexopyranoside (6).—Compound
3
+227° (c 0.50, MeOH); H NMR (400 MHz,
was prepared from
D-galactose (10.21 g, 55.1
D2O): l 2.09 (ddd, 1 H, J1,2eq =J2eq,3 3.6,
J2ax,2eq 12.6 Hz, H-2eq), 2.16 (ddd, 1 H, J1,2ax
4.8, J2ax,3 12.0 Hz, H-2ax), 3.66 (dd, 1 H, J5,6a
4.4, J6a,6b 12.1 Hz, H-6a), 3.72 (dd, 1 H, J5,6b
7.8 Hz, H-6b), 3.90–3.94 (m, 2 H, H-4 and
H-5), 4.32 (ddd, 1 H, J3,4 6.0 Hz, H-3), 7.28
(d, 2 H, J 9.2 Hz, aromatic H), 8.26 (d, 2 H,
aromatic H); MS: 286 [MH+]: Anal. Calcd for
C12H15O7: C, 50.53; H, 5.30; N, 4.91. Found:
C, 50.33; H, 5.25; N, 4.90.
mmol) according to Method A. To a solution
of pentaacetate 3 in 200 mL CH2Cl2 was
added 25% HBr–AcOH (67 mL). The mixture
was stirred for 4 h and poured into water (250
mL). The organic layer was washed with wa-
ter (2×250 mL), satd NaHCO3, and brine,
and then concentrated after drying over
Na2SO4. The product was purified by column
chromatography on silica gel (2:1 hexane–
EtOAc) to give 4 (19.9 g, 87.1%). A solution
of compound 4 (21.6 g, 55.2 mmol) in benzene
(100 mL) was added dropwise over 14 h to a
stirred solution of refluxing benzene (50 mL)
and Bu3SnH (19 mL, 66.3 mmol) [30]. After
Methyl 4,6-O-benzylidene-h- -galactopyra-
D
noside (9).—Compound 8 (25 g, 117.8 mmol)
was dissolved in a mixture of formic acid (125
mL) and benzaldehyde (125 mL). The mixture
was stirred for 20 min at rt and poured into

112
W. Hakamata et al. / Carbohydrate Research 324 (2000) 107–115
800 mL of 3.12 M K2CO3 solution and
petroleum ether (1 L). The crystalline product
in the solution was recrystallized from
CH2Cl2 –hexane to afford 22.4 g (67.3%) of 9:
mp 171.0–171.5 °C; [h]D +141° (c 0.59,
and then Bu3SnH (1.5 mL, 5.58 mmol) was
added to the solution. The mixture was
refluxed for 15 h and the solvent was evapo-
rated. The product was dissolved in MeCN
(80 mL) and washed with hexane (2×40 mL)
to remove the remaining Bu3SnH. After evap-
oration of the solvent, the product was
purified by column chromatography on silica
gel (2:1 hexane–EtOAc) to afford 12 (1.18 g,
96.4%): mp 115.5–116.0 °C; [h]D +91° (c
1
CHCl3); H NMR (400 MHz, CDCl3): l 3.46
(s, 3 H, ꢀOCH3), 3.71 (ddd, 1 H, J4,5 =J5,6b
1.4, J5,6a 2.0 Hz, H-5), 3.90 (dd, 1 H, J2,3 9.9,
J3,4 3.7 Hz, H-3), 3.93 (dd, 1 H, J1,2 3.0 Hz,
H-2), 4.09 (dd, 1 H, J6a,6b 12.7 Hz, H-6a), 4.28
(dd, 1 H, H-4), 4.30 (dd, 1 H, H-6b), 4.94 (d,
1 H, H-1), 5.56 (s, 1 H, PhCHꢀ), 7.36–7.51
(m, 5 H, ꢀPh); MS: 283 [MH+].
1
0.61, CHCl3); H NMR (400 MHz, CDCl3): l
2.25 (ddd, 1 H, J2,3eq 5.0, J3ax,3eq 13.2, J3ax,4 3.0
Hz, H-3eq), 2.36 (ddd, 1 H, J2,3ax 12.6, J3ax,4
3.4 Hz, H-3ax), 3.47 (s, 3 H, OCH3), 3.72 (m,
1 H, H-5), 4.12 (dd, 1 H, J5,6a 1.8, J6a,6b 12.6
Hz, H-6a), 4.24 (m, 1 H, H-4), 4.29 (dd, 1 H,
J5,6b 1.2 Hz, H-6b), 5.13 (d, 1 H, J1,2 3.2 Hz,
H-1), 5.47 (ddd, 1 H, H-2), 5.57 (s, 1 H,
PhCHꢀ), 7.35–8.07 (m, 10 H, aromatic H);
MS: 371 [MH+].
Methyl 2-O-benzoyl-4,6-O-benzylidene-h- -
D
galactopyranoside (10).—Aqueous NaOH
(40%, 10 mL) and Bu4NCl (0.28 g, 0.78 mmol)
were added to a solution of 9 (4.40 g, 15.59
mmol) in CH2Cl2 (60 mL) at rt, followed by a
dropwise addition of BzCl (1.9 mL, 16.37
mmol). The mixture was stirred for 20 min at
rt and poured into CH2Cl2 (300 mL) and
water (200 mL), the organic layer was washed
with satd NaHCO3 and brine, and then con-
centrated after drying over Na2SO4. The
product was purified by column chromatogra-
phy on silica gel (3:2 toluene–Et2O) to afford
10 (3.03 g, 39.6%): mp 192–193 °C, lit. 202–
204 °C [26], lit. 206–207 °C [16]; [h]D +146°
(c 1.1, CHCl3), lit. +146° [26], lit. +153°
Methyl
2,4,6-tri-O-benzoyl-3-deoxy-h- -
D
xylo-hexopyranoside (13).—Compound 12
(1.45 g, 3.92 mmol) was dissolved in a mixture
of MeOH (60 mL) and 1 M HCl (12 mL). The
mixture was stirred for 3.5 h at 50 °C, and
then satd NaHCO3 was added to neutrality.
The solution was evaporated and the product
was extracted with EtOAc from the residue.
After evaporation of the solvent, pyridine (60
mL) was added, followed by a dropwise addi-
tion of BzCl (2.1 mL, 17.5 mmol). The mix-
ture was stirred overnight and concentrated,
and the solution was washed with 1 M HCl,
satd NaHCO3, and brine, and then concen-
trated after drying over Na2SO4. The product
was purified by column chromatography on
silica gel (3:1 hexane–EtOAc) to afford 13
(1.57 g, 81.7%), which was recrystallized from
1
[16]; H NMR (400 MHz, CDCl3): l 3.44 (s, 3
H, OCH3), 3.81 (d, 1 H, H-5), 4.13 (dd, 1 H,
J5,6a 1.8, J6a,6b 12.6 Hz, H-6a), 4.29 (dd, 1 H,
J2,3 10.0, J3,4 3.6 Hz, H-3), 4.34 (dd, 1 H, J5,6b
1.4 Hz, H-6b), 4.36 (dd, 1 H, J4,5 1.2 Hz, H-4),
5.12 (d, 1 H, J1,2 3.6 Hz, H-1), 5.38 (d, 1 H,
H-2), 5.61 (s, 1 H, PhCHꢀ), 7.39–8.11 (m, 10
H, aromatic H); MS: 387 [MH+].
Methyl 2-O-benzoyl-3-deoxy-4,6-O-benzyli-
1
dene-h-
D
-xylo-hexopyranoside (12).—To a so-
hot EtOH: [h]D +69° (c 1.3, CHCl3); H
lution of 10 (1.27 g, 3.30 mmol) in CH2Cl2 (25
mL) was added phenylchlorothionoformate
[31] (0.7 mL, 4.94 mmol) at rt, followed by
dropwise addition of pyridine (2 mL). The
mixture was stirred for 19 h and poured into
CH2Cl2 (200 mL) and 1 M HCl (100 mL),
washed with satd NaHCO3 and brine, and
then concentrated after drying over Na2SO4.
The product was purified by column chro-
matography on silica gel (2:1 hexane–EtOAc).
The unstable thiocarbonyl derivative 11 was
immediately dissolved in toluene (100 mL),
NMR (400 MHz, CDCl3): l 2.38 (ddd, 1 H,
J2,3eq 4.6, J3ax,3eq 13.6, J3ax,4 3.4 Hz, H-3eq),
2.47 (ddd, 1 H, J2,3ax 13.0, J3ax,4 3.0 Hz, H-
3ax), 3.49 (s, 3 H, OCH3), 4.42 (ddd, 1 H, J4,5
1.4, J5,6a 5.0, J5,6b 10.0 Hz, H-5), 4.44 (dd, 1 H,
J6a,6b 8.4 Hz, H-6a), 4.56 (dd, 1 H, H-6b), 5.17
(d, 1 H, J1,2 3.6 Hz, H-1), 5.43 (ddd, 1 H,
H-2), 5.57 (ddd, 1 H, H-4), 7.40–8.14 (m, 15
H, aromatic H); MS: 419 [MH+].
p-Nitrophenyl 2,4,6-tri-O-benzoyl-3-deoxy-
h- -xylo-hexopyranoside (15).—Compound
D
15 was prepared from 13 (1.26 g, 2.57 mmol)

W. Hakamata et al. / Carbohydrate Research 324 (2000) 107–115
113
according to Methods B and C. The product
was purified by column chromatography on
silica gel (4:1 hexane–EtOAc) to afford 15
(0.95 g, (74.6%), which was recrystallized from
EtOH: mp 133.5–134.0 °C; [h]D +76° (c 0.60,
H-6b), 5.15 (d, 1 H, J1,2 3.6 Hz, H-1), 5.27
(dd, 1 H, J2,3 10.2 Hz, H-2), 5.80 (dd, 1 H, J3,4
10.2 Hz, H-3), 7.35–8.11 (m, 15 H, aromatic
H); MS: 507 [MH+].
Methyl
2,3,6-tri-O-benzoyl-4-deoxy-h- -
D
1
CHCl3); H NMR (400 MHz, CDCl3): l 2.56
xylo-hexopyranoside (20).—To a solution of
18 (10.9 g, 21.5 mmol) in CH2Cl2 (300 mL)
was added 4-dimethylaminopyridine (7.89 g,
64.5 mmol) at rt, followed by dropwise addi-
tion of phenylchlorothionoformate [31] (3.6
mL, 25.8 mmol). The mixture was stirred for 4
days and the solvent was evaporated. The
product was purified by column chromatogra-
phy on silica gel (3:2 hexane–EtOAc). The
unstable thiocarbonyl derivative 19 (11.1 g,
17.2 mmol) was immediately dissolved in
toluene (700 mL), and then Bu3SnH (5.8 mL,
20.6 mmol) was added to the solution. After
refluxing the mixture overnight, the solvent
was evaporated and the product was purified
by column chromatography on silica gel (3:1
hexane–EtOAc) to afford 20 (7.95 g, 94.3%):
mp 115.5–117.0 °C; [h]D +132° (c 1.1,
(ddd, 1 H, J2,3eq =J3eq,4 4.0, J3ax,3eq 13.8 Hz,
H-3eq), 2.66 (ddd, 1 H, J2,3ax 12.4, J3ax,4 3.0
Hz, H-3ax), 4.41–4.53 (m, 3 H, H-5, H-6a,
and H-6b), 5.60 (ddd, 1 H, J1,2 3.6 Hz, H-2),
5.62 (ddd, 1 H, J4,5 5.2 Hz, H-4), 6.06 (d, 1 H,
H-1), 7.19–8.15 (m, 19 H, aromatic H); MS:
598 [MH+].
p-Nitrophenyl 3-deoxy-h- -xylo-hexopyran-
D
oside (16).—Compound 16 was prepared from
15 (0.67 g, 1.13 mmol) according to Method
E. The product was purified by column chro-
matography on silica gel (8:1 CH2Cl2–MeOH)
to afford 16 (0.22 g, 67.4%), which was recrys-
tallized from hot EtOH: mp 162.0–163.5 °C,
lit. 163.5–164.5 °C [27]; [h]D +221° (c 0.56,
1
MeOH), lit. +219° [27]; H NMR (400 MHz,
D2O): l 2.13 (ddd, 1 H, J2,3eq=J3eq,4 4.4,
J3ax,3eq 13.6 Hz, H-3eq), 2.23 (ddd, 1 H, J2,3ax
13.0, J3ax,4 3.0 Hz, H-3ax), 3.65–3.67 (m, 2 H,
H-6a and H-6b), 3.89 (ddd, 1 H, J4,5 1.1, J5,6a
5.5, J5,6b 6.6 Hz, H-5), 4.13 (m, 1 H, H-4), 4.28
(ddd, 1 H, J1,2 3.6 Hz, H-2), 5.79 (d, 1 H,
H-1), 7.33 (d, 2 H, J 9.2 Hz, aromatic H), 8.28
(d, 2 H, aromatic H); MS: 286 [MH+]: Anal.
Calcd for C12H15O7: C, 50.53; H, 5.30; N,
4.91. Found: C, 50.37; H, 5.35; N, 4.91.
1
CHCl3); H NMR (400 MHz, CDCl3): l 1.90
(ddd, 1 H, J3,4ax=J4ax,5 =J4ax,4eq 11.9 Hz, H-
4ax), 2.48 (ddd, 1 H, J3,4eq 5.4, J4eq,5 2.2 Hz,
H-4eq), 3.46 (s, 3 H, ꢀOCH3), 4.36–4.50 (m, 3
H, H-5, H-6a and H-6b), 5.18 (d, 1 H, J1,2 3.6
Hz, H-1), 5.32 (dd, 1 H, J2,3 10.0 Hz, H-2),
5.79 (ddd, 1 H, H-3), 7.36–8.10 (m, 15 H,
aromatic H); MS: 419 [MH+].
p-Nitrophenyl 2,3,6-tri-O-benzoyl-4-deoxy-
Methyl 2,3,6-tri-O-benzoyl-h- -glucopyran-
D
h- -xylo-hexopyranoside (22).—Compound
D
oside (18).—Compound 17 (7.65 g, 39.4
mmol) and bistributyltin oxide [(Bu3Sn)2O, 25
mL, 59.1 mmol] were dissolved in toluene (750
mL). After refluxing the mixture overnight,
the solvent was cooled to rt, and BzCl (20.6
mL, 177.4 mmol) was added. The mixture was
stirred for 4 days at rt and the solvent was
evaporated. The residue was dissolved in
MeCN (400 mL) and washed with hexane
(2×200 mL) to remove the remaining
(Bu3Sn)2O. After evaporation of the solvent,
the product was purified by column chro-
matography on silica gel (3:1 hexane–EtOAc)
to afford 18 (10.9 g, 54.5%): [h]D +140° (c
21 was prepared from 20 (7.95 g, 16.2 mmol)
according to Method B. The residue was
purified by column chromatography on silica
gel (4:1 hexane–EtOAc) to afford 21 (6.96 g,
82.8%). Compound 22 was prepared from 21
(1.44 g, 2.78 mmol) according to Method C.
The product was purified by column chro-
matography on silica gel (4:1 hexane–EtOAc)
to afford 22 (1.17 g, 70.4%), which was recrys-
tallized from hot EtOH: mp 173.0–176.0 °C;
1
[h]D +148° (c 1.0, CHCl3); H NMR (400
MHz, CDCl3): l 1.99 (ddd, 1 H, J3,4ax
=
J
4ax,5=J4ax,4eq 12.0 Hz, H-4ax), 2.55 (ddd, 1
H, J3,4eq 5.0, J4eq,5 5.2 Hz, H-4eq), 4.36–4.47
(m, 3 H, H-5, H-6a and H-6b), 5.50 (dd, 1 H,
J1,2 3.6, J2,3 10.4 Hz, H-2), 5.99 (ddd, 1 H,
H-3), 6.06 (d, 1 H, H-1), 7.39–8.08 (m, 15 H,
aromatic H); MS: 598 [MH+].
1
1.0, CHCl3); H NMR (400 MHz, CDCl3): l
3.46 (s, 3 H, ꢀOCH3), 3.89 (m, 1 H, H-4), 4.12
(ddd, 1 H, J5,6a 2.2, J5,6b 4.6 Hz, H-5), 4.64
(dd, 1 H, J6a,6b 12.2 Hz, H-6a), 4.79 (dd, 1 H,

114
W. Hakamata et al. / Carbohydrate Research 324 (2000) 107–115
p-Nitrophenyl 4-deoxy-h-
D
-xylo-hexopyran-
Assay of enzyme hydrolytic acti6ity.—Pure-
grade a-galactosidases from M. 6inacea (Seik-
agaku Ind.), A. niger (Sigma Chemical Co.),
and green coffee bean (Bo¨ehringer Mannheim
oside (23).—Compound 23 was prepared from
22 (0.30 g, 0.51 mmol) according to Method
E. The product was purified by column chro-
matography on silica gel (7:1 CH2Cl2–MeOH)
to afford 23 (0.13 g, 87.9%), which was recrys-
tallized from EtOH: mp 152.5–155.0 °C; [h]D
Co.) were used in this study. PNP a- -galac-
D
topyranoside was purchased from Tokyo Ka-
sei Ind., and recrystallized from EtOH.
1
Hydrolytic activity of a-galactosidase was
assayed by measuring the release of p-nitro-
phenol from each glycosidic derivative after
incubation at the following conditions: M.
6inacea in 50 mM sodium phosphate buffer
(pH 5.9) at 40 °C, A. niger in 50 mM sodium
acetate buffer (pH 4.0) at 25 °C, green coffee
bean in 50 mM sodium phosphate buffer (pH
7.2) at 25 °C. The enzyme reaction was
stopped by the addition of 0.3 M Na2CO3,
and the p-nitrophenol released by the reaction
was measured spectrophotometically at 405
nm. One unit of enzyme activity was defined
as the amount of enzyme required to liberate 1
mmol of p-nitrophenol/min at each assay con-
dition. Kinetic studies on the hydrolysis of
+243° (c 0.51, MeOH); H NMR (400 MHz,
D2O): l 1.58 (ddd, 1 H, J3,4ax 14.1, J4ax,4eq
=
J4ax,5 12.1 Hz, H-4ax), 2.05 (m, 1 H, H-4eq),
3.52–3.64 (m, 2 H, H-6a and H-6b), 3.71 (dd,
1 H, J1,2 3.4, J2,3 10.4 Hz, H-2), 3.99 (m, 1 H,
H-5), 4.21 (ddd, 1 H, J3,4eq 5.4 Hz, H-3), 5.86
(d, 1 H, H-1), 7.27–8.29 (m, 4 H, aromatic
H); MS: 286 [MH+]; Anal. Calcd for
C12H15O7: C, 50.53; H, 5.30; N, 4.91. Found:
C, 50.81; H, 5.43; N, 4.75.
p-Nitrophenyl
2,3,4-tri-O-acetyl-h- -fu-
D
copyranoside (26).—Compound 25 was pre-
pared from 24 (1.01 g, 5.91 mmol) according
to Method A. The product was purified by
column chromatography on silica gel (2:1 hex-
ane–EtOAc) to afford 25 (1.95 g, 99.3%).
Compound 26 was prepared from 25 (4.55 g,
13.7 mmol) according to Method C. The
product was purified by column chromatogra-
phy on silica gel (3:1 hexane–EtOAc) to af-
ford 26 (1.20 g, 21.3%), which was
recrystallized from EtOH: mp 171.5–172.5 °C;
PNP a- -galactopyranoside and its deoxy
D
derivatives by the a-galactosidases were per-
formed at concentrations between 0.8 and 2.4
mM, and values of Km and Vmax were calcu-
lated from reciprocal plots of the reaction
curves.
Protein in the enzyme solution was deter-
mined by the method of Lowry using bovine
serum albumin as a standard.
1
[h]D +225° (c 1.0, MeOH); H NMR (400
MHz, CDCl3): l 1.13 (d, 3 H, J5,6 6.8 Hz,
H-6), 2.04, 2.07, 2.21 (3 s, 9 H, 3×OCOCH3),
4.19 (dq, 1 H, J4,5 1.0 Hz, H-5), 5.31 (dd, 1 H,
J2,3 11.2, J3,4 3.6 Hz, H-3), 5.37 (dd, 1 H, H-4),
5.57 (dd, 1 H, J1,2 3.5 Hz, H-2), 5.86 (dd, 1 H,
H-1), 7.15–8.23 (m, 4 H, aromatic H); MS:
412 [MH+].
Acknowledgements
The authors express their sincere thanks to
Dr N. Miyata of the National Institute of
p-Nitrophenyl h- -fucopyranoside (27).—
D
1
Health Science for recording the H NMR
Compound 27 was prepared from 26 (1.00 g,
2.44 mmol) according to Method D. The
product was recrystallized from hexane–
EtOAc to afford 27 (0.624 g, 92.3%): mp
194.5–195.5 °C; [h]D +249° (c 1.0, MeOH);
1H NMR (400 MHz, D2O): l 1.16 (d, 3 H, J5,6
6.8 Hz, H-6), 4.19 (dd, 1 H, J3,4 3.2 Hz, H-4),
4.01 (dd, 1 H, J1,2 3.8, J2,3 10.4 Hz, H-2),
4.11–4.17 (m, 2 H, H-3 and H-5), 5.82 (d, 1
H, H-1), 7.27–8.29 (m, 4 H, aromatic H); MS:
286 [MH+]; Anal. Calcd for C12H15O7: C,
50.53; H, 5.30; N, 4.91. Found: C, 50.36; H,
5.27; N, 4.85.
spectra, Dr T. Nakata of the Institute of
Physical and Chemical Research (RIKEN) for
the elemental analysis, and Ms J. Nakate for
the measurement of FAB mass spectra. This
work was supported in part by Special Coor-
dination Funds of the Science and Technology
Agency of the Japanese Government.
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