Complementary and synergistic roles in enzyme-catalyzed regioselective and complete hydrolytic deprotection of O-acetylated β-d-glucopyranosides of N-arylacetohydroxamic acids

Article abstract of DOI:10.1021/jo202123s

An efficient chemoenzymatic synthesis of β-d-glucopyranosides of N-arylacetohydroxamic acids 3a-c was achieved by the chemoselective O-deacetylation of 1a-c under mild, neutral conditions, with no accompanying N-deacetylation. Lipase AS Amano from Aspergi

Full text of DOI:10.1021/jo202123s

Article
pubs.acs.org/joc
Complementary and Synergistic Roles in Enzyme-Catalyzed
Regioselective and Complete Hydrolytic Deprotection of O-
Acetylated β-D-Glucopyranosides of N-Arylacetohydroxamic Acids
Akiko Baba and Tadao Yoshioka*
Hokkaido Pharmaceutical University School of Pharmacy, 7-1 Katsuraoka-cho, Otaru, Hokkaido 047-0264, Japan
S
* Supporting Information
ABSTRACT: An efficient chemoenzymatic synthesis of β-D-
glucopyranosides of N-arylacetohydroxamic acids 3a−c was
achieved by the chemoselective O-deacetylation of 1a−c under
mild, neutral conditions, with no accompanying N-deacetylation.
Lipase AS Amano from Aspergillus niger (LAS) and carboxylester-
ase from Streptomyces rochei (CSR) played complementary,
synergistic roles in the O-deacetylation of 1a and its partially O-
deacetylated intermediates. An intramolecular O-acetyl migration,
which proceeded simultaneously, also accelerated the overall reaction rate. Under weakly acidic conditions at pH 5.0, where the
intramolecular O-acetyl migration is markedly slower, LAS, CSR, and porcine liver esterase (PLE) exhibited different
regioselective O-deacetylation activities. LAS and PLE showed regioselective 3-O-deacetylation and 2-O-deacetylation activity,
respectively, for 1a and its tri-O-acetyl derivatives (4-7). CSR showed marked preferences for 3-O-deacetylation of 2,3,6-tri-O-
acetyl intermediate 5 and 4-O-deacetylation of 2,4,6-tri-O-acetyl intermediate 6. In contrast, CSR showed almost no O-
deacetylation activity toward the other tri-O-acetyl intermediates 4 and 7, which were efficiently O-deacetylated by LAS in a
complementary manner. Using these enzyme-catalyzed regioselective O-deacetylation as well as chemical methods, we could
synthesize all 14 partially O-acetylated intermediates (4−17) derived from 1a.
using an orthoester glycosidation method.12 The chemical O-
deacetylation of the glucopyranosides, using a catalytic amount
INTRODUCTION
Glucuronidation is a well-known metabolic conjugation
■
of barium methoxide in MeOH or in MeOH saturated with
reaction that is also thought to contribute to the toxic
bioactivation of some types of xenobiotics,1 particularly
carboxylic acid drugs and N-arylacetohydroxamic acids. The
NH3, afforded both O- and N-deacetylated derivatives,
suggesting that the N-acetyl group on the aglycone moiety is
alkali-labile. Consequently, the β-D-glucopyranosides of N-
arylacetohydroxamic acids were synthesized via complete O-
resulting glucuronides have been implicated as a cause of
toxicological adverse reactions to carboxylic acid drugs2 and the
and N-deacetylation followed by selective N-acetylation11 to
study their chemical13 and mutagenic activities.11,14 Recently,
we have developed a chemoenzymatic synthesis15 for β-D-
glucuronides of carboxylic acids from their acetylated methyl
ester derivatives in order to examine the quantitative structure−
activity relationships16 for the electrophilicity of the 1-β-O-acyl
linkages. The enzymatic hydrolytic deprotections of the sugar
protecting groups were performed at pH 5.0 to minimize the
nonenzymatic intramolecular acyl migration17 of the 1-β-O-acyl
groups. Two lipases from Aspergillus niger (LAS) and a
carboxylesterase from Streptomyces rochei (CSR) were the
biocatalysts of choice for the chemoenzymatic hydrolysis of the
O-acetyl groups. Subsequent hydrolysis of the glucuronide
methyl ester groups was achieved using either porcine liver
esterase (PLE) or lipase type B from Candida antarctica (CAL-
B).
carcinogenicity of N-arylacetohydroxamic acids.3 These glucur-
onides are alkali-labile, electrophilic metabolites that are capable
of covalently binding to proteins and nucleic acids, which could
elicit toxicological responses. However, the role of glucuronides
in adverse reactions to carboxylic acid drugs2 and the
carcinogenic properties4 of the O-glucuronide of N-arylaceto-
hydroxamic acids remain largely unknown.
In addition to carcinogenic N-arylacetohydroxamic acids, O-
glucuronides of biologically interesting hydroxamic acids have
been also reported. These include urinary glucuronides of the
anti-inflammatory drug bufexamac,5 glucuronides of the matrix
metalloprotease inhibitor trocade,6 and the histone deacetylase
inhibitor vorinostat.7−9 O-Deacetylation of the acetylated O-
glucuronide methyl ester of vorinostat was achieved in 67%
yield under standard basic conditions using sodium methoxide
in MeOH.8 The moderate yield might be due in part to the
alkali-labile nature of the N-acyl group on the hydroxamic acid
O-glycosidic linkage.
We report the application of the enzyme-catalyzed chemo-
selective O-deacetylation method to model substrates, 2,3,4,6-
We had previously reported the chemical synthesis of
acetylated β-D-glucuronide methyl esters10 and per-O-acety-
lated β-D-glucopyranosides11 of N-arylacetohydroxamic acids
Received: October 13, 2011
Published: January 24, 2012
© 2012 American Chemical Society
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tetra-O-acetyl-β-D-glucopyranosides of N-arylacetohydroxamic
acids 1a−c, to obtain the targets 3a−c directly, without N-
deacetylation (Scheme 1). This enzymatic O-deacetylation
RESULTS AND DISCUSSION
■
Chemical Synthesis of 1a−c and Partially O-Acety-
lated Glucopyranosides 4−17. 2,3,4,6-Tetra-O-acetyl-β-D-
glucopyranosides of N-arylacetohydroxamic acids 1a−c, which
have previously been synthesized by a two-step synthesis via
orthoester intermediates,12 were prepared directly from
acetobromglucose and the corresponding potassium N-
arylacetohydroxamates. The potassium salts of 1a−c, prepared
by the addition of a stoichiometric amount of KOH, showed far
higher nucleophilic reactivity toward acetobromglucose than
1a−c. However, the basic conditions could compete with the
decomposition of acetobromglucose, which might lead to the
poor yields between 28% and 32% (Scheme 1).
Scheme 1. Chemical and Chemoenzymatic Synthesis of β-D-
a
Glucopyranosides of N-Arylacetohydroxamic Acids 3a−c
The basic conditions of chemical O-deacetylation of 1a−c,
using a catalytic amount of barium methoxide in MeOH or
MeOH saturated with ammonia, proceeded with accompanying
N-deacetylation to afford 2a−c. When the O-deacetylation of 1
with barium methoxide in MeOH was performed at 0 °C until
the starting material was consumed, HPLC monitoring showed
a mixture of 2 and 3 as well as some mono-O-acetyl
intermediates. The following products were obtained: 2a
(20%) and 3a (70%) from 1a; 2b (54%) and 3b (37%) from
1b; and 2c (25%) and 3c (70%) from 1c. These results led us
to investigate the chemoenzymatic synthesis of 3a−c without
N-deacetylation. Compound 1a was chosen as a model
substrate for studying the enzyme-catalyzed O-deacetylation
activity, and the 14 partially O-deacetylated derivatives (4−17)
were synthesized as standard samples to investigate the O-
deacetylation sequence. The mono-O-acetyl derivatives 16 and
17 were synthesized by 4,6-O-orthoesterification19 of 3a
followed by the acidic opening of the orthoester group and
deprotection (Scheme 2). Derivatives 14 and 15 were
synthesized by 4,6-O-acetonidation of 3a followed by mono-
O-acetylation with acetic anhydride in pyridine20 and then
deprotection (Scheme 2). 2,3,4-Tri-O-acetyl and 2,3,6-tri-O-
acetyl derivatives 4 and 5 were also synthesized from 3a by 4,6-
O-orthoesterification19 and acetylation followed by deprotec-
tion (Scheme 2). In addition to 5, the tri-O-acetyl derivatives 6
and 7 were prepared via intramolecular O-acetyl migration of 4
under weakly alkaline conditions (Scheme 3). Although 2,3-di-
O-acetyl derivative 8 was synthesized through 4,6-O-acetoni-
dation, the other di-O-acetyl derivatives (9−13) were prepared
a
Reaction conditions: (i) aq KOH, EtOH, 25 °C, 32% (1a), 30% (1b),
28% (1c); (ii) LAS and CSR, phosphate buffer (pH 7.2), DMSO, 40
°C, 92% (3a), 90% (3b), 88% (3c); (iii)11 MeOH saturated with NH3,
0 °C; (iv)11 AcCl, dioxane, NaHCO3, 25 °C.
method may be useful as an alternative to the chemical method,
particularly for removing O-acetyl groups on glycosides with an
alkali-labile glycosidic bond or functional groups on the
aglycone moiety. Furthermore, we have synthesized all the
partially O-deacetylated derivatives of 1a (4−17) and analyzed
the kinetics of both the enzyme-catalyzed O-deacetylation of 1a
and the nonenzymatic intramolecular O-acetyl migration of the
resultant O-deacetylated intermediates. LAS has recently been
shown to exhibit regioselective 3-O-deacetylation activity;18
therefore, the enzymatic regioselective O-deacetylation was also
examined using 1a and its partially O-deacetylated intermedi-
ates (4−13). LAS, CSR, and PLE exhibit different regiose-
lective O-deacetylation activities, and thus several partially O-
deacetylated compounds, including di-O-acetyl derivatives 9−
13, that were difficult to synthesize regioselectively using
chemical methods, could be easily prepared chemoenzymati-
cally.
a
Scheme 2. Chemical Synthesis of Tri-O-acetyl (4 and 5) and Mono-O-acetyl (14−17) Compounds
a
Reaction conditions: (i) CH3CH(OMe)3, TsOH (cat.), CH3CN, 25 °C; (ii) H2O, 25 °C; (iii) Ac2O, pyridine, 25 °C then 80% (v/v) aq AcOH, 25
°C; (iv) dimethoxypropane, acetone, TsOH (cat.), 37 °C; (v) Ac2O, pyridine, 4 °C then 80% (v/v) aq AcOH, 25 °C.
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a
Scheme 3. Chemical Synthesis of Tri-O-acetyl Compounds 6 and 7 and Di-O-acetyl Compounds 8−13
a
Reaction conditions: (i) MOPS buffer (pH 7.5), CH3CN, 37 °C; (ii) dimethoxypropane, acetone, TsOH (cat.); (iii) Ac2O, pyridine, 25 °C then
80% (v/v) aq AcOH, 25 °C; (iv) 4-methoxytrityl chloride, pyridine, 25 °C; (v)50% (v/v) aq pyridine, 50 °C then 80% (v/v) aq AcOH.
a
Scheme 4. Chemoenzymatic Synthesis of Di-O-acetyl Compounds 9−13
a
Reaction conditions: (i) LAS, NaOAc buffer (pH 5.0), EtOH, 40 °C; (ii) PLE, NaOAc buffer (pH 5.0), EtOH, 40 °C; (iii) PLE, NaOAc buffer (pH
5.0), EtOH, 40 °C; (iv) CSR, NaOAc buffer (pH 5.0), EtOH, 40 °C; (v) PLE, NaOAc buffer (pH 5.0), EtOH, 40 °C.
by using intramolecular O-acetyl migrations of 8 and its 6-O-(4-
methoxy)trityl derivative under weakly alkaline conditions
(Scheme 3).
acetylation of glucopyranosides has been reported, with the
exception of the 3,6-di-O-acetylation26 and 2,6-di-O-acetyla-
tion.29 As described later, tri-O-acetyl 6 and 7 and di-O-acetyl
derivatives 9-13 (Scheme 4) were then individually synthesized
by enzymatic regioselective O-deacetylation of their corre-
sponding precursor substrates.
The acetonide and 4-methoxytrityl protecting groups were
successfully removed by using 80% (v/v) aq AcOH with no
accompanying hydrolysis or intramolecular O-acetyl migration.
1
The structures of compounds 4−17 were characterized by H
Enzyme Screening and Characterization for O-
Deacetylation Activity toward 1a. To minimize the
intramolecular O-acetyl migration, enzyme screening was
performed under mild acidic conditions with 0.75 mM of 1a
in 85 mM sodium acetate buffer (pH 5.0), which contained
15% (v/v) DMSO, at 40 °C for 10 min. Of the 15 enzymes
tested, LAS showed the highest O-deacetylation activity,
followed by PLE, lipase AP6 from Aspergillus niger, CSR, and
CAL-B. Acylase I showed weak activity and the other enzymes
did not show any hydrolytic activity toward 1a even when they
were incubated at pH 7.2. Because LAS and lipase AP6 showed
a similar HPLC pattern of hydrolytic products, the O-
deacetylation activity of four enzymes, namely, LAS, PLE,
CSR, and CAL-B, was examined more closely at pH 5.0 and at
pH 7.2. DMSO was the best cosolvent, followed by 2-
methoxyethanol, bis(2-methoxyethyl) ether, and EtOH. LAS
and 13C NMR and MS, together with analyses of the chemical
shifts of the pyranose moiety based on the acylation and
deacylation shifts.21 The crystallographic data and ORTEP
plots for 4 and 5 are provided in the Supporting Information.
Although regioselective chemical O-acetylation of C3-OH,23 C4-
OH,24 and C6-OH25 has been extensively exploited for α-D- or
β-D-glucopyranosides,22 the mono-O-acetyl derivatives 14, 15
and 16, 17 were synthesized and purified by chromatography
with no problems. The synthesis of 3,6-di-O-acetyl derivative
12 from 3a via an organotin-mediated direct O-acetylation26
was unsuccessful because of N-deacetylation of 3a during the
reaction step of heating with dibutyltin oxide in MeOH at 70
°C. A mild and selective method for the cleaving both O-
acetyl27 and N-acetyl28 groups has been reported under these
conditions. To our knowledge, no regioselective chemical di-O-
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and PLE exhibited high O-deacetylation activity and different
regioselectivity at pH 5.0 (Table 1). LAS mainly gave the 3-O-
about 40-fold higher than that of CSR. On the basis of the k
values of the tri-O-acetyl compounds 4−7, LAS showed higher
activity for 4 and 7 than for 5 and 6, whereas CSR showed
higher activity for 5 and 6 than for 4 and 7 (Table 2). These
Table 1. Yields of Major Products Obtained from Enzyme-
Catalyzed O-Deacetylation of 1a
Table 2. First-order Rate Constant k Values and
Regioselectivity for O-Deacetylation of Compounds 4−7 at
pH 5.0
a
b
enzyme
pH
products and yields (%)
c
d
LAS
5.0 1a (nd ), 6 (47%), di-O-acetyl 9 + 10 + 12 + 13 (28%),
mono-O-acetyl 14 + 17 (25%)
d
a
b
7.2 1a (nd ), 6 (9%), di-O-acetyl 10 + 12 + 13 (31%), mono-
enzyme
LAS
substrate
k
product ratio
O-acetyl 17 (52%)
4
5
6
7
4
5
6
7
4
5
6
7
1.54 0.02
0.70 0.01
0.033 0.003
1.48 0.02
0.012 0.001
2.94 0.12
9.36 0.42
0.024 0.002
0.79 0.03
0.164 0.008
0.47 0.01
0.225 0.012
9:8 (7.1:1)
c
CSR
5.0 1a (58%), 5 (19%), 7 (7%), di-O-acetyl 10 + 12 + 13
(11%), mono-O-acetyls 17 (5%)
10:12 (6.3:1)
13:10 (1.4:1)
13:12 (2.5:1)
7.2 1a (4%), 7 (5%), di-O-acetyl 10 + 12 + 13 (14%), mono-
O-acetyl 17 (45%), 3a (33%)
c
d
c
PLE
5.0 1a (nd ), 7 (23%), di-O-acetyl 11 + 12 + 13 (57%),
CSR
PLE
nd
mono-O-acetyl 15 + 16 (19%)
10:12 (20:1)
10:13 (35:1)
13:12 (3.8:1)
7.2 di-O-acetyl 12 + 13 (28%), mono-O-acetyl 14 + 15 + 16+
17 (65%), 3a (7%)
c
CAL-B
5.0 1a (64%), di-O-acetyl (29%)
7.2 1a (56%), di-O-acetyl (36%)
d
11
d
12
c
LAS +
c
5.0 di-O-acetyl 10 (27%), mono-O-acetyl 14 + 17 (48%), 3a
d
13
CSR
(25%)
13:12 (2.0:1)
7.2 mono-O-acetyl 14 + 17 (39%), 3a (60%)
a
a
Incubation was performed at pH 5.0 and 40 °C and k values are
Incubation was performed at the indicated pH and incubation time at
b
shown as h−1 (mg/mL)−1. Product ratio was calculated from the
b
c
40 °C for 2.0 h. Yields based on HPLC analysis. The concentration
of LAS and CSR was 5.0 mg/mL of the incubation mixture and that of
HPLC peak areas of the O-deacetylation products in the early stages of
c
d
the enzyme-catalyzed reaction. Ratio was not determined because of
PLE and CAL-B was 2.5 mg/mL of the incubation mixture. nd: not
detected.
d
low activity. Obtained effectively as the sole product.
results demonstrate that LAS and CSR played complementary,
synergistic roles in the O-deacetylation of 1a to give 3a via
intermediates 4−7, which explains the enhancement of O-
deacetylation of 1a by the combination of LAS and CSR (Table
1). The product ratios showed that LAS catalyzed the
regioselective 3-O-deacetylation of 4, 5, and 7 and PLE
efficiently catalyzed the regioselective 2-O-deacetylation of 4−
6. CSR preferentially catalyzed the 3-O-deacetylation of 5 and
the 4-O-deacetylation of 6, but did not catalyze the 3-O-
deacetylation or the 4-O-deacetylation of 4 and 7. The
elimination rate constant k values of 3,4-di-O-acetyl compound
11 with LAS and CSR were 0.65 0.04 and 0.039 0.002 h−1
(mg/mL)−1, respectively. This indicates that substrates
possessing a 3,4-di-O-acetyl moiety such as 1a, 4, 7, and 11
would be poor substrates for CSR, whereas the opposite
substrate preference for LAS was observed. Di-O-acetyl
compounds 9−13 were individually synthesized in yields
between 55% and 92%, using the enzyme-catalyzed regiose-
lective O-deacetylation activity of LAS, CSR, and PLE (Scheme
4). This method provides a convenient chemical and
chemoenzymatic route for the synthesis of all 14 O-acetyl
compounds (4−17).
Kinetic Analysis of Nonenzymatic Intramolecular O-
Acyl Migration at pH 7.2. Because the reaction rate at pH 7.2
was higher than at pH 5.0 (Table 1), the elimination rate
constant k values of 1a were determined at pH 7.2 as 2.24
0.13 h−1 (mg/mL)−1 for LAS, 9.23 0.32 h−1 (mg/mL)−1 for
PLE, and 0.300 0.003 h−1 (mg/mL)−1 for CSR. Although the
k value for LAS was as same as that at pH 5.0, the k values for
CSR and PLE were 20-fold larger and 4-fold larger than those
at pH 5.0, respectively. This may explain the enhancement of
the k values observed at pH 7.2 compared with those at pH 5.0.
Because intramolecular O-acyl migration has been observed at
neutral or slightly alkaline pH,17 kinetic studies on the
intramolecular O-acetyl migration were then performed for
deacetylated product 6 (60% and 47% yields after 0.5 and 2.0 h,
respectively), as we have previously observed.18 In contrast,
PLE largely afforded the 2-O-deacetylated product 7 (63% and
23% yields after 0.5 and 2.0 h, respectively). The
regioselectivity of LAS was calculated as 92% and the
regioselectivity of PLE was 85%, after incubation for 0.5 h. At
preparative scales, compounds 6 and 7 were successfully
isolated in yields of 56% and 53%, respectively, under the
optimized conditions. Conversely, CSR and CAL-B showed
lower O-deacetylation activity at pH 5.0, leaving about 60% of
the starting material 2 unreacted after 2.0 h incubation. The
activity of CAL-B was low at both pH 7.2 and pH 5.0, whereas
LAS, CSR, and PLE showed higher activity at pH 7.2. CSR was
a particularly good catalyst at pH 7.2; the major products
formed after 2.0 h were the mono-O-acetyl compound 17
(45%) and 3a (33%). LAS and PLE showed higher activity
toward 1a and the tri-O-acetyl intermediates, and hence the
simultaneous use of CSR and either LAS or PLE was
investigated. The combination of CSR and LAS resulted in
synergistic O-deacetylation to afford 3a, and a higher
conversion yield was observed at pH 7.2 than at pH 5.0
(Table 1). The combination of CSR and LAS was as good as or
better than the combination of CSR and PLE (data not
shown).
Kinetic Analysis of Regioselective O-Deacetylation
and Complementary Roles of LAS and CSR. To confirm
the enzyme-catalyzed regioselective O-deacetylation and to
explain the synergistic effect of LAS and CSR, kinetic analyses
were performed on the O-deacetylation of 1a and the tri-O-
acetyl compounds 4−7 using LAS, CSR, and PLE. The analyses
were performed at pH 5.0 to minimize O-acetyl migration.17
The first-order elimination rate constant k values for 1a with
LAS, PLE, and CSR were 2.27 0.03, 2.30 0.10, and 0.051
0.004 h−1 (mg/mL)−1, respectively, indicating that the O-
deacetylation activity of LAS was as high as that of PLE and
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the tri-O-acetyl and mono-O-acetyl intermediates. The O-acetyl
migrations obeyed consecutive reversible first-order reaction
kinetics; the k values are summarized in Table 3.
O-acetyl migration to compounds 5 and 7 followed by LAS-
and CSR-catalyzed complementary O-deacetylation to 10
which was O-deacetylated to 3a via mono-O-acetyl inter-
mediates 14 and 17. Compounds 1a−c were successfully
converted to 3a−c using the combination of LAS and CSR at
pH 7.2 and 40 °C, with excellent chemoselectivity for O-
deacetylation. Compound 3a was obtained in 92% yield after
incubation for 5.0 h, compound 3b was obtained in 90% yield
after incubation for 4.0 h, and compound 3c was obtained in
88% yield after incubation for 4.0 h.
In summary, highly chemoselective O-deacetylation of alkali-
labile per-acetylated O-glucopyranosides 1a−c was achieved by
the simultaneous use of enzymes LAS and CSR that exhibited
complementary and synergistic roles in the enzyme-catalyzed
O-deaceylation. This enzymatic O-deacetylation, under mild
and neutral conditions, may be useful as an alternative to the
chemical method, particularly for removing O-acetyl groups of
alkali-labile glycosides. LAS, CSR, and PLE exhibit different
regioselective O-deacetylation activities, Furthermore, several
partially O-deacetylated derivatives of 1a could be easily
synthesized by using LAS, CSR, or PLE that exhibited different
regioselective O-deacetylation activity.
Table 3. First-order Rate Constant k Values for
a
Intramolecular O-Acetyl Migration at pH 7.2
migration
k (h−1
)
migration
k (h−1
)
4 to 5
5 to 4
5 to 6
6 to 5
6 to 7
7 to 6
4.26 0.03
0.11 0.02
0.83 0.01
0.86 0.02
0.67 0.03
0.63 0.04
16 to 17
17 to 16
15 to 16
16 to 15
14 to 15
15 to 14
1.51 0.10
0.12 0.03
0.23 0.01
0.34 0.07
0.17 0.02
0.14 0.01
a
Incubation was performed under the same conditions as the
enzymatic O-deacetylation at pH 7.2 and 40 °C.
The k values were highest for the O-acetyl migration from
the 4-O-position to the 6-O-position, which gave 2,3,6-tri-O-
acetyl compound 5 from 2,3,4-tri-O-acetyl compound 4 and 6-
O-acetyl compound 17 from 4-O-acetyl compound 16.
However, the k values for the corresponding reverse migrations
were the lowest. The k values for O-acetyl migrations between
the secondary hydroxyl groups were similar; the tri-O-acetyl
compounds had values of 0.63−0.86 h−1, and the mono-O-
acetyl compounds had values of 0.14−0.23 h−1. Because the O-
acetyl migration from the 4-O-position to the 6-O-position was
the most favorable step, the major products in equilibrium were
those with a 6-O-acetyl group: tri-O-acetyl compounds 5−7
and mono-O-acetyl compound 17. For the di-O-acetyl
compounds, 10, 12, and 13 were the major migration products
in equilibrium (data not shown).
When 1a was incubated with the combination of LAS and
CSR at pH 5.0, 2,6-di-O-acetyl 10 remained after 2.0 h (Table
1), indicating the lower O-deacetylation activity of the enzymes
toward 2-O- and 6-O-acetyl groups. However, at pH 7.2, no di-
O-acetyl compounds were detected after 2 h (Table 1),
probably because of the spontaneous O-acetyl migration in 10
which gave 3,6-di-O-acetyl 12 and 4,6-di-O-acetyl 13, which
subsequently underwent LAS- and CSR-catalyzed O-deacetyla-
tion to 6-O-acetyl compound 17. CSR showed the highest O-
deacetylation activity followed by LAS for mono-O-acetyl
compounds 14−17, and PLE exhibited the lowest activity
(Table 4).
EXPERIMENTAL SECTION
■
Materials and Methods. Compounds 3a−c and 2a−c were
prepared according to the reported procedures.11,30 Compounds 1a−c
were prepared by a slightly modified method of the reported
procedure.11 Fifteen commercially available enzymes, used in the
screening for O-deacetylation activity toward 1a, were as follows and
used as received: lipase AS Amano (from Aspergillus niger) (LAS),
carboxylesterase (from Streptomyces rochei, crude) (CSR), lipase type B
(from Candida antarctica) (CAL-B), porcine liver esterase (PLE),
lipase AP6 (from Aspergillus niger), lipase G Amano 50 (from
Penicillium camembertii), lipase AYS Amano (from Candida rugosa),
lipase PS Amano (from Burkholderia cepacia), lipase F-AP15 (from
Rhizopus oryzae), lipase AK Amano (from Pseudomonas fluorescens),
acylase I (from Aspergillus sp.), lipase from porcine pancreas, and three
kinds of lipases from Candida cylindracea (lipases MY and OF from
Meito Sangyo Co. Ltd. and CCL of 37 u/mg from Fluka). Amberlite
XAD-4 was used after grinding (80−200 mesh). Column chromatog-
raphy and preparative TLC were performed using silica gel 60 and
silica gel plate, respectively. All other chemicals used were commercial
1
products. H and 13C NMR spectra were recorded in DMSO-d6 with
and without D2O, and chemical shifts are presented by δ values with
reference to the residual solvent signal of DMSO; that is, 2.49 and
31
1
39.50 ppm for H and 13C NMR, respectively. Further analysis by
2D NMR (COSY, HMBC, HMQC) was performed to confirm the
NMR peak assignments. MS spectra were recorded with a positive
ionization mode. HPLC analyses were performed using a revered-
phase column of Symmetry (C18, 5 μm, 4.6 × 150 mm) with detection
at 240 nm at 30 °C.
Table 4. First-Order Rate Constant k Values of O-
a
Deacetylation of 14−17 at pH 7.2
b
b
b
substrate
CSR
LAS
PLE
14
15
16
17
0.17 0.01
4.8 0.1
0.09 0.01
0.92 0.04
0.15 0.01
0.01 0.00
0.08 0.01
0.19 0.01
0.05 0.01
0.01 0.00
General Incubation Conditions for Enzyme-Catalyzed O-
Deacetylation. Unless otherwise indicated, the incubation was
performed with substrate at initial concentration of 0.75 mM in 85
mM sodium acetate buffer (pH 5.0) containing 15% (v/v) DMSO as a
1.4 0.1
0.14 0.01
cosolvent and at 40.0
0.1 °C, in the presence of an appropriate
a
b
Incubation was performed at pH 7.2 and 40 °C. k values are shown
amount of enzyme(s). The progress of the reaction, monitored using
HPLC, obeyed according to first-order reaction kinetics, from which
the values of rate constant (k) were determined.
as h−1 (mg/mL)−1.
Enzyme-Catalyzed Complete O-Deacetylation of 1a−
c to 3a−c. The combination of LAS and CSR at pH 7.2
resulted in the enhancement of O-deacetylation of 1a to 3a
because of the complementary, synergistic roles of the enzymes
and the nonenzymatic O-acetyl migrations. The sequence of
LAS- and CSR-catalyzed O-deacetylation of 1a to 3a was as
follows: LAS-catalyzed O-deacetylation to 6 and nonenzymatic
Kinetics of Intramolecular O-Acetyl Migration of 2,3,4-Tri-O-
acetyl Compound 4 and 3-O-Acetyl Compound 15 and
Enzyme-Catalyzed O-Deacetylation Reaction. Kinetic experi-
ments of intramolecular O-acetyl migration of 4 and 15 at the initial
concentration of 0.75 mM were performed at 40.0 0.1 °C in 85 mM
sodium phosphate buffer (pH 7.2) containing 15% (v/v) DMSO as a
cosolvent. The progress of the reaction was monitored using HPLC.
The values of individual O-acetyl migration rate constant (k) were
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computed on the assumption that the reactions obey according to first-
order kinetics. The hydrolysis of O-acetyl groups was negligible during
the reaction periods. The kinetics of the O-acetyl migration reactions
of tri-O-acetyl compounds 4−7 are, therefore, represented by the
following equations, where k4→6, for example, means the rate constant
for the O-acetyl migration from the position 4-OH to 6-OH of the
glucopyranoside ring.
and C6-H′), 4.94 (t, J = 9.6 Hz, 1H, C4-H), 4.95 (dd, J = 8.5 and 9.6
Hz, 1H, C2-H), 5.31 (t, J = 9.6 Hz, 1H, C3-H), 5.34 (d, J = 8.5 Hz, 1H,
C1-H), 7.36 (d, J = 8.9 Hz, 2H, Ar-H), 7.50 (d, J = 8.9 Hz, 2H, Ar-H);
13C NMR (100 MHz, DMSO-d6) δ 20.18, 20.23, 20.3, 21.8, 61.5 (C6),
67.8 (C2 or C4), 69.3 (C2 or C4), 70.9 (C5), 71.7 (C5), 102.6 (C1),
126.2, 128.6, 131.3, 138.4, 168.9, 169.2, 169.4, 169.8, 171.4. MS (FAB,
positive) m/z 516 [M + H]+, 331, 169 (base). Anal. Calcd for
C22H26NClO11: C, 51.22; H, 5.08; N, 2.72. Found: C, 51.42; H, 4.99;
N, 2.80. 2,3,4,6-Tetra-O-acetyl-β-D-glucopyranoside of N-(4-
methyl)phenylacetohydroxamic acid (1c): white solid (3.9 g,
d[4]/dt = −k
[4] + k
4→6
[5], d[5]/dt
6→4
= k
= k
[4] + k
[6] − (k
4→3
+ k
3→4
)[5], d[6]/dt
28%); mp 100−103 °C; IR (Nujol) cm−1 1750, 1690; [α]20 −45
4→6
6→4
4→3
D
(c 0.56, EtOH); 1H NMR (400 MHz, DMSO-d6) δ 1.93 (s, 6H), 1.98
(s, 3H), 2.02 (s, 3H), 2.15 (s, 3H), 2.32 (s, 3H), 4.05−4.16 (m, 3H,
C5-H, C6-H and C6-H′), 4.90 (dd, J = 8.2 and 9.6 Hz, 1H, C2-H), 4.93
(t, J = 9.4 Hz, 1H, C4-H), 5.30 (d, J = 8.2 Hz, 1H, C1-H), 5.32 (t, J =
9.6 Hz, 1H, C3-H), 7.20 (d, J = 8.5 Hz, 2H, Ar-H), 7.24 (d, J = 8.5 Hz,
2H, Ar-H); 13C NMR (100 MHz, DMSO-d6) δ 20.2, 20.3, 20.4, 20.6,
21.8, 61.5 (C6), 67.9 (C2 or C4), 69.3 (C2 or C4), 70.9 (C5), 71.7 (C5),
102.5 (C1), 125.5, 129.2, 137.0, 137.1, 168.9, 169.2, 169.2, 169.4,
169.9, 171.2; MS (FAB, positive) m/z 496 [M + H]+, 331, 169 (base).
Anal. Calcd for C23H29NO11: C, 55.75; H, 5.90; N, 2.83. Found: C,
55.68; H, 6.00; N, 2.78.
[5] + k [7] − (k
3→2
+ k
)[6], d[7]/dt
3→4
2→3
= k [6] − k [7]
2→3 3→2
The kinetics of the O-acetyl migration reactions of mono-O-acetyl
compounds 14−17 are similarly represented by the following
equations.
d[14]/dt = − k
[14] + k
[15], d[15]/dt
+ k )[15], d[16]/dt
2→3
3→2
= k
[14] + k
[16] − (k
3→2
2→3
4→3
6→4
3→4
+ k
4→3
= k
= k
[15] + k
[17] − (k
[17]
)[16], d[17]/dt
4→6
3→4
4→6
2,3,4-Tri-O-acetyl-β-D-glucopyranoside of N-phenylacetohy-
droxamic Acid (4). To a solution of 3a (510 mg, 1.63 mmol) in
pyridine (6.0 mL) was added 4-methoxytrityl chloride (610 mg, 1.98
mmol). After 16 h at room temperature, acetic anhydride (0.56 mL,
5.9 mmol) was added to the reaction mixture. After 27 h, methanol
was added to quench the excess reagents, and then the reaction
mixture was evaporated under reduced pressure. The resultant residue
was dissolved in 80% (v/v) aq AcOH (20 mL) and then kept at room
temperature for 3 h. After evaporation of the reaction mixture, the
residue was purified by silica gel column chromatography with ethyl
acetate: benzene (1:1, v/v) to afford 4 (501 mg) in 70% overall yield
from 3a: mp 148−149 °C (colorless fine needles from ethyl acetate−
[16] − k
6→4
HPLC Analysis Conditions. All mobile phases, at a flow rate of 0.7
mL/min, were containing 0.1% (v/v) AcOH to minimize intra-
molecular acetyl migrations. The mobile phase used for analysis of tri-
O-acetyl derivatives was EtOH/2-PrOH/H2O (3:1:14, v/v) and the
retention times of 4, 5, 6, and 7 as well as 1a were 13.3, 15.1, 9.8, 12.1,
and 31.1 min, respectively. The mobile phase used for analysis of di-O-
acetyl derivatives was EtOH/2-PrOH/H2O (1:1:12, v/v) and the
retention times of 8, 9, 10, 11, 12, and 13 were 16.4, 9.3, 13.3, 8.8,
10.9, and 11.8 min, respectively. The mobile phase used for analysis of
3a and mono-O-acetyl derivatives was 2-PrOH:H2O (1:17, v/v) and
the retention times of 14, 15, 16, and 17 as well as 3a were 19.0, 10.8,
11.7, 18.1, and 6.1 min, respectively. The mobile phases used for
analysis of 3b and 3c were EtOH/2-PrOH/H2O (3:1:10, v/v) and
EtOH/2-PrOH/H2O (3:1:12, v/v), respectively. The retention times
of 3b and 3c were 6.1 and 4.6 min, respectively.
hexane); IR (Nujol) cm−1 3440, 1750, 1670; [α]20 −50 (c 0.52,
D
EtOH); 1H NMR (400 MHz, DMSO-d6) δ 1.90 (s, 3H), 1.91 (s, 3H),
1.96 (s, 3H), 2.24 (s, 3H), 3.45 (dd, J = 5.4 and 12.0 Hz, 1H, C6-H),
3.55 (ddd, J = 2.4, 5.4, and 12.0 Hz, 1H, C6-H′), 3.81 (ddd, J = 2.4, 5.4,
and 9.5 Hz, 1H, C5-H), 4.72 (t, J = 5.4 Hz, 1H, C6-OH), 4.88 (dd, J =
8.5 and 9.3 Hz, 1H, C2-H), 4.92 (t, J = 9.5 Hz, 1H, C4-H), 5.23 (d, J =
8.5 Hz, 1H, C1-H), 5.25 (t, J = 9.5 Hz, 1H, C3-H), 7.29−7.34 (m, 3H,
Ar-H), 7.41−7.45 (m, 2H, Ar-H); 13C NMR (100 MHz, DMSO-d6) δ
20.1, 20.3, 22.0, 59.9 (C6), 68.1 (C4), 69.5 (C2), 72.2 (C3), 73.9 (C5),
102.6 (C1), 124.9, 127.1, 128.6, 139.4, 168.7, 169.0, 169.3, 171.6; MS
(FAB, positive) m/z 440 [M + H]+, 289 (base), 229, 169. Anal. Calcd
for C20H25NO10: C, 54.67; H, 5.73; N, 3.19. Found: C, 54.54; H, 5.67;
N, 3.09.
General Synthesis of 2,3,4,6-Tetra-O-acetyl-β-D-glucopyra-
nosides of N-Arylacetohydroxamic Acids (1a−c). Besides our
previous procedure,11 the compounds 1a−c were synthesized as
follows. To a solution of N-arylacetohydroxamic acid (35 mmol) in
EtOH (25 mL) and water (7 mL) containing KOH (35 mmol) was
added acetobromglucose (28 mmol). After being stirred for 1 h, the
reaction mixture was evaporated under reduced pressure, and the
residue was dissolved in Et2O and the organic layer was washed
thoroughly with 1 M NaOH (about six times), dried over Na2SO4, and
filtered. The filtrate was evaporated under reduced pressure to afford a
crude product, which was purified by recrystallization from ethyl
acetate−hexane to afford the compounds 1a−c. 2,3,4,6-Tetra-O-
acetyl-β-D-glucopyranoside of N-phenylacetohydroxamic acids
(1a): colorless fine needles (4.3 g, 32%); mp 109−110 °C (lit.11
2,3,6-Tri-O-acetyl-β-D-glucopyranoside of N-Phenylacetohy-
droxamic Acid (5). To a stirred suspension of 3a (1.02 g, 3.26 mmol)
in CH3CN (16 mL) and trimethyl orthoacetate (2.0 mL, 16 mmol)
was added a catalytic amount of TsOH (25 mg), and the reaction
mixture was kept for 0.5 h at room temperature. After addition of
pyridine (1 mL), the reaction mixture was evaporated under reduced
pressure, and the resultant residue was acetylated with acetic anhydride
(1.2 mL, 13 mmol) and pyridine (10 mL) for 2 h at room
temperature. After addition of MeOH (2 mL), the reaction mixture
was evaporated under reduced pressure. The residue was dissolved in
ethyl acetate and the solution was washed with 50 mM HCl and then
water, dried with Na2SO4, and filtered. The filtrate was evaporated
under reduced pressure to afford the corresponding crude 2,3-di-O-
acetyl-4,6-orthoacetate derivative, which was recrystallized from ethyl
acetate−hexane to afford the pure compound (1.09 g) in 74% overall
mp 109−110 °C); IR (Nujol) cm−1 1760, 1740, 1690; [α]20 −56 (c
D
0.52, EtOH); 1H NMR (400 MHz, DMSO-d6) δ 1.92 (s, 3H), 1.93 (s,
3H), 1.97 (s, 3H), 2.00 (s, 3H), 2.19 (s, 3H), 4.04−4.15 (m, 3H, C5-
H, C6-H and C6-H′), 4.93 (dd, J = 8.3 and 9.3 Hz, 1H, C2-H), 4.94 (t, J
= 9.5 Hz, 1H, C4-H), 5.32 (t, J = 9.5 Hz, 1H, C3-H), 5.33 (d, J = 8.3
Hz, 1H, C1-H), 7.29−7.36 (m, 3H, Ar-H), 7.41−7.49 (m, 2H, Ar-H);
13C NMR (100 MHz, DMSO-d6) δ 20.2, 20.3, 20.4, 20.4, 21.9, 61.5
(C6), 67.8 (C2 or C4), 69.3 (C2 or C4), 70.8 (C5), 71.7 (C5), 102.5
(C1), 124.9, 127.2, 128.5, 139.4, 168.7, 169.0, 169.2, 169.7, 171.2; MS
(FAB, positive) m/z 482 [M + H]+, 331, 169 (base), 109. Anal. Calcd
for C22H27NO11: C, 54.88; H, 5.65; N, 2.91. Found: C, 55.03; H, 5.59;
N, 2.84. 2,3,4,6-Tetra-O-acetyl-β-D-glucopyranoside of N-(4-
chloro)phenylacetohydroxamic acids (1b): white solid (4.3 g,
1
yield: H NMR (270 MHz, DMSO-d6) δ 1.33 (s, 3H), 1.90 (s, 3H),
1.97 (s, 3H), 2.17 (s, 3H), 3.19 (s, 3H), 3.70−3.72 (m, 2H), 3.80−
3.85 (m, 2H), 4.90 (t, J = 8.5 Hz, 1H), 5.19 (t, J = 9.5 Hz, 1H), 5.24
(d, J = 8.0 Hz, 1H), 7.27−7.34 (m, 3H, Ar-H), 7.41−7.46 (m, 2H, Ar-
H); 13C NMR (67.5 MHz, DMSO-d6) δ 20.2, 20.4, 21.3, 21.9, 50.2,
60.3, 64.9, 69.3, 70.3, 70.9, 102.9, 111.9, 125.0, 127.3, 128.7, 139.4,
168.9, 169.5, 171.4. Anal. Calcd for C21H27NO10: C, 55.62; H, 6.00; N,
3.09. Found: C, 55.57; H, 6.04; N, 3.09. To the solution of this
30%); mp 117−119 °C; IR (Nujol) cm−1 1750, 1700; [α]20 −44
D
(c 0.48, EtOH); 1H NMR (400 MHz, DMSO-d6) δ 1.94 (s, 3H), 1.98
(s, 3H), 1.99 (s, 3H × 2), 2.21 (s, 3H), 4.03−4.14 (m, 3H, C5-H, C6-H
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compound (789 mg, 1.74 mmol) in AcOH (10 mL) was added H2O
(2.0 mL), and the resultant solution was kept at room temperature for
15 min. After evaporation of the solvent under reduced pressure, the
residue was purified by silica gel column chromatography with ethyl
acetate/benzene (1:1, v/v) to afford the desired product 5 (540 mg,
68%) along with 4 (150 mg, 19%). The analytical data of 5 are as
PLE (117 mg) in 100 mM sodium acetate buffer (pH 5.0) (20 mL)
was incubated at 40 °C for 15 min and then was extracted thoroughly
with ethyl acetate. After evaporation of the combined organic layer, the
residue was purified by preparative TLC.
2,3-Di-O-acetyl-β-D-glucopyranoside of N-Phenylacetohy-
droxamic Acid (8). To a suspension of 3a (2.59 g, 8.27 mmol) in
acetone (20 mL) and dimethoxypropane (30 mL) was added a
catalytic amount of TsOH (20 mg). After being stirred for 1 h at 37
°C, the resultant clear reaction mixture was evaporated under reduced
pressure, the residue was dissolved in pyridine (6 mL), and then acetic
anhydride (2.5 mL, 26 mmol) was added. After 2 h at room
temperature, methanol was added to quench the excess anhydride, and
then the reaction mixture was evaporated under reduced pressure to
afford crude 2,3-di-O-acetyl-4,6-isopropylidene derivative, which was
purified by recrystallization from ethyl acetate−hexane (2.49 g, 69%).
The compound (2.17 g, 4.96 mmol) was dissolved in 80% (v/v) aq
AcOH (12 mL) and then kept at 37 °C for 2 h. Evaporation of the
follows: mp 124−125 °C (colorless block from ethyl acetate−hexane);
1
IR (Nujol) cm−1 3380, 1750, 1685; [α]20 −73 (c 0.61, EtOH); H
D
NMR (400 MHz, DMSO-d6) δ 1.90 (s, 3H), 1.97 (s, 3H), 2.01 (s,
3H), 2.18 (s, 3H), 3.49 (dd, J = 5.6 and 9.5 Hz, 1H, C4-H), 3.75 (ddd,
J = 2.0, 6.3, and 9.5 Hz, 1H, C5-H), 4.10 (dd, J = 6.3 and 12.0 Hz, 1H,
C6-H), 4.28 (dd, J = 2.0 and 12.0 Hz, 1H, C6-H′), 4.77 (dd, J = 8.3 and
9.5 Hz, 1H, C2-H), 5.02 (d, J = 9.5 Hz, 1H, C3-H), 5.19 (d, J = 8.3 Hz,
1H, C1-H), 5.68 (d, J = 5.6 Hz, 1H, C4-OH), 7.28−7.33 (m, 3H, Ar-
H), 7.39−7.43 (m, 2H, Ar-H); 13C NMR (100 MHz, DMSO-d6) δ
20.1, 20.4, 20.5, 21.7, 62.5 (C6), 67.4 (C4), 69.8 (C2), 73.4 (C3), 74.4
(C5), 102.8 (C1), 124.9, 127.1, 128.5, 139.6, 168.8, 169.4, 169.9, 171.3;
MS (FAB, positive) m/z 440 [M + H]+, 289 (base), 229. Anal. Calcd
for C20H25NO10: C, 54.67; H, 5.73; N, 3.19. Found: C, 54.86; H, 5.84;
N, 3.20.
reaction mixture afforded 8 (a white powder, 1.77 g, 90%): IR (Nujol)
1
cm−1 3420, 1760, 1730, 1690; [α]20 −78 (c 0.43, EtOH); H NMR
D
(270 MHz, DMSO-d6) δ 1.89 (s, 3H), 1.96 (s, 3H), 2.25 (s, 3H),
3.47−3.50 (m, 3H), 3.68−3.72 (m, 1H), 4.63 (t, J = 5.6 Hz, 1H, C6-
OH), 4.73 (dd, J = 8.3 and 9.5 Hz, 1H, C2-H), 4.97 (t, J = 9.5 Hz, 1H,
C3-H), 5.08 (d, J = 8.3 Hz, 1H, C1-H), 5.49 (d, J = 4.9 Hz, C4-OH),
7.27−7.33 (m, 3H, Ar-H), 7.40−7.45 (m, 2H, Ar-H); 13C NMR (67.5
MHz, DMSO-d6) δ 20.3, 20.6, 22.2, 60.3 (C6), 67.2 (C4), 70.0 (C2),
74.8 (C3), 76.7 (C5), 102.8 (C1), 124.9, 127.1, 128.6, 139.4, 169.0,
169.6, 171.8; HRMS (FAB, positive) calcd for C18H24NO9 [M + H]+
m/z = 398.1451, found 398.1425 (error −2.6 mmu). Anal. Calcd for
C18H23NO9: C, 54.41; H, 5.83; N, 3.52. Found: C, 54.14; H, 5.96; N,
3.78.
2,4,6-Tri-O-acetyl-β-D-glucopyranoside (6) and 3,4,6-Tri-O-
acetyl-β-D-glucopyranoside (7) of N-Phenylacetohydroxamic
Acid. (A) Intramolecular O-Acetyl Migration Method. To a
solution of 4 (735 mg, 1.67 mmol) in CH3CN (21 mL) was added 50
mM MOPS−NaOH (pH 7.5) buffer (400 mL), and the reaction
mixture was kept at 37 °C for 4 h to afford the mixture composed
mainly of 5, 6, and 7. After the reaction mixture was extracted three
times with ethyl acetate, the combined organic layers were dried over
Na2SO4 and then filtered. The filtrate was evaporated under reduced
pressure and the residue was purified by silica gel column
chromatography with ethyl acetate−benzene (2:3, v/v), and the final
purification was performed by preparative TLC developed with
CHCl3−MeOH (20:1, v/v) to afford 5 (133 mg, 18%), 6 (133 mg,
19%), and 7 (160 mg, 22%). The analytical data of 6 are as follows:
mp 157−159 °C (colorless fine needles from ethyl acetate−hexane);
IR (Nujol) cm−1 3380, 1760, 1740, 1660; [α]20D −86 (c 0.50, EtOH);
1H NMR (400 MHz, DMSO-d6) δ 1.99 (s, 3H), 2.00 (s, 3H), 2.03 (s,
2,6-Di-O-acetyl-β-D-glucopyranoside (10), 3,6-di-O-Acetyl-β-
D-glucopyranoside (12), and 4,6-Di-O-acetyl-β-D-glucopyrano-
side (13) of N-Phenylacetohydroxamic Acid. To a solution of 8
(267 mg, 0.672 mmol) in CH3CN (10 mL) was added 100 mM
MOPS−NaOH (pH 7.5) buffer (190 mL), and the reaction mixture
was kept at 37 °C for 8 h. The reaction mixture composed mainly of
the title compounds was thoroughly extracted with ethyl acetate, and
the combined organic layers were evaporated under reduced pressure.
The residue was purified by silica gel column chromatography with
CHCl3−MeOH (20:1, v/v), and the final purification was performed
by preparative TLC developed with CHCl3−MeOH (10:1, v/v) to
afford 10 (a white powder, 75 mg, 28%), 12 (a white powder, 85 mg,
32%), and 13 (a white powder, 55 mg, 21%). The analytical data of 10
are as follows: IR (Nujol) cm−1 3420, 1745, 1680; [α]20D −65 (c 0.46,
EtOH); 1H NMR (270 MHz, DMSO-d6) δ 1.97 (s, 3H), 2.00 (s, 3H),
2.17 (s, 3H), 3.17−3.25 (m, 1H), 3.36−3.45 (m, 1H), 3.52−3.56 (m,
1H), 4.05 (dd, J = 6.9 and 12.0 Hz, 1H, C6-H), 4.28 (dd, J = 2.0 and
12.0 Hz, 1H, C6-H′), 4.62 (t, J = 9.0 Hz, 1H, C2-H), 4.93 (d, J = 8.5
Hz, 1H, C1-H), 5.46 (d, J = 5.6 Hz, OH), 5.49 (d, J = 6.3 Hz, 1H,
OH), 7.25−7.31 (m, 3H, Ar-H), 7.38−7.43 (m, 2H, Ar-H); 13C NMR
(67.5 MHz, DMSO-d6) δ 20.6, 20.8, 21.9, 62.9 (C6), 69.7, 71.9, 73.4,
73.7, 103.2 (C1), 124.6, 126.9, 128.4, 139.4, 168.9, 169.9, 171.2.
HRMS (FAB, positive) calcd for C18H24NO9 [M + H]+ m/z =
398.1451, found 398.1464 (error 1.3 mmu). The analytical data of 12
3H), 2.19 (s, 3H), 3.73 (dt, J = 5.9 and 9.5 Hz, 1H, C3-H), 3.84−3.89
(m, 1H, C5-H), 4.01 (dd, J = 2.4 and 12.3 Hz, 1H, C6-H), 4.07 (dd, J =
5.6 and 12.3 Hz, 1H, C6-H′), 4.69−4.75 (m, 2H, C2-H and C4-H), 5.07
(d, J = 8.5 Hz, 1H, C1-H), 5.70 (d, J = 5.9 Hz, 1H, C3-OH), 7.29−7.32
(m, 3H, Ar-H), 7.41−7.45 (m, 2H, Ar-H); 13C NMR (100 MHz,
DMSO-d6) δ 20.5, 20.6, 20.7, 21.9, 61.8 (C6), 70.2 (C2 or C4), 70.7
(C3), 71.1 (C5), 71.7 (C2 or C4), 103.0 (C1), 124.8, 127.2, 128.7,
139.5, 169.0, 169.5, 169.9, 171.4; MS (FAB, positive) m/z 440 [M +
H]+, 289 (base). Anal. Calcd for C20H25NO10: C, 54.67; H, 5.73; N,
3.19. Found: C, 54.58; H, 5.80; N, 3.18. The analytical data of 7 (a
white solid) are as follows: mp 84−86 °C (colorless fine needles from
ethyl acetate−hexane); IR (Nujol) cm−1 3380, 1760, 1740, 1670;
1
[α]20 −53 (c 0.47, EtOH); H NMR (400 MHz, DMSO-d6) δ 1.95
D
(s, 3H), 1.98 (s, 3H), 1.99 (s, 3H), 2.26 (s, 3H), 3.52−3.58 (m, 1H,
C2-H), 3.93−4.01 (m, 2H, C5-H and C6-H), 4.09 (dd, J = 5.4 and 12.0
Hz, 1H, C6-H′), 4.80 (t, J = 9.5 Hz, 1H, C4-H), 4.95 (d, J = 8.3 Hz, 1H,
C1-H), 5.07 (t, J = 9.5 Hz, 1H, C3-H), 6.04 (d, J = 5.4 Hz, 1H, C2-
OH), 7.25 (t, J = 7.6 Hz, 1H, Ar-H), 7.39 (t, J = 7.6 Hz, 2H, Ar-H),
7.50 (dd, J = 1.2 and 7.6 Hz, 2H, Ar-H); 13C NMR (100 MHz,
DMSO-d6) δ 20.39, 20.41, 20.6, 22.1, 61.6 (C6), 68.2 (C4), 69.7 (C2),
70.7 (C5), 74.1 (C3), 104.4 (C1), 123.5, 126.3, 128.4, 139.3, 169.4,
169.5, 169.9, 171.6; MS (FAB, positive) m/z 440 [M + H]+ (base),
398, 289, 229. Anal. Calcd for C20H25NO10: C, 54.67; H, 5.73; N, 3.19.
Found: C, 54.78; H, 5.88; N, 3.18.
are as follows; IR (Nujol) cm−1 3490, 1720, 1660; [α]20 −72° (c
D
0.30, EtOH); 1H NMR (270 MHz, DMSO-d6) δ 1.98 (s, 3H), 2.02 (s,
3H), 2.24 (s, 3H), 3.28−3.41 (m, 2H), 3.54−3.60 (m, 1H, C5-H), 4.07
(dd, J = 6.3 and 12.0 Hz, 1H, C6-H), 4.21 (dd, J = 2.2 and 12.0 Hz,
1H, C6-H′), 4.79 (d, J = 8.3 Hz, 1H, C1-H), 4.80 (t, J = 10.0 Hz, 1H,
C3-H), 5.48 (d, J = 5.8 Hz, OH), 5.79 (d, J = 5.4 Hz, 1H, OH), 7.20−
7.26 (m, 1H, Ar-H), 7.34−7.39 (m, 2H, Ar-H), 7.49 (dd, J = 1.3 and
8.8 Hz, 2H, Ar-H); 13C NMR (67.5 MHz, DMSO-d6) δ 20.5, 21.0,
22.1, 62.7 (C6), 67.6 (C4), 69.9 (C2), 73.4 (C5), 76.9 (C3), 104.8 (C1),
123.6, 126.3, 128.4, 139.4, 169.6, 170.1, 171.6. HRMS (FAB, positive)
calcd for C18H24NO9 [M + H]+ m/z = 398.1451, found 398.1433
(error −1.8 mmu). Anal. Calcd for C18H23NO9·H2O requires C, 52.05;
H, 6.07; N, 3.37. Found: C, 52.10; H, 6.10; N, 3.40. The analytical data
of 13 are as follows; IR (Nujol) cm−1 3410, 1750, 1680; [α]20D −108°
(c 0.39, EtOH); 1H NMR (270 MHz, DMSO-d6) δ 1.96 (s, 3H), 2.00
(s, 3H), 2.25 (s, 3H), 3.26−3.31 (m, 1H, C2-H, overlapped with H2O
Enzymatic Regioselective O-Deacetylation Method. Com-
pound 6 was obtained in 56% yield as follows: the reaction mixture
consisting of 1a (16.6 mg, 34.5 μmol) in EtOH (3.5 mL) and LAS
(117 mg) in 100 mM sodium acetate buffer (pH 5.0) (20 mL) was
incubated at 40 °C for 20 min and then was extracted thoroughly with
ethyl acetate. After evaporation of the combined organic layers, the
residue was purified by preparative TLC with EtOAc/benzene (1:1, v/
v). The compound 7 was obtained in 53% yield as follows: the reaction
mixture consisting of 1a (16.8 mg, 34.9 μmol) in EtOH (3.5 mL) and
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peak), 3.45 (t, J = 9.5 Hz, 1H, C3-H), 3.68−3.75 (m, 1H, C5-H), 3.92
(dd, J = 2.4 and 12.2 Hz, 1H, C6-H), 4.04 (dd, J = 5.9 and 12.2 Hz,
1H, C6-H′), 4.62 (t, J = 9.5 Hz, 1H, C4-H), 4.72 (d, J = 8.0 Hz, 1H, C1-
H), 5.47 (brs, 1H, OH), 5.77 (brs, 1H, OH), 7.22 (t, J = 7.4 Hz, 1H,
Ar-H), 7.37 (t, J = 7.3 Hz, 2H, Ar-H), 7.50 (d, J = 7.8 Hz, 2H, Ar-H);
13C NMR (67.5 MHz, DMSO-d6) δ 20.4, 20.8, 22.1, 61.9 (C6), 70.5
(C4), 71.0 (C5), 72.0 (C2), 73.2 (C3), 104.7 (C1), 123.3, 126.1, 128.4,
139.2, 169.6, 169.9, 171.5. HRMS (FAB, positive) calcd for
C18H24NO9 [M + H]+ m/z = 398.1451, found 398.1432 (error −1.9
mmu).
min) from the substrate 6 (2.0 mM) with CSR (2.5 mg/mL), 3,4-di-
O-acetyl compound 11 (60% after 20 min) from the substrate 4 (1.5
mM) with PLE (5.0 mg/mL), 3,6-di-O-acetyl ompound 12 (55% after
3.5 h) from the substrate 5 (1.5 mM) with PLE (5.0 mg/mL) and 4,6-
di-O-acetyl ompound 13 (72% after 80 min) from the substrate 6 (1.5
mM) with PLE (5.0 mg/mL).
2-O-acety-β-D-glucopyranoside 14 and 3-O-acetyl-β-D-glu-
copyranoside 15 of N-phenylaceto-hydroxamic acid. As shown
above for the synthesis of 8, acetonidation of 3a (1.53 g, 4.88 mmol)
gave the corresponding 4,6-isopropylidene derivative, which was
acetylated with acetic anhydride (0.42 mL, 4.38 mmol) in pyridine (2
mL) at 4 °C for 3 h. After evaporation of the reaction mixture, the
residue was purified by silica gel column chromatography with ethyl
acetate-benzene (1:3, v/v) to afford 4,6-isopropylidene derivatives of
14 (220 mg, 13%) and 15 (425 mg, 25%). The 4,6-isopropylidene
derivatives of 14 (207 mg, 0.524 mmol) and 15 (200 mg, 0.506 mmol)
were each deprotected with 80% (v/v) aq AcOH (3.0 mL) at 25 °C
for 3 h. Evaporation of the reaction mixtures gave the title compounds
14 (175 mg, 94%) and 15 (167 mg, 93%). The analytical data of 14 (a
white powder) are as follows; IR (Nujol) cm−1 3400, 1740, 1660;
[α]20D −68° (c 0.47, EtOH); 1H NMR (400 MHz, DMSO-d6) δ 1.97
(s, 3H), 2.25 (s, 3H), 3.19−3.22 (m, 1H, C4-H), 3.27−3.39 (m, 2H,
overlapped with H2O peak, C3-H and C5-H), 3.45−3.51 (m, 1H, C6-
H′), 3.70 (dd, J = 5.4 and 12.0 Hz, 1H, C6-H), 4.55 (t, J = 5.6 Hz, 1H,
C6-OH), 4.62 (t, J = 9.3 Hz, 1H, C2-H), 4.81 (d, J = 8.5 Hz, 1H, C1-
H), 5.23 (brs, 1H, OH), 5.37 (brs, 1H, OH), 7.28−7.33 (m, 3H, Ar-
H), 7.41−7.45 (m, 2H, Ar-H); 13C NMR (100 MHz, DMSO-d6) δ
20.8, 22.3, 60.7 (C6), 69.5 (C4), 72.1 (C2), 73.8 (C3), 77.3 (C5), 103.3
(C1), 124.7, 127.0, 128.6, 139.4, 169.2, 171.8. HRMS (FAB, positive)
calcd for C16H22NO8 [M + H]+ m/z = 356.1345, found 356.1337
(error −0.9 mmu). Anal. Calcd for C16H21NO8·1/2 H2O: C, 52.74; H,
6.09; N, 3.84. Found: C, 52.88; H, 6.03; N, 3.83. The analytical data of
15 (a white powder) are as follows; IR (Nujol) cm−1 3390, 1760,
1660; [α]20D −73° (c 0.30, EtOH); 1H NMR (400 MHz, DMSO-d6) δ
2.03 (s, 3H), 2.30 (s, 3H), 3.26−3.38 (m, 2H, overlapped with H2O
peak, C2-H and C4-H), 3.45−3.50 (m, 1H, C6-H′), 3.65 (dd, J = 5.4
and 12.0 Hz, 1H, C6-H), 4.52 (t, J = 5.6 Hz, 1H, C6-OH), 4.67 (d, J =
8.0 Hz, 1H, C1-H), 4.77 (t, J = 9.3 Hz, 1H, C3-H), 5.24 (d, J = 5.8 Hz,
1H, OH), 5.75 (d, J = 5.4 Hz, 1H, OH), 7.24 (t, J = 7.6 Hz, 1H, Ar-H),
7.38 (t, J = 7.6 Hz, 2H, Ar-H), 7.54 (d, J = 7.6 Hz, 2H, Ar-H); 13C
NMR (100 MHz, DMSO-d6) δ 21.1, 22.6, 60.4 (C6), 67.3, 70.0, 76.8,
77.3 (C3), 104.6 (C1), 123.3, 126.1, 128.4, 139.0, 169.6, 171.7; HRMS
(FAB, positive) calcd for C16H22NO8 [M + H]+ m/z = 356.1345,
found 356.1347 (error 0.2 mmu). Anal. Calcd for C16H21NO8: C,
54.08; H, 5.96; N, 3.94. Found: C, 54.11; H, 6.03; N, 3.92.
2,4-Di-O-acetyl-β-D-glucopyranoside (9) and 3,4-di-O-acetyl-
β-D-glucopyranoside (11) of N-phenylacetohydroxamic acid.
To a solution of 8 (1.32 g, 3.32 mmol) in pyridine (20 mL) was added
4-methoxytrityl chloride (1.69 g, 5.47 mmol) and the reaction mixture
was kept at room temperature for 15 h. After addition of MeOH to
quench the excess reagent, the reaction mixture was evaporated under
reduced pressure. The residue was purified by silica gel column
chromatography with ethyl acetate-benzene (1:2, v/v) to afford 6-(4-
methoxytrityl) derivative (1.90 g, 85%). The compound (827 mg, 1.23
mmol) was dissolved in 50% (v/v) aq pyridine (20 mL) and the
solution was kept at 50 °C for 24 h to afford a mixture composed
mainly of the 6-(4-methoxytrityl) derivatives of 8, 9 and 11. After
evaporation of the reaction mixture under reduced pressure, the
residue was purified by silica gel column chromatography with ethyl
acetate-benzene (1:2, v/v) to afford 6-(4-methoxytrityl) derivatives of
9 (a white powder, 178 mg, 22%) and 11 (a white powder, 218 mg,
26%), which were then deprotected with 80% (v/v) aq AcOH (7 mL)
at 30 °C for 3 h. Evaporation of the each reaction mixture gave 9 and
11, which were purified by silica gel column chromatography with
CHCl3-MeOH (40:1, v/v). The analytical data of 9 (a white powder)
are as follows; IR (Nujol) cm−1 3390, 1740, 1660; [α]20 −84° (c
D
0.38, EtOH); 1H NMR (270 MHz, DMSO-d6) δ 1.97 (s, 3H), 2.00 (s,
3H), 2.24 (s, 3H), 3.34−3.38 (m 1H, C6-H), 3.43−3.47 (m, 1H, C6-
H′), 3.55−3.60 (m, 1H, C5-H), 3.67 (dt, J = 5.8 and 9.3 Hz, 1H, C3-
H), 4.66 (t, J = 9.5 Hz, 1H, C4-H), 4.69 (t, J = 8.5 Hz, 1H, C2-H), 4.71
(d, J = 5.4 Hz, 1H, OH), 4.96 (d, J = 8.5 Hz, 1H, C1-H), 5.58 (d, J =
5.6 Hz, 1H, OH), 7.27−7.32 (m, 3H, Ar-H), 7.40−7.45 (m, 2H, Ar-
H); 13C NMR (67.5 MHz, DMSO-d6) δ 20.7, 20.8, 22.2, 60.4 (C6),
70.7 (C4), 71.0 (C3), 71.9 (C2), 74.4 (C5), 103.1 (C1), 124.8, 127.1,
128.7, 139.4, 169.0, 169.4, 171.8. HRMS (FAB, positive) calcd for
C18H24NO9 [M + H]+ m/z = 398.1451, found 398.1461 (error 1.0
mmu). Anal. Calcd for C18H23NO9 requires C, 54.41; H, 5.83; N, 3.52.
Found: C, 54.24; H, 5.85; N, 3.49. The analytical data of 11 (a white
powder) are as follows; IR (Nujol) cm−1 3470, 1750, 1650; [α]20
D
−48° (c 0.45, EtOH); 1H NMR (270 MHz, DMSO-d6) δ 1.93 (s, 3H),
1.96 (s, 3H), 2.29 (s, 3H), 3.36−3.52 (m, 3H, C2-H, C6-H, C6-H′),
3.60−3.65 (m, 1H, C5-H), 4.71 (brs, 1H, OH), 4.85 (d, J = 8.1 Hz,
1H, C1-H), 4.80 (t, J = 9.5 Hz, 1H, C3-H), 5.00 (t, J = 9.5 Hz, 1H, C4-
H), 5.99 (d, J = 5.3 Hz, 1H, OH), 7.23 (t, J = 7.3 Hz, 1H, Ar-H), 7.39
(t, J = 7.5 Hz, 2H, Ar-H), 7.52−7.55 (m, 2H, Ar-H); 13C NMR (67.5
MHz, DMSO-d6) δ 20.5, 20.6, 22.5, 60.0 (C6), 68.5 (C3), 69.8 (C2),
73.8 (C5), 74.6 (C4), 104.3 (C1), 123.4, 126.2, 128.4, 139.1, 169.2,
169.6, 171.7. HRMS (FAB, positive) calcd for C18H24NO9 [M + H]+
m/z = 398.1451, found 398.1479 (error 2.8 mmu). Anal. Calcd for
C18H23NO9: C, 54.41; H, 5.83; N, 3.52. Found: C, 54.20; H, 5.81; N,
3.48.
4-O-Acetyl-β-D-glucopyranoside 16 and 6-O-Acetyl-β-D-glu-
copyranoside 17 of N-Phenylacetohydroxamic Acid. To a
suspension of 3a (1.00 g, 3.19 mmol) in CH3CN (20 mL) and
trimethyl orthoacetate (1.2 mL, 9.6 mmol) was added a catalytic
amount of p-TsOH (35 mg). After the mixture was stirred for 3 h at
room temperature, water (20 mL) was added to hydrolyze the 4,6-
orthoacetate group. After 1.5 h, the reaction mixture was evaporated
under reduced pressure, and the residue was purified by preparative
HPLC on a column of LiChrosorb RP-8 (7 μm, 10 × 250 mm) with
MeOH/H2O (1:4, v/v) to afford the title compounds 16 (410 mg,
33%) and 17 (417 mg, 33%). The analytical data of 16 (a white
powder) are as follows: IR (Nujol) cm−1 3420, 1740, 1670; [α]20
Enzymatic regioselective O-deacetylation for the synthesis
of di-O-acetyl compounds 9−13. In general, the formation of each
di-O-acetyl compound in the reaction mixture was analyzed and
identified with the standard sample by using reversed-phase HPLC, as
described below at the section of HPLC analysis conditions.
Incubation mixture was composed of the appropriate precursor
substrate in 85 mM sodium acetate buffer (pH 5.0) containing 15 (v/
v) % EtOH and appropriate enzyme. Purification and isolation were
performed by preparative TLC of the ethyl acetate extracts of the
incubation mixtures. The precursor substrate and enzyme with their
initial concentrations in the incubation mixture and the isolation yield
of each di-O-acetyl compound were as follows: 2,4-di-O-acetyl
compound 9 (78% after 40 min) from the substrate 4 (3.0 mM)
with LAS (2.5 mg/mL), 2,6-di-O-acetyl compound 10 (92% after 6
D
−102 (c 0.50, EtOH); 1H NMR (400 MHz, DMSO-d6) δ 2.01 (s, 3H),
2.30 (s, 3H), 3.26−3.34 (m, 2H, overlapped with H2O), 3.37−3.44
(m, 3H), 4.58−4.63 (m 2H), 4.63 (d, J = 8.3 Hz, 1H, C1-H), 5.36 (d, J
= 5.6 Hz, 1H, OH), 5.70 (d, J = 5.1 Hz, 1H, OH), 7.23 (t, J = 7.6 Hz,
1H, Ar-H), 7.39 (t, J = 7.6 Hz, 2H, Ar-H), 7.56 (d, J = 7.6 Hz, 2H, Ar-
H); 13C NMR (100 MHz, DMSO-d6) δ 20.9, 22.6, 60.6 (C6), 71.0
(C4), 72.2, 73.5, 74.3, 104.6 (C1), 123.1, 126.0, 128.4, 139.0, 169.5,
171.7; HRMS (FAB, positive) calcd for C16H22NO8 [M + H]+ m/z =
356.1345, found 356.1349 (error 0.3 mmu). Anal. Calcd for
C16H21NO8: C, 54.08; H, 5.96; N, 3.94. Found: C, 54.11; H, 6.03;
N, 3.92. The analytical data of 17 (a white powder) are as follows: IR
(Nujol) cm−1 3400, 1750, 1660; [α]20D −91 (c 0.60, EtOH); 1H NMR
(400 MHz, DMSO-d6) δ 1.99 (s, 3H), 2.24 (s, 3H), 3.11−3.21 (m,
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3H, C2-H, C3-H and C4-H), 3.37−3.41 (m, 1H, C5-H), 4.07 (dd, 1H, J
= 6.6 and 12.0 Hz, C6-H), 4.22 (dd, 1H, J = 2.2 and 12.0 Hz, C6-H′),
4.59 (d, J = 7.8 Hz, 1H, C1-H), 5.22 (d, J = 5.4 Hz, 1H, OH), 5.26 (d, J
= 5.4 Hz, 1H, OH), 5.55 (d, J = 5.4 Hz, 1H, OH), 7.23 (t, J = 7.6 Hz,
1H, Ar-H), 7.38 (t, J = 7.6 Hz, 2H, Ar-H), 7.51 (d, J = 7.6 Hz, 2H, Ar-
H); 13C NMR (100 MHz, DMSO-d6) δ 20.5, 22.2, 63.0 (C6), 69.8,
72.0, 73.7, 76.1, 104.9 (C1), 123.3, 126.1, 128.4, 139.2, 170.1, 171.5;
HRMS (FAB, positive) calcd for C16H22NO8 [M + H]+ m/z =
356.1345, found 356.1342 (error −0.3 mmu).
Equipment Management Center, Creative Research Institution,
Hokkaido University, Japan) for measurement of NMR spectra
of 1b and 1c. We also thank Koji Wada and Megumi Mizukami
for measurement of HRMS analyses and Hiroaki Ohno and
Ryosuke Tatsunami for measurement of some NMR spectra.
REFERENCES
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General Chemoenzymatic Synthesis of 3a−c from 1a−c
Using LAS and CSR at pH 7.2. To a solution of 1a−c (80 μmol) in
DMSO (10 mL) was added a solution of LAS (335 mg; 5 mg/mL of
incubation mixture) and CSR (335 mg, 5 mg/mL of incubation
mixture) in 100 mM sodium phosphate (pH 7.2) buffer (57 mL) and
the mixture stirred for 5.0 h with 1a and for 4.0 h with 1b and 1c at 40
°C. After filtration, the filtrate was loaded onto a XAD-4 column (7 g,
1.6 × 10 cm), which had been washed thoroughly with acetone and
then equilibrated with water. The column was washed with water (100
mL), and then the desired products 3a, 3b, and 3c were eluted with 5,
10, and 7.5% (v/v) aqueous CH3CN, respectively. The product-
containing fractions were pooled and concentrated under reduced
pressure. The residue was purified by preparative TLC developed with
CHCl3−acetone−H2O (4:18:1, v/v) to afford 3a−c. β-D-Glucopyr-
anoside of N-phenylacetohydroxamic acid (3a): yield of 3a (a white
powder) (92%); IR (Nujol) cm−1 3360, 1670; [α]20 −52 (c 0.36,
D
1
H2O); H NMR (400 MHz, DMSO-d6/D2O) δ 2.29 (s, 3H), 3.10−
3.22 (m, 4H), 3.39−3.47 (m, 1H, overlapped with DHO peak), 3.65
(d, J = 11.4 Hz, 1H), 4.50 (d, J = 7.8 Hz, 1H, C1-H), 7.24 (t, J = 7.3
Hz, 1H, Ar-H), 7.39 (t, J = 7.6 Hz, 2H, Ar-H), 7.55 (dd, J = 1.2 and 7.6
Hz, 2H, Ar-H); 13C NMR (100 MHz, DMSO-d6/D2O) δ 22.6, 60.8
(C6), 69.6, 72.1, 76.4, 77.2, 104.8 (C1), 123.0, 125.9, 128.4, 139.0,
171.7; HRMS (FAB, positive) calcd for C14H20NO7 [M + H]+ m/z =
314. 1240, found 314.1241 (error 0.1 mmu). β-D-Glucopyranoside of
N-(4-chloro)phenylacetohydroxamic acid (3b): yield of 3b (a white
(4) (a) Irving, C. C.; Wiseman, R. Jr. Cancer Res. 1971, 31, 1645−
1648. (b) Wang, C. Y.; Imaida, K.; Zukowski, K.; Lee, M.-S. Cancer
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(5) (a) Roncucci, R.; Simon, M. J.; Lambelin, G.; Thiriaux, J.; Buu-
Hoi, N. P. Biochem. Pharmacol. 1968, 17, 187−194. (b) Boreham, D.
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164−168.
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8829−8834.
(7) Du, L.; Musson, D. G.; Wang, A. Q. J. Pharm. Biomed. Anal. 2006,
42, 556−564.
(8) Thomas, M.; Rivault, F.; Tranoy-Opalinski, I.; Roche, J.; Gesson,
J.-P.; Papot, S. Bioorg. Med. Chem. Lett. 2007, 17, 983−986.
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4262−4264.
powder) (90%); IR (Nujol) cm−1 3380, 1650; [α]20 −46 (c 0.42,
D
1
H2O); H NMR (400 MHz, DMSO-d6/D2O) δ 2.31 (s, 3H), 3.11−
3.24 (m, 4H), 3.44−3.47 (m, 1H), 3.64−3.67 (m, 1H), 4.52 (d, J = 7.8
Hz, 1H, C1-H), 7.44 (d, J = 8.8 Hz, 2H, Ar-H), 7.57 (d, J = 8.8 Hz, 2H,
Ar-H); 13C NMR (100 MHz, DMSO-d6/D2O) δ 22.8, 61.0 (C6), 69.8,
72.3, 76.4, 77.4, 105.4 (C1), 125.4, 128.8, 130.7, 138.2, 172.5; HRMS
(FAB, positive) calcd for C14H19NO7Cl [M + H]+ m/z = 348.0850,
found 348.0833 (error 0.1 mmu). β-D-Glucopyranoside of N-(4-
methyl)phenylacetohydroxamic acid (3c): yield of 3c (a white
powder) (88%); IR (Nujol) cm−1 3290, 1650; [α]20 −45 (c 0.38,
D
H2O); 1H NMR (400 MHz, DMSO-d6/D2O) δ 2.26 (s, 3H), 2.31 (s,
3H), 3.13−3.18 (m, 4H), 3.43−3.46 (m, 1H), 3.65−3.68 (m, 1H),
4.48 (d, J = 7.3 Hz, 1H, C1-H), 7.19 (d, J = 8.3 Hz, 2H, Ar-H), 7.39 (d,
J = 8.3 Hz, 2H, Ar-H); 13C NMR (100 MHz, DMSO-d6/D2O) δ 20.8,
22.6, 60.9 (C6), 69.7, 72.2, 76.4, 77.3, 104.8 (C1), 124.3, 129.2, 136.2,
136.6, 171.8; HRMS (FAB, positive) calcd for C15H22NO7 [M + H]+
m/z = 328. 1396, found 328.1389 (error 0.1 mmu).
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ASSOCIATED CONTENT
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S
* Supporting Information
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1H and 13C NMR spectra of 1a−c, 3a−c, and 4−17 and
crystallographic data and table of compounds 4 and 5. This
material is available free of charge via the Internet at http://
pubs.acs/org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +81-134-62-1894. Fax: +81-134-62-5161. E-mail:
yoshioka@hokuyakudai.ac.jp.
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ACKNOWLEDGMENTS
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(18) Baba, A.; Yoshioka, T. J. Mol. Catal. B: Enzym. 2011, 69, 74−82.
We are deeply grateful to Rigaku X-ray Research Laboratory of
Rigaku Corp. (Tokyo, Japan) for X-ray structure analyses. We
thank Tomohiro Hirose (Instrumental Analysis Division,
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NOTE ADDED AFTER ASAP PUBLICATION
■
There were errors in the text on the fourth and fifth pages and
in Table 1 in the version published ASAP on January 24, 2012;
the correct version reposted on February 1, 2012.
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