An improved chemo-enzymatic synthesis of 1-β-O-acyl glucuronides: Highly chemoselective enzymatic removal of protecting groups from corresponding methyl acetyl derivatives

Article abstract of DOI:10.1021/jo701547b

(Chemical Equation Presented) An improved and widely applicable chemo-enzymatic method for the synthesis of a series of 1-β-O-acyl glucuronides 5a-f has been developed from the corresponding methyl acetyl derivatives 3a-f, which were stereospecifically synthesized from cesium salts of carboxylic acids 1a-f and methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-α-D- glucopyranuronate (2). Chemoselectivity of lipase AS Amano (LAS) in the hydrolytic removal of O-acetyl groups of 3a-f to provide methyl esters 4a-f was influenced by the nature of their 1-β-O-acyl groups; high selectivity was evident only for 3b and 3f. Carboxylesterase from Streptomyces rochei (CSR), newly screened as an alternative to LAS, showed much greater chemoselectivity toward the O-acetyl groups than LAS; 3a, 3d, and 3e were chemoselectively hydrolyzed only by CSR. The combination of CSR with LAS yielded better results in the hydrolysis of 3c and 3f than did single usage of CSR. Final deprotection of the methyl ester groups of 4a-f to provide 5a-f was chemoselectively achieved by using lipase from Candida antarctica type B (CAL-B) as well as esterase from porcine liver (PLE), although CAL-B possessed higher chemoselectivity and catalytic efficiency than did PLE. CSR also exhibited high chemoselectivity in the synthesis of (S)-naproxen 1-β-O-acyl glucopyranoside (7) from its 2,3,4,6-tetra-O-acetyl derivative 6.

Full text of DOI:10.1021/jo701547b

An Improved Chemo-Enzymatic Synthesis of 1-â-O-Acyl
Glucuronides: Highly Chemoselective Enzymatic Removal of
Protecting Groups from Corresponding Methyl Acetyl Derivatives
Akiko Baba and Tadao Yoshioka*
Hokkaido Pharmaceutical UniVersity School of Pharmacy, 7-1 Katsuraoka-cho, Otaru,
047-0264, Hokkaido, Japan
yoshioka@hokuyakudai.ac.jp
ReceiVed July 26, 2007
An improved and widely applicable chemo-enzymatic method for the synthesis of a series of 1-â-O-acyl
glucuronides 5a-f has been developed from the corresponding methyl acetyl derivatives 3a-f, which
were stereospecifically synthesized from cesium salts of carboxylic acids 1a-f and methyl 2,3,4-tri-O-
acetyl-1-bromo-1-deoxy-R-D-glucopyranuronate (2). Chemoselectivity of lipase AS Amano (LAS) in the
hydrolytic removal of O-acetyl groups of 3a-f to provide methyl esters 4a-f was influenced by the
nature of their 1-â-O-acyl groups; high selectivity was evident only for 3b and 3f. Carboxylesterase
from Streptomyces rochei (CSR), newly screened as an alternative to LAS, showed much greater
chemoselectivity toward the O-acetyl groups than LAS; 3a, 3d, and 3e were chemoselectively hydrolyzed
only by CSR. The combination of CSR with LAS yielded better results in the hydrolysis of 3c and 3f
than did single usage of CSR. Final deprotection of the methyl ester groups of 4a-f to provide 5a-f
was chemoselectively achieved by using lipase from Candida antarctica type B (CAL-B) as well as
esterase from porcine liver (PLE), although CAL-B possessed higher chemoselectivity and catalytic
efficiency than did PLE. CSR also exhibited high chemoselectivity in the synthesis of (S)-naproxen 1-â-
O-acyl glucopyranoside (7) from its 2,3,4,6-tetra-O-acetyl derivative 6.
Introduction
Recent articles have reviewed bioactivation pathways for drug
metabolism to electrophilic metabolites, after covalent binding
to target macromolecules (proteins and/or DNA), which has
toxicological consequences.8-13 These chemically reactive
In drug discovery and development and for drug safety, it is
important to understand drug metabolism and the underlying
mechanism of drug-induced toxicity, including pharmacological
actions, necrotic activity, immune-mediated response, idiosyn-
cratic reactions, and potential cancer-inducing properties.1-7
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1569.
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344-369.
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* To whom correspondence should be addressed. Fax: +81-134-62-5161.
Phone: +81-134-62-1894.
2005, 35, 325-361.
(9) Zhou, S.; Chan, E.; Duan, W.; Huang, M.; Chen, Y.-Z. Drug Met.
(1) Evans, D. C.; Watt, A. P.; Nicoll-Griffith, D. A.; Baillie, T. A. Chem.
Res. Toxicol. 2004, 17, 3-16.
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10.1021/jo701547b CCC: $37.00 © 2007 American Chemical Society
Published on Web 11/07/2007
J. Org. Chem. 2007, 72, 9541-9549
9541

Baba and Yoshioka
CHART 1. Three NSAIDs Used in the Preceding Study20
CHART 2. Wide-Ranging Carboxylic Acids Used in This
Study
SCHEME 1. Chemo-Enzymatic Synthetic Method for
1-â-O-Acyl Glucuronides
range of structures (Chart 2), while focusing on the validation
and improvement of the synthetic method, especially for
chemoselectivity in the enzymatic hydrolytic deprotection of
methyl acetyl derivatives of 1-â-O-acyl glucuronides 3a-f and
4a-f.
The enzymatic deprotection of these compounds has been
achieved by using newly screened enzymes, carboxylesterase
from Streptomyces rochei (CSR) and lipase from Candida
antarctica type B (CAL-B), as well as LAS and PLE. In
addition, because of recent reports on a glucosidation pathway
for carboxylic acid drugs,22-24 we applied this chemo-enzymatic
method for the synthesis of 1-â-O-acyl glucopyranoside deriva-
tive 7 of NP, starting from its per-O-acetylated compound 6,
as a model compound.
metabolites include 1-â-O-acyl glucuronides, associated with
many carboxylate drugs such as nonsteroidal anti-inflammatory
drugs (NSAIDs). Because these acyl glucuronides are, in
general, electrophilic species,9,14-19 covalent binding of acyl
glucuronides to tissue proteins is believed to cause hypersen-
sitivity and idiosyncratic reactions. Some acyl glucuronides have
been implicated in the adverse effects of some NSAIDs which
have been withdrawn from the market,1,8,10,12 although their
toxicological relevance is not well understood.
A previous study20 has reported a facile chemo-enzymatic
synthesis of 1-â-O-acyl glucuronides from commercially avail-
able methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-R-D-glucopy-
ranuronate 2 and three NSAIDs [diclofenac (DF), mefenamic
acid (MF), and (S)-naproxen (NP)] as model carboxylate drugs
(Chart 1), which provides a useful synthetic method for
toxicological research on 1-â-O-acyl glucuronides. Two char-
acteristics of this method, among the synthetic methods reported
for 1-â-O-acyl glucuronides,19,21 include stereospecific glucu-
ronidation with nearly exclusive â-configuration and highly
chemoselective enzymatic removal of sugar ester-type protection
groups without affecting 1-â-O-acyl functions by using lipase
AS Amano (LAS) and porcine liver esterase (PLE) (Scheme
1).
Results and Discussion
Synthesis of Methyl Acetyl Derivatives of 1-â-O-Acyl
Glucuronides 3a-f. Diverse carboxylic acids 1a-f, having a
wide range of size and numbers of methyl substituents at the
R-carbon of 4-biphenylyl carboxylic acid derivatives 1d-f, were
chosen for validating the chemo-enzymatic synthetic strategy.
The condensation of 1a-f with methyl 2,3,4-tri-O-acetyl-1-
bromo-1-deoxy-R-D-glucopyranuronate (2) was performed by
using a previously reported procedure20 to provide the corre-
sponding 1-â-O-acyl glucuronides 3a-f in moderate yields,
without production of R-anomers (based on 1H NMR data). The
In the present study, we extended this synthetic chemo-
enzymatic method using carboxylic acids 1a-f with a wide
1
yields of 3a-f and their H-chemical shifts and J values for
(14) Faed, E. M. Drug Metab. ReV. 1984, 15, 1213-1249.
(15) Spahn-Langguth, H. Drug Metab. ReV. 1992, 24, 5-48.
(16) Shipkova, M.; Armstrong, V. W.; Oellerich, M.; Wieland, E. Ther.
Drug Monit. 2003, 25, 1-16.
(17) Mullangi, R.; Bhamidipati, R. K.; Srinivas, N. R. Curr. Pharm. Anal.
2005, 1, 251-264.
(18) Yang, X.-X.; Hu, Z.-P.; Boelsterli, U. A.; Zhou, S.-F. Curr. Pharm.
Anal. 2006, 2, 259-277.
(19) (a) Perrie, J. A.; Harding, J. R.; Holt, D. W.; Johnston, A.; Mealth,
P.; Stachulski, A. V. Org. Lett. 2005, 13, 2591-2594. (b) Stachulski, A.
V.; Harding, J. R.; Lindon, J. C.; Maggs, J. L.; Park, B. K.; Wilson, I. D.
J. Med. Chem. 2006, 49, 6931-6945. (c) Bowkett, E. R.; Harding, J. R.;
Maggs, J. L.; Park, B. K.; Perrie, J. A.; Stachulski, A. V. Tetrahedron 2007,
63, 7596-7605.
(20) Baba, A.; Yoshioka, T. Org. Biomol. Chem. 2006, 4, 3303-3310.
(21) (a) Becker, B.; Barua, A. B.; Olson, J. A. Biochem. J. 1996, 314,
249-252. (b) Juteau, H.; Gareau, Y.; Labelle, M. Tetrahedron Lett. 1997,
38, 1481-1484.
anomeric protons are summarized in Table 1. The â-configu-
ration of 3a-f was confirmed by J values of 7.8-8.3 Hz.25,26
Compound 3e, obtained from the racemic carboxylic acid 1e,
was a 1:1 mixture of diastereoisomers (2R)-3e and (2S)-3e, based
(22) Tang, C.; Ma, B. Drug Metab. Dispos. 2005, 33, 1796-1802.
(23) Tang, C.; Hochman, J. H.; Ma, B.; Subramanian, R.; Vyas, K. P.
Drug Metab. Dispos. 2003, 31, 37-45.
(24) Shipkova, M.; Strassburg, C. P.; Braun, F.; Streit, F.; Gro¨ne, H.-J.;
Armstrong, V. W.; Tukey, R. H.; Oellerich, M.; Wieland, E. Br. J.
Pharmacol. 2001, 132, 1027-1034.
(25) (a) Root, Y. Y.; Wagner, T. R.; Norris, P. Carbohydr. Res. 2002,
337, 2343-2346. (b) Trynda, A.; Madaj, J.; Konits, A.; Wis´niewski, A.
Carbohydr. Res. 2000, 329, 249-252.
(26) Marchettini, N.; Ulgiati, S.; Rossi, C. Magn. Reson. Chem. 1989,
27, 223-226.
9542 J. Org. Chem., Vol. 72, No. 25, 2007

Chemo-Enzymatic Synthesis of 1-â-O-Acyl Glucuronides
TABLE 1. Stereospecific Synthesis of Methyl Acetyl Derivatives of
providing 1d (data not shown). The effect of cosolvents other
than DMSO on chemoselectivity toward 3d was investigated
with use of acetone, t-BuOH, CH3CN, dioxane, DMF, diglyme,
MeOH, methyl cellosolve, methyl carbitol, and THF at a
concentration of 20% (v/v). Except for THF and methyl carbitol,
in which LAS was significantly inactivated, carboxylic acid 1d
was released in 30% yield or greater, indicating chemoselectivity
of LAS toward 3d was not improved by the nature of the
cosolvent. Compounds 3c and 3e provided products 4c and 4e
in moderate yields (57% and 41%, respectively) but also released
carboxylic acids 1c (21%) and 1e (35%), although in lower
yields than those from 3a and 3d. High chemoselectivity with
LAS was found only for 3b and 3f. While 3b provided 4b in
95% yield after 6.0 h, 3f provided 4f in only 14% yield after
1.5 h because of an accumulation of the corresponding partially
O-deacetylated intermediates (85%); however, the corresponding
carboxylic acid 1f was not detected. These results indicate that
high chemoselectivity of LAS can be achieved, especially for
substrates that have rather bulky groups near 1-â-O-acyl
functions, such as the o-phenylamino substituent (of 3b) and
R,R-dimethyl substituents (of 3f).
1-â-O-Acyl Glucuronides 3a-f
yield
(%)a
C1-H
(δ)b
J
R-CO2H
product
(Hz)c
1a
1b
1c
1d
1e
3a
3b
3c
3d
3e
50
67
55
58
52
6.25
6.24
6.28
6.04
5.99
6.05
5.99
6.05
6.00
8.1
7.8
7.8
8.2
8.3
8.3
8.3
8.3
8.3
(2R)-1e
(2S)-1e
1f
(2R)-3e
(2S)-3e
3f
44
45
52
a Isolated yields, as recrystallized form, based on 2 as the limiting reagent.
b Chemical shifts of the anomeric protons measured in DMSO-d6. c Coupling
constants of the anomeric protons observed as doublets.
TABLE 2. Enzymatic Hydrolysis of 3a-f with LAS
Enzyme Screening for Chemoselective Hydrolysis of 3d
as a Model Substrate. It is important to remove the protecting
groups of sugar moieties without affecting their 1-â-O-acyl
linkage. Therefore, enzymes were newly screened to achieve
the chemoselective deprotection of O-acetyl groups of the
substrates other than 3b. Screening was performed with 0.1 mM
3d, as a model substrate, in 20 mM sodium citrate buffer (pH
5.0) containing 20% (v/v) DMSO at 40 °C for 2 h in the
presence of each enzyme at an amount of 10 mg/mL of
incubation mixture. Of 17 enzymes tested, a carboxylesterase
from Streptomyces rochei (CSR) showed the highest chemose-
lectivity. Compound 3d provided 4d in 65% yield along with
partially O-deacetylated intermediates (20%), unreacted 3d
(10%), and the released carboxylic acid 1d in only 4% yield.
Equine liver acetone powder provided 4d (35%) as well as 1d
(30%). Acylase Amano, lipase AP6, lipase AYS Amano, acylase
I, and lipase from Candida cylindracea showed higher catalytic
activity but lower chemoselectivity, resulting in the release of
1d in 60-95% yields. Ten additional enzymes tested showed
much lower catalytic actitivity toward 3d, leaving 60-90% of
3d unreacted.
concn
time
(h)b
products and yields
(%)c
substrate
(mM)a
3a
3b
3c
3d
3e
3f
1.00d
0.50d
0.40
2.0
6.0
4.5
0.25
1.5
1.5
4a (17), 1a (80), otherse (3)
4b (95), 1b (3)
4c (57), 1c (21), others (21)
4d (3), 1d (92), others (6)
4e (41), 1e (35), 3e (11), others (13)
4f (14), 1f (ndf), others (85)
0.20
0.50d
0.50d
a Initial concentration of substrate in 20 mM sodium citrate buffer (pH
5.0) containing 20% (v/v) DMSO. b Incubation time at 40 °C. c Yields based
on HPLC analysis. d The reaction mixture was a suspension. e Others )
partially O-deacetylated compounds. f nd ) not detectable.
on comparison of 1H NMR spectra. The methyl protons of the
2-O-acetyl group of (2R)-3e, assigned to a singlet peak at 1.52
ppm by the HMBC method (data not shown), are markedly
shifted ca. 0.4 ppm to higher field compared to that of the (2S)-
3e isomer whose 2-O-acetyl methyl protons resonate at 1.91
ppm. This upfield shift may be due in part to the anisotropic
effect of the aromatic ring of (2R)-3e.
Chemoselective Enzymatic Removal of O-Acetyl Groups
of 3a-f. In a preceding paper,20 enzyme screening revealed that
lipase AS Amano (LAS) showed high chemoselectivity toward
the O-acetyl groups of methyl acetyl derivatives of 1-â-O-acyl
glucuronides of MF, DF, and NP. Therefore, the chemoselec-
tivity of LAS toward a new series of the substrates 3a-f was
first examined under the same conditions to verify the scope of
the chemoselectivity of LAS. Incubation was conducted in 20
mM sodium citrate buffer (pH 5.0) containing 20% (v/v) DMSO
as a cosolvent at 40 °C by the addition of 10 mg/mL LAS in
the incubation mixture. Results shown in Table 2 indicate that
the chemoselectivity of LAS was influenced by the nature of
the substrate 1-O-acyl groups.
CSR-Catalyzed Removal of O-Acetyl Groups of 3a-f.
Newly screened CSR was examined for chemoselectivity and
catalytic efficiency toward 3a-f. The catalytic efficiency of CSR
toward 3d was optimal at 50-55 °C; the efficiency at 50 °C
was about 3-fold greater than that at 40 °C; activity was
deactivated at 60 °C (Figure 1).
Therefore, incubation with CSR was performed at the optimal
temperature of 50 °C. Table 3 summarizes the results, obtained
in 20 mM sodium citrate buffer (pH 5.0) containing 20% (v/v)
DMSO as a cosolvent after addition of CSR at 10 mg/mL of
incubation mixture. Compounds 3a, 3d, and 3e, which did not
yield good results with LAS, were chemoselectively hydrolyzed
to 4a, 4d, and 4e, respectively, using CSR. Compound 3a, at
an initial amount of 1.00 µmol/mL of the incubation mixture,
was chemoselectively hydrolyzed to provide 4a in 92% yield
after 4 h (run 1). When the amount of 3a was increased to 2.00
µmol/mL of incubation mixture for preparative scale (run 2),
the incubation mixture became a suspension. CSR-catalyzed
hydrolysis of the resultant suspension was prolonged for 9.0 h,
probably due in part to the dissolution of 3a being a rate-limiting
Compounds 3a and 3d released the corresponding carboxylic
acids 1a and 1d in 80% and 92% yields, respectively, indicating
much lower chemoselectivity of LAS toward both 3a and 3d.
Compound 4d also was confirmed to be hydrolyzed by LAS,
J. Org. Chem, Vol. 72, No. 25, 2007 9543

Baba and Yoshioka
FIGURE 2. Time courses of enzymatic hydrolysis of 3c to 4c with
CSR (9), LAS (b), or both CSR and LAS ([) in 20 mM sodium citrate
buffer (pH 5.0) containing 20% (v/v) DMSO at 40 °C.
FIGURE 1. Effect of incubation temperature on CSR-catalyzed
hydrolysis of 3d to 4d in 20 mM sodium citrate buffer (pH 5.0)
containing 20% (v/v) DMSO at pH 4.0 and 55 °C (∆), at pH 4.5 and
55 °C (0), at pH 5.0 and 40 °C ([), at pH 5.0 and 50 °C (2), at pH
5.0 and 55 °C (9), or at pH 5.0 and 60 °C (b).
TABLE 4. Enzymatic Hydrolysis of 3a-f with Both CSR and LAS
TABLE 3. Enzymatic Hydrolysis of 3a-f with CSR
concn time
products and yields
(%)c
substrate run (mM)a (h)b
3c
1
2
1
1
0.40
2.0
9.0
3.0
3.0
4c (94), 1c (4), otherse (2)
4c (93), 1c (6)
(2R)-4e (84), (2R)-1e (11), others (5)
4f (98)
2.00d
1.00d
1.00d
concn
time
(h)b
products and yields
(%)c
(2R)-3e
3f
substrate run (mM)a
3a
1
2
1
1
1
2
1
2
1
2
1
2
1
1.00
4.0 4a (92), 1a (6)
9.0 4a (82), 1a (16)
4.0 4b (13), 1b (1), 3b (84)
2.0 4c (10), 1c (nde), 3c (89), othersf (2)
4.5 4d (92), 1d (7)
7.5 4d (86), 1d (10)
3.0 4e (95), 1e (2)
6.0 4e (96), 1e (4)
4.0 (2R)-4e (95), (2R)-1e (3)
a Initial concentration of substrate in 20 mM sodium citrate buffer (pH
5.0) containing 20% (v/v) DMSO. b Incubation time at 40 °C. c Yields based
on HPLC analysis. d The reaction mixture was a suspension. e Others )
partially O-deacetylated compounds.
2.00d
0.50d
0.40
3b
3c
3d
0.80
1.50d
0.20
3e
In contrast, the ortho-substituted compound 3b, which was
chemoselectively hydrolyzed by LAS (Table 2), was minimally
hydrolyzed by CSR. Similarly, CSR-catalyzed hydrolysis of both
3c and 3f was very slow. For these cases, a considerable amount
of substrates 3b, 3c, and 3f remained unreacted; however,
significant amounts of the corresponding carboxylic acids 1b,
1c, and 1f were not released. For 3c and 3f, therefore, concurrent
use of CSR and LAS was examined next at 40 °C, because
LAS was deactivated at 50 °C. Figure 2 shows the time course
of formation of 4c from 3c through enzymatic hydrolysis with
CSR, LAS, or both CSR and LAS (each 10 mg/mL); catalytic
activity of CSR for hydrolysis of 3c to 4c was lower than that
of LAS. In the CSR-catalyzed reaction no significant accumula-
tion of the corresponding partially O-deacetylated intermediates
occurred, whereas these intermediates accumulated in the LAS-
catalyzed reaction (data not shown). This indicates that catalytic
activity of CSR toward the intermediates is much greater than
that of LAS. Therefore, concurrent use of the enzymes
synergistically accelerated the rate of formation of 4c and
resulted in 94% yield in 2 h (Table 4, run 1).
Similarly, 3f effectively provided 4f (98%) by concurrent use
of both enzymes (Table 4). Furthermore, concurrent usage of
CSR and LAS shortened the reaction time required for the
hydrolysis of (2R)-3e; (2R)-4e was readily obtained in 84% yield
in only 3 h (Table 4).
Products 4a-f were isolated easily through EtOAc extraction
from the incubation mixture or by passage through an XAD-4
column, as reported previously.20 The structures of 4d (isolated
0.50d
0.20
(2R)-3e
(2S)-3e
3f
1.00d
0.20
30
(2R)-4e (89), (2R)-1e (11)
2.0 (2S)-4e (96), (2S)-1e (2)
3.0 (2S)-4e (98), (2S)-1e (2)
1.5 4f (14), 1f (nd), 3f (83), others (4)
1.00d
0.50d
a Initial concentration of substrate in 20 mM sodium citrate buffer (pH
5.0) containing 20% (v/v) DMSO. b Incubation time at 50 °C. c Yields based
on HPLC analysis. d The reaction mixture was a suspension. e nd ) not
detectable. f Others ) partially O-deacetylated compounds.
step. The initial concentration of substrate 3a should be
approximately 1.0 mM, near saturated concentration under the
incubation conditions. Compounds 3d and 3e, at initial amounts
of 0.80 and 0.50 µmol/mL, respectively, also successfully
underwent chemoselective hydrolysis to provide 4d and 4e in
yields of 92% and 96%, respectively. Both (2R)-3e and (2S)-
3e also were chemoselectively hydrolyzed in high yields,
although (2S)-3e was a better substrate than (2R)-3e. A
remarkable difference in the rates of CSR-catalyzed hydrolysis
of (2R)-3e and (2S)-3e was observed when the reaction was
started from a suspension at a concentration of 1.00 µmol/mL
(run 2 in each case). Similarly, starting from a suspension of
3d at an initial amount of 1.50 µmol/mL (run 2), the reaction
time again was prolonged for 7.5 h and the yield of 4d was
decreased to 86% with an increased production of carboxylic
acid 1d (10%). These results indicate that the initial concentra-
tion of the substrates should be near their saturated concentra-
tions.
9544 J. Org. Chem., Vol. 72, No. 25, 2007

Chemo-Enzymatic Synthesis of 1-â-O-Acyl Glucuronides
TABLE 5. Enzymatic hydrolysis of 4a-f with PLE or CAL-B
SCHEME 2. Chemo-Enzymatic Synthesis of (S)-Naproxen
(NP) 1-â-O-Acyl Glucopyranoside 6
enzyme
(mg/mL of
incubation
mixture)
concn
time
(h)b
products and yields
(%)c
from CAL-B-catalyzed hydrolysis of (2R)-4e and (2S)-4e,
respectively. No significant epimerization was observed for the
final products (2R)-5e and (2S)-5e, based on HPLC analysis
and NMR spectra (see the Supporting Information).
Because these four enzymes are commercially available and
inexpensive, the overall reaction could be readily scaled up to
millimole amounts of starting material.
substrate
(mM)a
4a
4b
4c
4d
CAL-B (2)
CAL-B (1)
CAL-B (1)
PLE (0.4)
CAL-B (0.4)
CAL-B (1)
PLE (0.4)
CAL-B (0.4)
CAL-B (0.4)
CAL-B (0.4)
CAL-B (0.4)
CAL-B (0.8)
1.00
1.00
1.00
0.100
0.100
1.00
0.100
0.100
1.00
1.00
2.00
2.00
2.0
2.5
9.0
1.0
0.25
2.0
1.5
0.5
0.75
1.0
2.0
3.0
5a (87), 1a (8)
5b (98), 1b (ndd)
5c (93), 1c (nd)
5d (68), 1d (17), 4d (15)
5d (97)
5d (99)
5e (82), 1e (nd), 4e (15)
5e (99)
4e
Chemo-Enzymatic Synthesis of 7 from Its 2,3,4,6-Tetra-
O-acetyl Derivative 6. As shown in Scheme 2, the chemo-
enzymatic method was applied to the synthesis of (S)-naproxen
1-â-O-acyl glucopyranoside 6 as the model compound.
In carbohydrate chemistry, many ester-hydrolytic enzymes
have been reported as catalysts for mainly regioselective
synthetic reactions.29 Recently, CAL-B was reported as a
biocatalyst for the chemoselective deacetylation of acylglu-
copyranosides.30 Therefore, CAL-B and PLE as well as LAS
and CSR were examined for activity in the chemoselective
deacetylation of 6. LAS and PLE showed low chemoselectivity
under the optimum conditions used for abovementioned 1-â-
O-acyl glucuronides, releasing a considerable amount of NP.
CAL-B was chemoselective for 6, but showed low catalytic
activity. CSR-catalyzed deacetylation of 6 in 20 mM MES-
NaOH buffer (pH 5.5) containing 20% (v/v) DMF at 50 °C for
40 h provided 7 in 64% yield, along with mono-O-acetates
(34%). Modification of the incubation conditions is needed for
a better result and is now in progress.
In conclusion, our chemo-enzymatic method was improved
for the synthesis of 1-â-O-acyl glucuronides by using CSR and
CAL-B as well as LAS and PLE as catalysts. This efficient
method is applicable to a wide range of carboxylic acids,
providing 1-â-O-acyl glucuronides with complete â-selectivity
and in good yields. Differences in chemoselectivity and substrate
specificity between CSR and LAS are currently under investiga-
tion.
5e (99)
(2R)-4e
(2S)-4e
4f
(2R)-5e (99)
(2S)-5e (99)
5f (97)
a Initial concentration of substrate in 20 mM sodium citrate buffer (pH
5.0) containing 20% (v/v) DMSO. b Incubation time at 40 °C. c Yields based
on HPLC analysis. d nd ) not detectable.
by an XAD-4 column method) and 4e (isolated by the EtOAc
extraction method) were confirmed by 1H and 13C NMR studies
(see the Experimental Section). 1H NMR of 4e showed it to be
a 1:1 mixture of diastereoisomers of (2R)-4e and (2S)-4e.
Enzyme Screening for Chemoselective Hydrolysis of the
Methyl Ester of 4d as a Model Substrate. Chemoselective
hydrolysis of the methyl esters of 4a-f was investigated. Porcine
liver esterase (PLE), an effective enzyme for the hydrolysis of
methyl esters of 1-â-O-acyl glucuronides20 of DF, MF, and NP,
was applied first to the hydrolysis of the methyl ester of 4d. As
shown in Table 5, the product 5d was obtained in 68% yield
with concomitant formation of the corresponding carboxylic acid
1d (17%), indicating that chemoselectivity of PLE was influ-
enced by the structure of the 1-â-O-acyl group. Lipases have
been used for the enzymatic removal of carboxylic methyl
esters,27 and the lipase from Candida antarctica type B (CAL-
B) has been utilized in the chemo-enzymatic synthesis of 1-â-
O-acyl glucuronide.28 Therefore, screening was performed by
using 0.1 mM 3d as a model substrate, in 20 mM sodium citrate
buffer (pH 5.0) containing 20% (v/v) DMSO at 40 °C for 1.5
h in the presence of 10 mg/mL of each enzyme. Of the 17
enzymes tested, CAL-B showed the highest chemoselectivity,
leading to the formation of 4d in 97% yield within 15 min.
Acylase Amano and acylase I showed the worst chemoselec-
tivity for our purpose, resulting in the release of 1d in almost
quantitative yield. The other 14 enzymes were either low in
chemoselectivity or showed almost no catalytic activity toward
4d. Table 5 summarizes the results of hydrolytic cleavage of
4a-f with CAL-B or PLE.
Experimental Section
Materials. Optically active 2-(4-biphenylyl)propionic acids, (2R)-
2e and (2S)-2e, with optical purities being 98.2% and 99.2%
(reported by HPLC method), respectively, were kindly gifted from
Nagase ChemteX Corporation (Osaka, Japan) and were used without
further purification. Racemic 2-(4-biphenylyl)propionic acid (2e),31
methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-R-D-glucopyranuronate
(1),32 and 2, 3, 4, 6-tetra-O-acetyl-1-bromo-1-deoxy-R-D-glucopy-
(29) (a) Kadeleit, D.; Reents, R.; Jeyaraj, D. A.; Waldmann, H. Enzyme
catalysis in organic synthesis; Drauz, K., Waldmann, H., Eds.; Willey-
VCH, Weinheim, Germany, 2002; Vol. III, pp 1333-1417. (b) Kadereit,
D.; Waldmann, H. Chem. ReV. 2001, 101, 3367-3396. (c) Bornscheuer,
U. T.; Kazlauskas, R. J. Hydrolases in organic synthesis; Willey-VCH,
Weinheim, Germany, 1999; pp 131-172. (d) Ferrero, M.; Gotor, V. Chem.
ReV. 2000, 100, 4319-4347. (e) Bashir, N. B.; Phythian, S. J.; Reason, A.
J.; Roberts, S. M. J. Chem. Soc., Perkin Trans. I 1995, 2203-2222. (f)
Drueckhammer, D. G.; Hennen, W. J.; Pederson, R. L.; Barbas, C. F., III;
Gautheron, C. M.; Krach, T.; Wong, C.-H. Synthesis 1991, 499-525.
(30) Schramm, S.; Dettner, K.; Unverzagt, C. Tetrahedron Lett. 2006,
47, 7741-7743.
PLE showed high chemoselectivity toward 4b and 4f,
although the catalytic activity was lower than that of CAL-B
(data not shown). CAL-B-catalyzed hydrolysis of 4e provided
a 1:1 mixture of (2R)-5e and (2S)-5e, which were readily isolated
by preparative-HPLC with a C18 column (see the Experimental
Section); the retention times of these compounds isolated were
identical with those of compounds (2R)-5e and (2S)-5e obtained
(27) Barbayianni, E.; Fotakopoulou, I.; Schmidt, M.; Constantinou-
Kokotou, V.; Bornscheuer, U. T.; Kokotos, G. J. Org. Chem. 2005, 70,
8730-8733.
(31) Fujii, K.; Nakao, K.; Yamauchi, T. Synthesis 1982, 456-457.
(32) Bollenback, G. N.; Long, J. W.; Benjamin, D. G.; Lindquist, J. A.
J. Am. Chem. Soc. 1955, 77, 3310-3315.
(28) Akin, A.; Curran, T. T. Synth. Commun. 2005, 35, 1649-1661.
J. Org. Chem, Vol. 72, No. 25, 2007 9545

Baba and Yoshioka
1
ranose33 were synthesized according to the reported procedures.
Amberlite XAD-4 was used after grinding (80-200 mesh). The
17 commercially available enzymes, used in the screening for their
chemoselective hydrolytic activities, were as follows. Liver acetone
powder from equine, lipase from porcine pancreas, acylase I (from
Aspergillus sp.), pectin esterase from orange peel (PE), acylase
Amano (from Aspergillus sp.), lipase CAL-B (from Candida
Antarctica type B), lipase M Amano 10 (from Mucor jaVanicus),
lipase MML (from Mucor miehei), lipase from Phycomyces nitens,
lipase AK Amano (from Pseudomonas fluorescence), lipase AYS
Amano (from Candida rugosa), CSR (carboxylesterase from
Streptomyces rochei, crude), lipase AP6 (from Aspergillus niger),
lipase PS Amano (from Burkholderia cepacia), lipase R Amano
(from Penicillium roqueforti), newlase (from Rhizopus niVeus), and
lipase (from Candida cylindracea). All other chemicals used were
analytical grade commercial products.
196 (base peak); H NMR (400 MHz, DMSO-d6) δ 9.14 (s, 1H),
7.80 (dd, 1H, J ) 1.5 and 7.8 Hz), 7.45 (dt, 1H, J ) 2.0 and 8.8
Hz), 7.37 (t, 2H, J ) 8.3 Hz), 7.27 (d, 2H, J ) 7.3 Hz), 7.19 (d,
1H, J ) 8.3 Hz), 7.12 (t, 1H, J ) 7.3 Hz), 6.82 (t, 1H, J ) 7.3
Hz), 6.24 (d, 1H, J ) 7.8 Hz), 5.63 (t, 1H, J ) 9.8 Hz), 5.22 (dd,
1H, J ) 7.8 and 9.3 Hz), 5.11 (1H, t, J ) 9.3 Hz), 4.77 (d, 1H, J
) 9.5 Hz), 3.61 (s, 3H), 2.012 (s, 3H), 2.008 (s, 3H), 1.995 (s,
3H); 13C NMR (100 MHz, DMSO-d6) δ 169.4, 169.3, 169.2, 167.0,
165.2, 147.9, 139.8, 135.7, 131.3, 129.5, 123.8, 122.3, 117.7, 114.1,
109.6, 91.0, 71.4, 70.4, 69.5, 68.8, 52.6, 20.30, 20.26, 20.20.
Methyl 2,3,4-tri-O-acetyl-1-â-O-(4-phenyl)benzoyl-D-glucopy-
ranuronate (3c): mp 211-212 °C (white needles from EtOH).
Found: C, 60.79; H, 5.06; C26H26O11 requires C, 60.70; H, 5.09.
m/z (EI) 514 (M+), 317, 257, 181 (base peak); 1H NMR (400 MHz,
DMSO-d6) δ 8.00 (d, 2H, J ) 8.8 Hz), 7.88 (d, 2H, J ) 8.8 Hz),
7.76 (d, 2H, J ) 7.3 Hz), 7.51 (t, 2H, J ) 7.3 Hz), 7.44 (t, 1H, J
) 7.3 Hz), 6.28 (d, 1H, J ) 7.8 Hz), 5.62 (t, 1H, J ) 9.3 Hz), 5.21
(dd, 1H, J ) 7.8 and 9.3 Hz), 5.11 (1H, t, J ) 9.3 Hz), 4.79 (d,
1H, J ) 9.8 Hz), 3.62 (s, 3H), 2.01 (s, 6H), 1.98 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) δ 169.4, 169.3, 169.2, 167.0, 163.6, 145.7,
138.5, 130.2, 129.1, 128.6, 127.2, 127.0, 126.7, 91.4, 71.4, 70.4,
69.7, 68.8, 52.6, 20.3, 20.24, 20.21.
HPLC Analysis. Enzymatic reactions were analyzed with a
Shimadzu HPLC system, equipped with a column of Symmetry
C18 (5 µm, 4.6 × 150 mm2, Waters). Mobile phases used were
aqueous CH3CN (except for 3a and 4a with MeOH) containing 50
mM ammonium acetate (pH 4.5) and 10 mM tetra-n-butylammo-
nium bromide, as reported previously.20
Methyl 2,3,4-tri-O-acetyl-1-â-O-(4-phenyl)phenylacetyl-D-glu-
copyranuronate (3d): mp 149-151 °C (white needles from
EtOAc/Hexane). Found: C, 61.04; H, 5.35; C27H28O11 requires C,
61.36; H, 5.34. m/z (EI) 528 (M+), 468, 317, 257, 194, 167 (base
peak); 1H NMR (400 MHz, DMSO-d6) δ 7.66-7.60 (m, 4H), 7.48-
7.43 (m, 1H), 7.38-7.31 (m, 4H), 6.04 (d, 1H, J ) 8.2 Hz), 5.49
(t, 1H, J ) 9.6 Hz), 5.05-4.94 (m, 2H), 4.68 (d, 1H, J ) 9.9 Hz),
3.79 (s, 2H), 3.63 (s, 3H), 1.98 (s, 3H), 1.95 (s, 3H), 1.84 (s, 3H);
13C NMR (100 MHz, DMSO-d6) δ 169.41, 169.38, 169.25, 168.8,
166.9, 139.7, 139.0, 132.6, 129.9, 128.9, 127.4, 126.7, 126.6, 90.8,
71.4, 70.8, 69.7, 68.8, 52.6, 20.24, 20.17, 20.1.The methlene carbon
of 3d was overlapping with the solvent peaks of DMSO-d6.
Methyl 2,3,4-tri-O-acetyl-1-â-O-(2-(4-biphenylyl))propionyl-
D-glucopyranuronate (3e) (mixture of diastereoisomers): mp
118-122 °C (white needles from EtOH). Found: C, 61.92; H, 5.57;
C28H30O11 requires C, 62.03; H, 5.58. m/z (EI) 542 (M+), 482, 317,
257, 181 (base peak); 1H NMR (400 MHz, DMSO-d6) (mixture of
diastereoisomers) δ 7.65-7.60 (m, 4H), 7.45 (t, 2H, J ) 7.8 Hz),
7.37-7.30 (m, 3H), 6.05 (d, 0.5H, J ) 8.3 Hz), 5.99 (d, 0.5H, J )
8.3 Hz), 5.51 (t, 0.5H, J ) 9.8 Hz), 5.44 (t, 0.5H, J ) 9.8 Hz),
5.00 (t, 0.5H, J ) 9.8 Hz), 4.99 (dd, 0.5H, J ) 7.8 and 9.3 Hz),
4.97 (t, 0.5H, J ) 9.8 Hz), 4.87 (dd, 0.5H, J ) 8.3 and 9.8 Hz),
4.67 (d, 1H, J ) 9.8 Hz), 3.95-3.88 (m, 1H), 3.63 (s, 1.5H), 3.61
(s, 1.5H), 1.97 (s, 1.5H), 1.97 (s, 1.5H), 1.96 (s, 1.5H), 1.93 (s,
1H), 1.90 (s, 1.5H), 1.52 (s, 1.5H), 1.42 (d, 1.5H, J ) 6.8 Hz),
1.40 (d, 1.5H, J ) 6.8 Hz); 13C NMR (100 MHz, DMSO-d6) δ
172.0, 171.9, 169.4, 169.3, 169.2, 168.9, 168.3, 166.8, 139.67,
139.65, 139.18, 139.14, 138.9, 138.4, 128.93, 128.90, 128.1, 127.8,
127.4, 127.0, 126.9, 126.6, 126.5, 91.0, 90.8, 71.4, 70.7, 69.7, 69.4,
68.8, 52.60, 52.57, 43.93, 43.7, 20.3, 20.19, 20.15, 19.6, 18.0, 17.7.
Synthesis of 2-(4-Biphenylyl)-2-methylpropionic Acid (1f). A
solution of 1d (1.25 g, 5.9 mmol) in MeOH (10 mL) was refluxed
in the presence of a catalytic amount of TsOH (50 mg) for 1 h to
provide the corresponding methyl ester in 96% isolated yield, which
was treated with NaH (60%, 0.64 g, 16 mmol) in THF (18 mL)
followed by the dropwise addition of CH3I (0.83 mL, 13.3 mmol)
and then the mixture was stirred for overnight. After concentration
in vacuo, the residue was worked up with water and then the miture
was extracted with EtOAc (2 × 25 mL) to give the corresponding
dimethylated ester in 83% isolated yield. The ester was refluxed
in MeOH (15 mL) and 2 M NaOH (20 mL) for 3 h. After
concentration in vacuo, 1 M HCl (15 mL) was added to the residue
and the mixture was extracted with EtOAc (2 × 25 mL). The
combined organic layer was dried over Na2SO4 and then concen-
trated to provide 1f (1.13 g) in 80% overall yield from 1d: mp
175-176 °C (a white solid from benzene/hexane) (lit.34 mp 175-
177 °C); 1H NMR (400 MHz, CDCl3) δ 7.58-7.55 (m, 4H), 7.48
(d, 2H, J ) 8.3 Hz), 7.42 (t, 2H, J ) 7.8 Hz), 7.36-7.31 (m, 1H),
1.64 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 183.0, 142.8, 140.7,
139.9, 128.8, 127.3, 127.2, 127.1, 126.3, 46.1, 26.3.
General Procedure for the Synthesis of Methyl 2,3,4-Tri-O-
acetyl-1-â-O-acyl-D-glucopyranuronates (3a-f). These com-
pounds were readily synthesized within 2 h at room temperature
by the reaction of cesium salts of the corresponding carboxylic acids
(1a-f) with commercially available methyl 2,3,4-tri-O-acetyl-1-
bromo-1-deoxy-R-D-glucopyranuronate (2, 0.9 equiv) in DMSO as
reported previously.20 Analytical and spectral data of the products
are as follows.
Methyl 2,3,4-tri-O-acetyl-1-â-O-benzoyl-D-glucopyranuronate
(3a): mp 141-143 °C (white needles from EtOH). Found: C,
54.80; H, 5.08; C20H22O11 requires C, 54.80; H, 5.06. m/z (EI) 438
(M+), 378, 317, 228, 186, 157, 106, 77 (base peak); 1H NMR (400
MHz, DMSO-d6) δ 7.93 (dd, 2H, J ) 1.2 and 8.3 Hz), 7.72 (tt,
1H, J ) 1.2 and 7.3 Hz), 7.57 (t, 2H, J ) 7.3 Hz), 6.25 (d, 1H, J
) 8.1 Hz), 5.60 (t, 1H, J ) 9.3 Hz), 5.19 (dd, 1H, J ) 8.1 and 9.3
Hz), 5.10 (1H, t, J ) 9.3 Hz), 4.77 (d, 1H, J ) 9.5 Hz), 3.61 (s,
3H), 2.01 (s, 6H), 1.97 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
δ 169.4, 169.2, 169.1, 166.9, 163.7, 134.4, 129.5, 129.0, 127.9,
91.3, 71.4, 70.4, 69.7, 68.7, 52.5, 20.3, 20.2.
Methyl
2,3,4-tri-O-acetyl-1-â-O-{(2R)-2-(4-biphenylyl)}-
propionyl-D-glucopyranuronate ((2R)-3e): mp 90-92 °C (white
needles from EtOH). Found: C, 61.89; H, 5.56; C28H30O11 requires
C, 62.03; H, 5.58. m/z (EI) 542 (M+), 482, 317, 257, 181 (base
peak); 1H NMR (400 MHz, DMSO-d6) δ 7.63-7.60 (m, 4H), 7.45
(t, 2H, J ) 7.7 Hz), 7.37-7.30 (m, 3H), 5.99 (d, 1H, J ) 8.3 Hz),
5.44 (t, 1H, J ) 9.5 Hz), 4.98 (t, 1H, J ) 9.8 Hz), 4.87 (dd, 1H,
J ) 8.3 and 9.5 Hz), 4.67 (d, 1H, J ) 10.0 Hz), 3.92 (q, 1H, J )
7.1 Hz), 3.63 (s, 3H), 1.97 (s, 3H), 1.90 (s, 3H), 1.52 (s, 3H), 1.40
(d, 3H, J ) 7.1 Hz); 13C NMR (100 MHz, DMSO-d6) δ 172.0,
169.3, 169.2, 168.3, 166.8, 139.6, 139.1, 138.8, 128.9, 127.8, 127.4,
127.0, 126.5, 90.8, 71.4, 70.7, 69.4, 68.8, 52.6, 43.7, 20.2, 20.1,
19.6, 17.6.
Methyl 2,3,4-tri-O-acetyl-1-â-O-(2-phenylamino)benzoyl-D-
glucopyranuronate (3b): mp 154-155.5 °C (pale yellow needles
from EtOH). Found: C, 59.04; H, 5.08; N, 2.57; C26H27O11N
requires C, 58.98; H, 5.14; N, 2.65. m/z (EI) 529 (M+), 469, 213,
Methyl
2,3,4-tri-O-acetyl-1-â-O-{(2S)-2-(4-biphenylyl)}-
propionyl-D-glucopyranuronate ((2S)-3e): mp 152-154 °C (white
needles from EtOH). Found: C, 62.03; H, 5.59; C28H30O11 requires
C, 62.03; H, 5.58. m/z (EI) 542 (M+), 482, 317, 257, 181 (base
(33) Lemieux, R. U. Methods Carbohydr. Chem. 1963, 2, 221-222.
(34) Cromwell, N. H.; Hess, P. H. J. Am. Chem. Soc. 1961, 83, 1237-
1240.
9546 J. Org. Chem., Vol. 72, No. 25, 2007

Chemo-Enzymatic Synthesis of 1-â-O-Acyl Glucuronides
peak); 1H NMR (400 MHz, DMSO-d6) δ 7.65-7.60 (m, 4H), 7.47-
7.43 (m, 2H), 7.37-7.34 (m, 3H), 6.05 (d, 1H, J ) 8.3 Hz), 5.50
(t, 1H, J ) 9.5 Hz), 5.01 (t, 1H, J ) 9.8 Hz), 4.99 (dd, 1H, J ) 8.3
and 9.5 Hz), 4.67 (d, 1H, J ) 9.8 Hz), 3.91 (q, 1H, J ) 7.1 Hz),
3.61 (s, 3H), 1.97 (s, 3H), 1.96 (s, 3H), 1.93 (s, 3H), 1.43 (d, 3H,
J ) 7.1 Hz); 13C NMR (100 MHz, DMSO-d6) δ 171.8, 169.3, 169.2,
168.9, 166.7, 139.7, 139.2, 138.4, 128.9, 128.0, 127.4, 126.8, 126.6,
91.0, 71.4, 70.8, 69.7, 68.8, 52.5, 43.9, 20.2, 20.1, 18.0.
Methyl 2,3,4-tri-O-acetyl-1-â-O-{2-(4-biphenylyl)-2-methyl}-
propionyl-D-glucopyranuronate (3f): mp 165-166.5 °C (white
needles from EtOH). Found: C, 62.37; H, 5.83; C29H32O11 requires
C, 62.62; H, 5.80. m/z (EI) 556 (M+), 496, 257, 195 (base peak);
1H NMR (400 MHz, DMSO-d6) δ 7.65-7.61 (m, 4H), 7.46 (t, 2H,
J ) 7.3 Hz), 7.37-7.34 (m, 3H), 6.00 (d, 1H, J ) 8.3 Hz), 5.48
(dd, 1H, J ) 9.3 and 9.6 Hz), 4.98 (t, 1H, J ) 9.3 Hz), 4.91 (dd,
1H, J ) 8.3 and 9.3 Hz), 4.67 (d, 1H, J ) 9.8 Hz), 3.63 (s, 3H),
1.97 (s, 3H), 1.93 (s, 3H), 1.74 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 173.9, 169.3, 169.2, 168.6, 166.8, 142.4, 139.5, 138.8,
128.9, 127.4, 126.7, 126.5, 126.0, 91.1, 71.4, 70.7, 69.4, 68.8 52.6,
45.8, 25.9, 25.4, 20.2, 20.1, 19.9.
Screening of Enzymes for Chemoselective Hydrolysis of the
Model Substrates 3d and 4d. Each incubation was carried out at
the initial concentration of 0.1 mM for 3d and 4d in 25 mM sodium
citrate buffer (pH 5.0) containing 20% (v/v) DMSO as a cosolvent
at 40 °C. Each enzyme of the commercially available 17 enzymes
was added to the incubation mixture at the final amount of 10 mg/
mL of incubation mixture, regardless of their solubilities in the
incubation mixture. For enzyme assays, an aliquot of the incubation
mixture was appropriately diluted with an HPLC carrier and then
analyzed by the abovementioned reversed-phase HPLC. The
concentration of CH3CN in the mobile phase was 55% (v/v) and
35% (v/v) for 3d and 4d, respectively.
General Procedure for the Enzymatic Hydrolysis of Sub-
strates 3d and 3e to 4d and 4e: Methyl 1-â-O-(4-phenyl)-
phenylacetyl-D-glucopyranuronate (4d). To a solution of 3d (20.7
mg, 39.2 µmol) in DMSO (10 mL) was added a solution of CSR
(500 mg; 10 mg/ mL of incubation mixture) in 25 mM sodium
citrate buffer (pH 5.0) (40 mL) then the solution was stirred for
4.5 h at 50 °C. The conversion yield to 4d was 92% by HPLC
analysis and the amount of the liberated acid 1d was 7%. The
product 4d was isolated by passing through a short XAD-4 column
(1.5 g, 1.6 × 3 cm2), which had been washed thoroughly with
acetone and then equilibrated with water. After the incubation
mixture was loaded onto the column, the column was washed with
water (30 mL) and then 20% (v/v) aqueous CH3CN (30 mL). The
product 4d was eluted with 75% (v/v) aqueous CH3CN (30 mL)
with a recovery yield of 98% (by HPLC analysis). 4d: 1H NMR
(400 MHz, MeOH-d4) δ 7.61-7.55 (m, 4H), 7.43-7.29 (m, 5H),
5.52 (d, 1H, J ) 7.8 Hz), 3.95 (t, 1H, J ) 9.5 Hz), 3.79 (s, 2H),
3.75 (s, 3H), 3.54 (t, 1H, J ) 9.3 Hz), 3.47-3.38 (m, 2H); 13C
NMR (100 MHz, MeOH-d4) δ 172.0, 170.8, 142.1, 141.4, 134.0,
131.1, 129.8, 128.3, 128.1, 127.9, 95.9, 77.33, 77.28, 73.6, 72.9,
52.9, 41.1; m/z (SIMS, positive) [M + Na]+ 425.1225 (error 1.4
mmu). C21H22NO8Na requires m/z 425.1211.
3.91-3.87 (m, 2H), 3.66 (s, 1.5 H), 3.62 (s, 1.5H), 3.38-3.27 (m,
2H), 3.18-3.15 (m, 1H), 1.45 (d, 1.5H, J ) 6.6 Hz), 1.43 (d, 1.5H,
J ) 6.9 Hz); 13C NMR (100 MHz, DMSO-d6) δ 172.7, 172.6, 168.9,
168.8, 139.8, 139.2, 139.0, 138.9, 128.9, 128.1, 127.4, 126.9, 126.8,
126.60, 126.57, 94.5 75.9, 75.5, 75.3, 72.1, 72.0, 71.2, 52.0, 51.9,
44.0, 18.7, 18.5; m/z (EI, positive) [M]+ 416.1490 (error 2.0 mmu).
C22H24O8 requires m/z 416.1470.
Consecutive Enzymatic Hydrolysis for the Synthesis of 1-â-
O-Acyl-D-glucopyranuronates (5a-f): 1-â-O-Benzoyl-D-glu-
copyranuronate (5a). To a solution of 3a (39.8 mg, 93.3 µmol)
in DMSO (9.1 mL) was added a solution of CSR (456 mg; 10 mg/
mL of incubation mixture) in 25 mM sodium citrate buffer (pH
5.0) (37 mL) then the solution was stirred for 9 h at 50 °C. The
initial concentration of 3a was 2.00 mM and the conversion yield
to 4a was 82% by HPLC analysis. The yield was increased to 92%
for 4 h of incubation, when starting at the initial concentration of
3a of 1.00 mM. The product 4a was extracted with EtOAc (3 ×
60 mL) in a recovery yield of 98%. After removal of the organic
solvent in vacuo, the residue was taken up into DMSO (7.4 mL)
and then a solution of CALB (148 mg; 2 mg/mL of incubation
mixture) in 25 mM sodium citrate buffer (pH 5.0) (67 mL) was
added and the resulting solution was stirred for 2 h at 40 °C. The
conversion yield to 5a was 87% by HPLC analysis. The isolation
of 5a was performed by passing through a XAD-4 column (5 g,
1.6 × 10 cm2) as described above. The incubation mixture was
acidified to pH around 2.5 by the addition of 1 M HCl and then
the solution was loaded onto the column, which was then washed
with water (80 mL) followed by elution with 20% (v/v) aqueous
CH3CN. The fractions containing 5a were pooled and concentrated
in vacuo to give 5a in recovery yield of 93%: 1H NMR (400 MHz,
MeOH-d4) δ 8.11-8.08 (m, 2H), 7.63 (tt, 1H, J ) 1.2 and 7.6
Hz), 7.51-7.47 (m, 2H), 5.75 (d, 1H, J ) 7.8 Hz), 3.96 (d, 1H, J
) 9.3 Hz), 3.67-3.50 (m, 3H); 13C NMR (100 MHz, MeOH-d4) δ
172.6, 166.6, 134.8, 131.0, 130.6, 129.6, 96.1, 77.6, 77.2, 73.7,
73.0; m/z (SIMS, positive) [M + H]+ 299.0782 (error 1.6 mmu).
C13H15O8 requires m/z 299.0766.
1-â-O-(2-Phenylamino)benzoyl-D-glucopyranuronate (5b). To
a solution of 3b (53.0 mg, 100 µmol) in DMSO (20 mL) was added
a solution of LAS (1.00 g; 10 mg/mL of incubation mixture) in 25
mM sodium citrate buffer (pH 5.0) (80 mL) then the solution was
stirred for 6 h at 40 °C. The conversion yield to 4b was 92% by
HPLC analysis. The product 4b was almost quantitatively extracted
with EtOAc (3 × 60 mL). After removal of the organic solvent in
vacuo, the residue was taken up into DMSO (5 mL) and then a
solution of CALB (35 mg; 1 mg/mL of incubation mixture) in 25
mM sodium citrate buffer (pH 5.0) (30 mL) was added and the
resulting solution was stirred for 2.5 h at 40 °C. The conversion
yield to 5b was 98% by HPLC analysis. After acidification of the
incubation mixture with 1 M HCl to pH around 2.5, 5b was
extracted quantitatively with EtOAc (2 × 35 mL). The isolation of
5b was performed by passing through a XAD-4 column (2.5 g, 1.0
× 12 cm2) as described above. After removal of the organic solvent
in vacuo, the residue was taken up into water (8 mL) and then the
solution was loaded onto the column, which was then washed with
water (20 mL)and 30% CH3CN (20 mL) followed by elution with
50% (v/v) aqueous CH3CN. The fractions containing 5b were
pooled and concentrated in vacuo to give 5b in recovery yield of
98%: 1H NMR (400 MHz, MeOH-d4) δ 8.07 (dd, 1H, J ) 1.7 and
8.1 Hz), 7.36-7.32 (m, 3H), 7.24-7.19 (m, 3H), 7.09 (dt, 1H, J
) 1.7 and 7.3 Hz), 6.75 (t, 1H, J ) 8.1 Hz), 5.77 (d, 1H, J ) 7.6
Hz), 3.99 (d, 1H, J ) 9.5 Hz), 3.64-3.51 (m, 3H); 13C NMR (100
MHz, MeOH-d4) δ 172.2, 168.1, 149.8, 141.8, 135.9, 133.1, 130.5,
125.0, 123.7, 118.2, 114.9, 112.0, 95.7, 77.6, 77.2, 73.7, 73.0; m/z
(SIMS, positive) [M + H]+ 390.1139 (error -0.6 mmu). C19H20-
NO8 requires m/z 390.1187.
Methyl 1-â-O-(2-(4-Biphenylyl))propionyl-D-glucopyranur-
onate (4e) (Mixtures of Diastereoisomers). To a solution of 3e
(27.1 mg, 50.0 µmol) in DMSO (20 mL) was added a solution of
CSR (1.00 g; 10 mg/ mL of incubation mixture) in 25 mM sodium
citrate buffer (pH 5.0) (80 mL) then the solution was stirred for 6
h at 50 °C. The conversion yield to 4e was 96% by HPLC analysis.
The incubation mixture was successfully extracted with EtOAc (2
× 100 mL) and the combined organic layer was dried over Na2-
SO4. After removal of the solvent in vacuo, the residue was purified
by recrystallization from CH3CN. 4e: 1H NMR (400 MHz, DMSO-
d6) (mixture of diastereoisomers) δ 7.65-7.60 (m, 4H), 7.47-7.33
(m, 5H), 5.47 (d, 0.5H, J ) 5.4 Hz, OH), 5.45 (d, 0.5H, J ) 8.3
Hz), 5.44 (d, 0.5H, J ) 8.1 Hz), 5.40 (d, 0.5H, J ) 5.6 Hz, OH),
5.39 (d, 0.5H, J ) 5.6 Hz, OH), 5.33 (d, 0.5H, J ) 5.6 Hz, OH),
5.27 (d, 0.5H, J ) 5.1 Hz, OH), 5.25 (d, 0.5H, J ) 5.4 Hz, OH),
1-â-O-(4-Phenyl)benzoyl-D-glucopyranuronate (5c). To a solu-
tion of 3c (41.2 mg, 80.0 µmol) in DMSO (8 mL) was added a
solution of LAS (400 mg; 10 mg/mL of incubation mixture) and
CSR (400 mg, 10 mg/mL of incubation mixture) in 25 mM sodium
J. Org. Chem, Vol. 72, No. 25, 2007 9547

Baba and Yoshioka
citrate buffer (pH 5.0) (32 mL) then the solution was stirred for 9
h at 40 °C. The conversion yield to 4c was 93% by HPLC analysis.
The product 4c was quantitatively extracted with EtOAc (2 × 50
mL). After removal of the organic solvent in vacuo, the residue
was taken up into DMSO (15 mL) and then a solution of CALB
(76 mg; 1 mg/mL of incubation mixture) in 25 mM sodium citrate
buffer (pH 5.0) (61 mL) was added and the resulting solution was
stirred for 9 h at 40 °C. The conversion yield to 5c was 93% by
HPLC analysis. After acidification of the incubation mixture with
1 M HCl to pH around 2.5, the solution was loaded onto a XAD-4
column (5 g, 1.6 × 10 cm2) as described above. The column was
washed with water (100 mL) and 30% CH3CN (50 mL) followed
by elution with 50% (v/v) aqueous CH3CN. The fractions containing
5c were pooled and concentrated in vacuo to give 5c in recovery
yield of 98%: 1H NMR (400 MHz, MeOH-d4) δ 8.17 (d, 2H, J )
8.8 Hz), 7.76 (d, 2H, J ) 8.8 Hz), 7.68 (d, 2H, J ) 8.8 Hz), 7.47
(d, 2H, J ) 7.4 Hz), 7.41-7.37 (m, 1H), 5.77 (d, 1H, J ) 7.8 Hz),
3.98 (d, 1H, J ) 9.5 Hz), 3.63-3.51 (m, 3H); 13C NMR (100 MHz,
MeOH-d4) δ 172.4, 166.4, 147.7, 141.1, 131.6, 130.1, 129.4, 129.3,
128.3, 128.1, 96.1, 77.6, 77.3, 73.8, 73.0; m/z (SIMS, positive) [M
+ H]+ 375.1093 (error 1.4 mmu). C19H19O8 requires m/z 375.1079.
1-â-O-(4-Phenyl)phenylacetyl-D-glucopyranuronate (5d). To
a solution of 3d (45.4 mg, 85.9 µmol) in DMSO (12 mL) was added
a solution of CSR (570 mg, 10 mg/mL of incubation mixture) in
25 mM sodium citrate buffer (pH 5.0) (45 mL) then the solution
was stirred for 7.5 h at 50 °C. The conversion yield to 4d was
86% and the ratio of liberated 1d was 10% by HPLC analysis.
The product 4d was quantitatively extracted with EtOAc (2 × 50
mL). After removal of the organic solvent in vacuo, the residue
was taken up into DMSO (15 mL) and then a solution of CALB
(59 mg; 0.8 mg/mL of incubation mixture) in 25 mM sodium citrate
buffer (pH 5.0) (59 mL) was added and the resulting solution was
stirred for 2 h at 40 °C to provide quantitatively 5d by HPLC
analysis. After acidification of the incubation mixture with 1 M
HCl to pH around 2.5, the solution was loaded onto a XAD-4
column (5 g, 1.6 × 10 cm2) as described above. The column was
washed with water (100 mL) and 20% CH3CN (100 mL) followed
by elution with 45% (v/v) aqueous CH3CN. The fractions containing
5d were pooled and concentrated in vacuo to provide quantitatively
5d: 1H NMR (400 MHz, MeOH-d4) δ 7.59-7.57 (m, 2H), 7.56-
7.54 (m, 2H), 7.40 (t, 2H, J ) 7.3 Hz), 7.37 (d, 2H, J ) 8.3 Hz),
7.30 (tt, 1H, J ) 1.2 and 7.3 Hz), 5.55 (d, 1H, J ) 7.8 Hz), 3.91
(d, 1H, J ) 9.8 Hz), 3.79 (s, 2H), 3.55 (t, 1H, J ) 9.0 Hz), 3.47
(t, 1H, J ) 9.0 Hz), 3.43 (t, 1H, J ) 9.0 Hz); 13C NMR (100 MHz,
MeOH-d4) δ 172.13, 172.06, 142.1, 141.3, 134.0, 131.1, 129.8,
128.3, 128.0, 127.9, 95.8, 77.5, 77.2, 73.6, 72.9, 41.0; m/z (SIMS,
positive) [M + H]+ 389.1262 (error 2.7 mmu). C20H21O8 requires
m/z 389.1235.
and both the compounds were fully separated to provide sufficiently
pure compounds (2R)-5e and (2S)-5e.
1-â-O-{(2R)-2-(4-Biphenylyl)}propionyl-D-glucopyranur-
onate ((2R)-5e). To a solution of (2R)-3e (40.0 mg, 73.7 µmol) in
DMSO (15 mL) was added a solution of CSR (740 mg; 10 mg/mL
of incubation mixture) in 25 mM sodium citrate buffer (pH 5.0)
(59 mL) then the solution was stirred for 30 h at 50 °C. The
conversion yield to (2R)-4e was 89% by HPLC analysis. The
product (2R)-4e was quantitatively extracted with EtOAc (2 × 75
mL). After removal of the organic solvent in vacuo, the residue
was taken up into DMSO (12.5 mL) and then a solution of CALB
(26 mg; 0.4 mg/mL of incubation mixture) in 25 mM sodium citrate
buffer (pH 5.0) (53 mL) was added and the resulting solution was
stirred for 1 h at 40 °C to provide quantitatively the product (2R)-
5e by HPLC analysis. After acidification of the incubation mixture
with 1 M HCl to pH around 2.5, (2R)-5e was quantitatively
extracted with EtOAc (2 × 50 mL). 1H NMR (400 MHz, MeOH-
d4) δ 7.54-7.49 (m, 4H), 7.37-7.33 (m, 4H), 7.27-7.23 (m, 1H),
5.46 (d, 1H, J ) 7.8 Hz), 3.85 (d, 1H, J ) 9.5 Hz), 3.83 (d, 1H,
J ) 7.3 Hz), 3.48 (t, 1H, J ) 8.8 Hz), 3.36 (t, 1H, J ) 9.0 Hz),
3.30 (dd, 1H, J ) 8.3 and 9.0 Hz), 1.48 (d, 3H, J ) 7.3 Hz); 13C
NMR (100 MHz, MeOH-d4) δ 174.8, 172.1, 142.0, 141.4, 140.5,
129.8, 129.2, 128.3, 128.2, 127.9, 95.8, 77.6, 77.2, 73.6, 72.9, 46.2,
19.1; m/z (SIMS, positive) [M + H]+ 403.1394 (error 0.2 mmu).
C21H23O8 requires m/z 403.1392.
1-â-O-{(2S)-2-(4-Biphenylyl)}propionyl-D-glucopyranur-
onate ((2S)-5e). To a solution of (2S)-3e (40.0 mg, 73.7 µmol) in
DMSO (15 mL) was added a solution of CSR (740 mg; 10 mg/mL
of incubation mixture) in 25 mM sodium citrate buffer (pH 5.0)
(59 mL) then the solution was stirred for 3 h at 50 °C. The
conversion yield to (2S)-4e was 98% by HPLC analysis. The
product (2R)-4e was quantitatively extracted with EtOAc (2 × 75
mL). After removal of the organic solvent in vacuo, the residue
was taken up into DMSO (7.2 mL) and then a solution of CALB
(14.4 mg; 0.4 mg/mL of incubation mixture) in 25 mM sodium
citrate buffer (pH 5.0) (29 mL) was added and the resulting solution
was stirred for 3.5 h at 40 °C to provide quantitatively the product
(2S)-5e by HPLC analysis. After acidification of the incubation
mixture with 1 M HCl to pH around 2.5, (2S)-5e was quantitatively
extracted with EtOAc (2 × 50 mL). 1H NMR (400 MHz, MeOH-
d4) δ 7.53-7.47 (m, 4H), 7.35-7.29 (m, 4H), 7.25-7.21 (m, 1H),
5.45 (d, 1H, J ) 7.8 Hz), 3.82 (d, 1H, J ) 7.1 Hz), 3.80 (d, 1H,
J ) 8.8 Hz), 3.43 (t, 1H, J ) 8.8 Hz), 3.37 (t, 1H, J ) 8.8 Hz),
3.30 (t, 1H, J ) 8.3 Hz), 1.45 (d, 3H, J ) 7.1 Hz); 13C NMR (100
MHz, MeOH-d4) δ 174.7, 172.2, 142.1, 141.4, 140.6, 129.8, 129.2,
128.3, 128.2, 127.9, 95.8, 77.5, 77.3, 73.6, 72.9, 46.2, 19.2; m/z
(SIMS, positive) [M + H]+ 403.1367 (error -2.5 mmu). C21H23O8
requires m/z 403.1392.
1-â-O-(2-(4-Biphenylyl))propionyl-D-glucopyranuronate (5e)
(Mixture of Diastereoisomers). To a solution of 3e (27.1 mg, 50.0
µmol) in DMSO (20 mL) was added a solution of CSR (1.00 g, 10
mg/mL of incubation mixture) in 25 mM sodium citrate buffer (pH
5.0) (80 mL) then the solution was stirred for 6.0 h at 50 °C to
provide 4e (96%) by HPLC analysis. The product 4e was
quantitatively extracted with EtOAc (2 × 100 mL). After removal
of the organic solvent in vacuo, the residue was taken up into
DMSO (10 mL) and then a solution of CAL-B (19 mg; 0.4 mg/
mL of incubation mixture) in 25 mM sodium citrate buffer (pH
5.0) (38 mL) was added and the resulting solution was stirred for
45 min at 40 °C to provide 5e (99%) by HPLC analysis. After
acidification of the incubation mixture with 1 M HCl to pH around
2.5, 5e was quantitatively extracted with EtOAc (2 × 50 mL). After
removal of the organic solvent in vacuo, the residue was loaded
onto a semipreparative column of Symmetry C18 (7 µm, 19 × 150
mm2, Waters) to isolate the diastereoisomers (2R)-5e and (2S)-5e,
with the HPLC mobile phase being 30% (v/v) CH3CN containing
10 mM ammonium acetate (pH 4.5) and 1 mM tetra-n-butylam-
monium bromide at a flow rate of 3 mL/min with detection at 260
nm. The retention time of (2S)-5e was shorter than that of (2R)-5e
1-â-O-{2-(4-Biphenylyl)-2-methyl}propionyl-D-glucopyranu-
ronate (5f). To a solution of 3f (40.0 mg, 71.9 µmol) in DMSO
(14 mL) was added a solution of CSR (720 mg; 10 mg/mL of
incubation mixture) and LAS (720 mg; 10 mg/mL of incubation
mixture) in 25 mM sodium citrate buffer (pH 5.0) (58 mL) then
the solution was stirred for 3 h at 40 °C. The conversion yield to
4f was 95% by HPLC analysis. The product 4f was quantitatively
extracted with EtOAc (2 × 70 mL). After removal of the organic
solvent in vacuo, the residue was taken up into DMSO (7 mL) and
then a solution of CALB (29 mg; 0.8 mg/mL of incubation mixture)
in 25 mM sodium citrate buffer (pH 5.0) (29 mL) was added and
the resulting solution was stirred for 3 h at 40 °C to provide
quantitatively 5f by HPLC analysis. After acidification of the
incubation mixture with 1 M HCl to pH around 2.5, (2S)-5e was
1
quantitatively extracted with EtOAc (2 × 35 mL). H NMR (400
MHz, MeOH-d4) δ 7.60-7.54 (m, 4H), 7.46-7.38 (m, 4H), 7.32-
7.28 (m, 1H), 5.54 (d, 1H, J ) 8.1 Hz), 3.90 (d, 1H, J ) 9.8 Hz),
3.52 (t, 1H, J ) 9.0 Hz), 3.43 (t, 1H, J ) 9.0 Hz), 3.34 (t, 1H, J
) 8.1 Hz), 1.64 (s, 3H), 1.61 (s, 3H); 13C NMR (100 MHz, MeOH-
d4) δ 176.9, 172.1, 144.7, 142.0, 140.9, 129.8, 128.3, 127.91,
127.86, 127.5, 96.0, 77.7, 77.3, 73.5, 72.9, 47.8, 27.2, 26.9; m/z
9548 J. Org. Chem., Vol. 72, No. 25, 2007

Chemo-Enzymatic Synthesis of 1-â-O-Acyl Glucuronides
(SIMS, positive) [M + H]+ 417.1522 (error -2.5 mmu). C22H25O8
requires m/z 417.1547.
EtOAc (3 × 40 mL) in recovery yield of 96%. After removal of
the organic solvent in vacuo, the residue was loaded onto a XAD-4
column (1 g, 1.0 × 8 cm2) as described above. The column was
washed with water (20 mL) and then eluted with 30% (v/v) aqueous
CH3CN. The fractions containing 7 were pooled and concentrated
in vacuo to provide quantitatively 7: 1H NMR (400 MHz, MeOH-
d4) δ 7.64-7.59 (m, 3H), 7.31 (dd, 1H, J ) 2.0 and 8.5 Hz), 7.10
(d, 1H, J ) 2.4 Hz), 7.01 (dd, 1H, J ) 2.4 and 9.0 Hz), 5.41 (d,
1H, J ) 8.1 Hz), 3.85 (1H, q, J ) 7.1 Hz), 3.80 (s, 3H), 3.64 (dd,
1H, J ) 2.0 and 12.2 Hz), 3.51 (dd, 1H, J ) 4.4 and 12.2 Hz),
3.33-3.20 (m, 4H), 1.47 (d, 3H, J ) 7.1 Hz); 13C NMR (100 MHz,
MeOH-d4) δ 175.1, 159.2, 136.6, 135.3, 130.4, 130.2, 128.3, 127.3,
127.2, 119.9, 106.6, 96.1, 78.9, 78.1, 74.0, 70.9, 62.2, 55.7, 46.5,
19.2; m/z (SIMS, positive) [M + H]+ 393.1543 (error -0.4 mmu).
C20H25O8 requires m/z 393.1547.
Assay of Purity of 1-â-O-Acyl Glucuronides 5a-f and 1-â-
O-Acyl Glucopyranoside 7. Compounds 5a-f and 7 were treated
with 0.1 M NaOH at 37 °C for 15 min to complete the hydrolysis
of the ester linkage and the amounts of the corresponding carboxylic
acids 1a-f and NP formed were determined by HPLC. In addition,
the same amounts of the glucuronides 5a-f and 7 were incubated
with â-glucuronidase (from bovine liver) and â-glucosidase (from
almond), respectively, in 100 mM AcOH/NaOH buffer (pH 5.0) at
37 °C for 30 min and the amounts of liberated carboxylic acids
were also determined as described previously.20 Purities of the
glucuronides 5a-f and 7 as 1-â-O-acyl glucuronides and 1-â-O-
acyl glucopyranoside, respectively, were calculated from the
amounts of the corresponding carboxylic acids formed under both
sets of conditions. The calculated purities (%) of these compounds
were 99.8 ( 1.1 for 5a, 98.3 ( 0.2 for 5b, 99.9 ( 0.9 for 5c, 96.6
( 0.5 for 5d, 94.2 ( 1.3 for (2R)-5e, 96.1 ( 1.8 for (2S)-5e, 98.2
( 0.4 for 5f, and 97.4 ( 1.2 for 7.
2,3,4,6-Tetra-O-acetyl-D-1-â-O-(6-methoxy-R-methyl-2-naph-
thaleneacetyl)- glucopyranoside (6). To a solution of (S)-naproxen
(247 mg, 1.07 mmol) in MeOH (2 mL) was added an aqueous 3
M Cs2CO3 solution (178 µL, 1.07 mmol) then the solution was
stirred for 5 min. After removal of the solvent in vacuo, the residue
was dissolved in DMSO (2.5 mL) and then 2,3,4,6-tetra-O-acetyl-
1-bromo-1-deoxy-R-D-glucopyranose (352 mg, 0.8 equiv) was
added then the resulting solution was stirred for 1 h at room
temperature and HPLC assay showed the reaction was complete.
The reaction mixture was diluted with EtOAc (50 mL) and the
organic layer was washed with water (30 mL) and then aqueous
2% Na2CO3 solution (2 × 30 mL). After being dried over Na2SO4,
the organic solvent was removed in vacuo to give a crude product,
which was purified by recrystallization. Yield 51%; mp 181-182 °C
(white needles from EtOH). Found: C, 60.05; H, 5.80; C28H32O12
requires C, 60.03; H, 5.76. m/z (EI) 560 (M+), 331, 272, 185 (base
1
peak); H NMR (400 MHz, DMSO-d6) δ 7.78 (d, 2H, J ) 8.8
Hz), 7.70 (d, 1H, J ) 1.7 Hz), 7.36 (dd, 1H, J ) 1.7 and 8.8 Hz),
7.30 (d, 1H, J ) 2.4 Hz), 7.16 (dd, 1H, J ) 2.4 and 8.8 Hz), 6.00
(d, 1H, J ) 8.3 Hz), 5.43 (t, 1H, J ) 9.5 Hz), 4.94 (t, 1H, J ) 9.5
Hz), 4.93 (dd, 1H, J ) 8.3 and 9.8 Hz), 4.21 (ddd, 1H, J ) 2.2,
4.6, and 12.2 Hz), 4.15 (dd, 1H, J ) 4.6 and 12.2 Hz), 4.00-3.94
(m, 1H), 3.97 (q, 1H, J ) 7.1 Hz), 3.86 (s, 3H), 1.97 (s, 3H), 1.96
(s, 3H), 1.93 (s, 3H), 1.87 (s, 3H), 1.47 (d, 3H, J ) 7.1 Hz); 13C
NMR (100 MHz, DMSO-d6) δ 172.1, 169.9, 169.4, 169.1, 168.9,
157.3, 134.4, 133.4, 129.1, 128.3, 127.0, 126.2, 125.9, 118.8, 105.7,
91.2, 71.6, 71.4, 70.0, 67.6, 61.3, 55.2, 44.2, 20.4, 20.3, 20.2, 20.1,
18.1.
1-â-O-(6-Methoxy-R-methyl-2-naphthaleneacetyl)-D-glucopy-
ranoside (7). To a solution of 6 (20.2 mg, 36.0 µmol) in DMF (9
mL) was added a solution of CSR (360 mg; 10 mg/mL of incubation
mixture) in 25 mM sodium citrate buffer (pH 5.0) (27 mL) then
the solution was stirred for 40 h at 50 °C. The conversion yield to
7 was 64% by HPLC analysis. Other products were the corre-
sponding mono-O-acetates (total 34%). After adding NaCl to
saturate the reaction mixture, compound 6 was extracted with
Supporting Information Available: 1H and 13C NMR spectra
of compounds 3a-f, 4d, 4e, 5a-f, 6, and 7. This material is
available free of charge via the Internet at http://pubs.acs.org.
JO701547B
J. Org. Chem, Vol. 72, No. 25, 2007 9549