Synthesis of 1-β-O-acyl glucuronides of diclofenac, mefenamic acid and (S)-naproxen by the chemo-selective enzymatic removal of protecting groups from the corresponding methyl acetyl derivatives

Article abstract of DOI:10.1039/b608755h

Using a straightforward chemo-enzymatic procedure, 1-β-O-acyl glucuronides of three non-steroidal anti-inflammatory drugs, diclofenac (DF)5, mefenamic acid (MF)6 and (S)-naproxen(NP)7, were prepared. Caesium salts of these carboxylic acid drugs reacted wi

Full text of DOI:10.1039/b608755h

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of 1-b-O-acyl glucuronides of diclofenac, mefenamic acid and
(S)-naproxen by the chemo-selective enzymatic removal of protecting groups
from the corresponding methyl acetyl derivatives
Akiko Baba and Tadao Yoshioka*
Received 20th June 2006, Accepted 5th July 2006
First published as an Advance Article on the web 26th July 2006
DOI: 10.1039/b608755h
Using a straightforward chemo-enzymatic procedure, 1-b-O-acyl glucuronides of three non-steroidal
anti-inflammatory drugs, diclofenac (DF) 5, mefenamic acid (MF) 6 and (S)-naproxen (NP) 7, were
prepared. Caesium salts of these carboxylic acid drugs reacted with commercially available methyl
2,3,4-tri-O-acetyl-1-bromo-1-deoxy-a-D-glucopyranuronate 4 to give exclusively the corresponding
1-b-O-acyl glucuronides 8–10 in moderate yields. The protecting acetyl (for –OH group) and methyl
ester (for –CO2H group) groups of each sugar moiety were easily removed to provide the corresponding
free 1-b-O-acyl glucuronides 1–3 in high yields. Deprotection was achieved through effective
enzyme-catalysed chemo-selective hydrolyses of the acetyl groups using lipase AS Amano (LAS), and
of the methyl ester group using esterase from porcine liver (PLE).
Introduction
Much attention has been focused on the metabolic activation
of drugs to reactive metabolites, on the covalent binding of
these metabolites to target macromolecules (proteins and/or
DNA), and on the toxicity of these metabolites under certain
physiological conditions such as necrotic injury, idiosyncratic
reaction and cancer.1–7 1-b-O-Acyl glucuronides are such chem-
Such toxicological and chemical studies require an efficient
and widely applicable synthetic methodology for preparing pure
1-b-O-acyl glucuronides. To our knowledge, however, there is
no convenient synthetic methodology to replace biosynthetic
methods using liver microsomes fortified with UDPGA,24,26 or
ically reactive metabolites, associated with carboxylic acids such
as non-steroidal anti-inflammatory carboxylic acid drugs. Several
isoforms of UDP-glucuronosyltransferase, which are responsible
for the formation of these reactive 1-b-O-acyl glucuronides, have
the purification of 1-b-O-acyl glucuronides from urinary27 and/or
biliary28 metabolites. Chemical synthetic methods reported to date
can be divided into three categories based on the source of the
glucuronosyl starting materials. The first method utilises benzyl
2,3,4-tri-O-benzylglucuronate,29 with which carboxylic acids are
conjugated primarily using either the Mitsunobu reaction30 or
trichloroacetimidate method.31 The benzyl group is easily removed
by hydrogenolysis without affecting the anomeric acyl functional
group. This method, however, requires a multi-step process for
preparing the starting benzylated glucuronate, and generally gives
a mixture of anomers of 1-O-acyl glucuronide. The second method
utilises unprotected glucuronic acid, which is acylated with N-
acylimidazoles.23,32 However, except for the specific case of a
retinoylation reaction,32 reported yields are relatively low. The
third method utilises allyl glucuronate, which is acylated with
carboxylic acids by the Mitsunobu reaction or the HATU–NMM
procedure.33,34 This approach produces the desired products with
high b/a ratios, but the overall yields are unsatisfactory.
been identified.8,9 1-b-O-Acyl glucuronides are generally labile at
physiological pH and are known not only to undergo ester-bond
hydrolysis and intramolecular acyl migration, but also to bind
covalently to proteins and other nucleophilic components.10–15
It has been postulated that the covalent binding of 1-b-O-acyl
glucuronides may disrupt the function of a critical protein and/or
cause hypersensitivity. This possibility is based largely on the
hapten hypothesis: some 1-b-O-acyl glucuronides are believed to
be responsible for the adverse effects, such as hypersensitivity and
cellular toxicity, of parent drugs.11,12,16–18 Two mechanisms have
been proposed to explain the covalent binding of 1-b-O-acyl glu-
curonides to proteins,19,20 although the toxicological consequences
of covalent binding remain ambiguous. 1-b-O-Acyl glucuronides
have therefore attracted considerable toxicological and chemical
interest regarding their structure–activity relationship.21–25 The
ability of 1-b-O-acyl glucuronides to bind to proteins is of
particular interest for providing further insights into their intrinsic
toxicities.
Herein we report a facile chemo-enzymatic approach for the
preparation of 1-b-O-acyl glucuronides 1, 2 and 3 from com-
mercially available methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-a-
D-glucopyranuronate 4 and the corresponding non-steroidal anti-
inflammatory drugs (NSAIDs), namely: diclofenac (DF) 5, mefe-
namic acid (MF) 6 and (S)-naproxen (NP) 7 (Fig. 1), respectively.
Department of Pharmacy, Hokkaido Pharmaceutical University School of
Pharmacy, 7-1 Katsuraoka-cho, Otaru, 047-0264, Hokkaido, Japan. E-mail:
yoshioka@hokuyakudai.ac.jp; Fax: +81 134 62 5161; Tel: +81 134 62 1894
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1
Table 1 Yields of 8–10 and H-NMR data of the anomeric protons of
the crude products
NSAID
Product
Yield (%)
C1-H/ppma (J/Hz)
5
6
7
8
9
10
79
74
46
5.81 (7.6)
5.99 (7.3)
5.75 (7.6)
a
1H-Chemical shifts of C1-H (anomeric protons) are presented as d values
in CDCl3.
the formation of a-anomers was detected, indicating that this
glucuronidation method gave exclusively the expected 1-b-O-acyl
glucuronide derivatives.
Fig. 1 Structures of NSAIDs used.
Similar stereo-selectivity, probably due to participation of the
neighbouring 2-O-acetyl group, has been reported in the reac-
tions of 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bromide with
caesium carboxylates38 in DMF and with sodium carboxylates39
in the presence of a phase transfer catalyst to yield 1-b-O-acyl-
2,3,4,6-tetra-O-acetyl-D-glucopyranosides. In addition, bromide
4 has been reacted with a carboxylic acid40 using Ag2O. In
this latter approach, the resulting methyl 1-b-O-acyl-2,3,4-tri-
O-acetyl-D-glucopyranuronate was then chemically deacetylated,
followed by an enzymatic hydrolysis of the methyl ester to give the
corresponding free 1-b-O-acyl glucuronide in an overall yield of
ca. 45%.40
In the first glucuronidation step, methyl acetyl derivatives of 1-b-O-
acyl glucuronides 8–10 were obtained easily and stereo-specifically.
In the second step, enzymatic deprotection was performed with
high chemo-selectivity using the commercially available lipase AS
Amano (LAS) and esterase from porcine liver (PLE), as shown in
Scheme 1.
Results and discussion
Synthesis of methyl acetyl derivatives of 1-b-O-acyl glucuronides
8–10
Enzymatic deprotection of 8–10
Complete anomeric stereocontrol is critical for the synthesis
of 1-b-O-acyl glucuronides 1–3. As indicated in reviews of
carbohydrates,35,36 the most reliable method for constructing 1,2-
trans-glycosidic linkages is to utilise neighbouring-group par-
ticipation from the 2-O-acyl functionality. Therefore, the glu-
curonidation of DF 5, MF 6 and NP 7 was attempted using
commercially available bromide 4. Reactivity of the sodium salt of
DF with 4 to provide 8 was first examined in several solvents
(DMSO, DMF, diglyme, HMPA, 1,4-dioxane and MeOH) at
30 ◦C. DMSO was found to be the solvent of choice, whereas
the reaction rate was very reduced in MeOH. Among the counter
cations examined (sodium, caesium and tetraethylammonium),
the caesium salt of DF in DMSO gave the best yield of the
corresponding methyl acetyl derivative 8. Thus, as shown in
Scheme 1, treatment of 4 with the caesium salts of the carboxylic
acids 5–7 (1.5 equivalents) in DMSO at 30 ◦C for 3 h gave the
corresponding methyl acetyl derivatives 8–10 in moderate yields
(Table 1).
1H-NMR spectra (in CDCl3) of these crude reaction products
were obtained in order to check the product ratio of a- to b-
anomers. 1H-Chemical shifts of the crude products are also listed
in Table 1; each anomeric proton was observed as a doublet
at around d 5.8, with J values of approximately 7.5 Hz. From
their coupling constants, the stereochemistry of products 8–10
was assigned to the b-configuration.37 No signal attributed to
The above reaction conditions yielded 1-b-O-acyl glucuronides 8–
10 exclusively in moderate yields. The important issue of how
to remove the protecting groups in these compounds without
affecting their 1-b-O-acyl linkages remained. Recently, enzymes
with high chemo-, regio- and/or stereo-selectivity have been
employed as biocatalysts in organic reactions and many reviews
of this research area have been published.41
Therefore, enzymes were screened in order to identify candidates
capable of chemo-selectively hydrolysing the O-acetyl groups
and/or the methyl ester group of 8–10. Various enzymes including
lipases and PLE were tested for the chemo-selective hydrolysis of 8
(chosen as the model substrate). Incubations were conducted using
0.5 mM 8 in 25 mM sodium citrate buffer (pH 5.0) containing
20% (v/v) DMSO as a co-solvent at 40
0.1 ◦C for 30 min.
Each commercially available enzyme was added to the incubation
mixture at a final concentration of 5 mg cm−3. The incubation
was performed at pH 5.0 because the 1-b-O-acyl linkages of the
corresponding partially and fully deprotected compounds derived
from 8 were much less labile to both hydrolysis and intramolecular
acyl migration at slightly acidic pH. Similar findings have been
reported for other 1-b-O-acyl glucuronides.15,42
Of 11 enzymes tested, LAS and PLE showed hydrolytic activity
toward 8, whereas other enzymes did not show any hydrolytic
activity toward 8 under the conditions used. LAS gave several
Scheme 1 Chemo-enzymatic synthesis of 1-b-O-acyl glucuronides.
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Table 2 Reaction conditions and yields of LAS-catalysed hydrolyses of
8–10 to provide 12–14
major peaks on reversed-phase HPLC analysis, probably corre-
sponding to partially and fully deacetylated compounds. After
4 h incubation, a major peak corresponding to methyl ester
glucuronide 12 was detected, whereas PLE gave a major peak
which was shown to correspond to tri-O-acetyl derivative 11, as
shown in Scheme 2. The hydrolytic activities of LAS and PLE
were examined in more detail. The effect of co-solvents, including
DMSO, on LAS-catalysed hydrolysis of 8 was first investigated at
a concentration of 20% (v/v) in the incubation mixture. CH3CN
and tetrahydrofuran were ineffective at solubilising the substrate.
2-Methoxyethanol, DMF and t-BuOH were less effective in
stimulating the hydrolytic reaction than DMSO. Fig. 2 shows
chromatograms for the LAS-catalysed hydrolysis of 8 using
DMSO as the co-solvent. The substrate (peak A, Fig. 2a) was
fully deacetylated to provide methyl ester glucuronide 12 (peak D,
Fig. 2b) after 5 h incubation; DF (peak E, Fig. 2b) was detected as a
Conditions
Substrate Yield (%) pH (buffer)
Co-solvent (%, v/v a)
8
94
90
90
5.0 (Cib/NaOH)
6.0 (Pib/NaOH)
DMSO (20)
DMSO (20)
9
10
5.5 (Ci–Pib/NaOH) 2-Methoxyethanol (20)
a Percentage of the co-solvent in the incubation mixture. b Ci = citrate, Pi =
phosphate, Ci–Pi = citrate–phosphate.
by-product at ca. 3% yield. These results indicated that DMSO was
the best co-solvent of all those examined for the LAS-catalysed
chemo-selective hydrolysis of 8 to 12. Similarly, LAS-catalysed
hydrolyses of 9 and 10 were performed under their respective
optimum conditions to yield the corresponding methyl esters 13
and 14 in excellent yields. Reaction conditions and yields of LAS-
catalysed hydrolytic products 12–14 are summarized in Table 2.
It is very important to choose a suitable co-solvent for LAS-
catalysed hydrolysis in order to obtain high chemo-selectivity. For
example, DMSO was a good co-solvent for the hydrolysis of 8
and 9, but for 10 a considerable amount of NP (ca. 30%) was
formed with the concomitant formation of the expected product
14, probably due to lowered chemo-selectivity (data not shown).
2-Methoxyethanol was found to be the co-solvent of choice for
the chemo-selective hydrolysis of the substrate 10. These methyl
esters 12–14 were easily purified by passage through an Amberlite
XAD-4 column, followed by recrystallisation. The structures of
1
these compounds were confirmed by H- and 13C-NMR, and by
mass spectrometry.
PLE, commercially available as a mixture of isozymes,43 is
known to be one of the most useful enzymes for the synthesis of
chiral synthons.41 As mentioned above, PLE was shown to catalyse
the hydrolysis of methyl ester of 8 to provide 11. Therefore, PLE
was examined for hydrolytic activity toward methyl esters 12–
14 in order to obtain the free 1-b-O-acyl glucuronides 1–3. In
PLE-catalysed hydrolysis of the methyl ester 12 (chosen as the
model compound), it is also very important to choose a suitable
co-solvent to obtain high chemo-selectivity. For example, using
DMSO as a co-solvent in the PLE-catalysed hydrolysis of 12
resulted in the formation of a considerable amount of DF (ca.
20%). When the co-solvent was changed to t-BuOH, the expected
product 1 was obtained in 92% yield. Fig. 3a shows a typical
HPLC chromatogram of the PLE-catalysed hydrolysis of 12 with t-
BuOH as the co-solvent, where the corresponding free glucuronide
1 (peak A) was a major product and the hydrolysis generating DF
(peak B) was suppressed almost completely. However, as shown
in Table 3, DMSO was a good co-solvent for the PLE-catalysed
hydrolysis of methyl esters 13 and 14. The methyl ester groups of
substrates 12–14 were chemo-selectively hydrolysed under specific
Fig. 2 HPLC chromatograms of LAS-catalysed hydrolysis of 8. (a) after
2 min incubation; (b) after 5 h incubation. Conditions: 0.5 mM 8, 20%
(v/v) DMSO, 25 mM citrate buffer (pH 5.0), 8 mg cm−3 LAS and at 40 ◦C:
A, 8; D, 12; E, 5; B and C, partially deacetylated intermediates.
◦
conditions (pH and co-solvent) at 40 C for 1 to 3 h, providing
1–3 in excellent yields (Table 3). In all cases, the incubation was
performed at lower pH (between 5.0 and 6.0) rather than at the
optimum pH for PLE (ca. 7 to 8) in order to prevent the non-
enzymatic hydrolysis and intramolecular acyl migration of the
1-b-O-acyl linkages of both the substrates 12–14 and the products
1–3.
Scheme 2 Hydrolytic products of methyl acetyl derivative of diclofenac
1-b-O-acyl glucuronide (R-CO2H = diclofenac) 1. Route A, with LAS and
then PLE; route B, with PLE and then LAS; route C, with both PLE and
LAS.
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Table 3 Reaction conditions and yields of PLE-catalysed hydrolyses of
12–14 to provide 1–3
Conditions
Substrate
Yield (%)
pH (buffer)
Co-solvent (%, v/va)
12
13
14
92
99
90
5.0 (Cib/NaOH)
6.0 (Pib/NaOH)
5.5 (Ci–Pib/NaOH)
t-BuOH (15)
DMSO (20)
DMSO (20)
a Percentage of the co-solvent in the incubation mixture. b Ci = citrate, Pi =
phosphate, Ci–Pi = citrate–phosphate.
Fig. 4 Time course of PLE-catalysed hydrolysis of 12. (᭺), 12; (᭹), 1;
(ꢀ), DF; (᭛), aggregate. Conditions: 0.5 mM 12, 15% (v/v) t-BuOH,
0.4 mg cm−3 PLE at 40 ◦C.
The free 1-b-O-acyl glucuronides 1–3 were easily isolated by
passage through an Amberlite XAD-4 column. The structures of
1
these glucuronides were confirmed by H- and 13C-NMR, and
by mass spectrometry. b-Glucuronidase is known to hydrolyse
1-b-O-acyl glucuronides but 2-, 3- or 4-O-acyl isomers formed
through intramolecular acyl migration are resistant to hydrolysis
by the enzyme.12 Therefore, the purities of 1, 2 and 3 as 1-b-O-acyl
glucuronides were also determined using b-glucuronidase to be
97 1, 99 2 and 100 1%, respectively. These results indicate
that the glucuronides 1–3 are almost pure 1-b-O-acyl glucuronides.
The results presented here show that 8–10 are hydrolysed by
both LAS and PLE; LAS hydrolyses the O-acetyl groups of 8–10
with high chemo-selectivity to provide 12–14 and PLE hydrolyses
the methyl ester groups of 12–14 with high chemo-selectivity to
provide 1–3, via route A as shown in Scheme 2. As other possible
routes, we examined a reverse combination of the enzymes (route
B in Scheme 2) as well as the concurrent use of both enzymes
(route C in Scheme 2). In route B, compound 11, which is easily
obtained by treatment of 8 with PLE though in an ‘unsatisfactory
yield’, was incubated with LAS under the conditions used for the
hydrolysis of 8 to 12. As shown in Fig. 3b, the reaction did not
go to completion and gave mixtures of 1 (peak A), 5 (peak B), a
mono-O-acetyl derivative (peak C) and other unknown products.
The 1H-NMR spectrum of the mono-O-acetyl derivative showed
a singlet peak at d 1.92 corresponding to the –OCOCH3 group. On
the other hand, the concurrent use of both enzymes using DMSO
and t-BuOH as the co-solvents resulted in improved results, as
shown in Figs. 3c and 3d, respectively. In both the cases, other
than the expected 1, some hydrolysis of the 1-b-O-acyl linkage to
provide DF (peak B) (especially remarkable in Fig. 3c) occurred
and partially deacetylated intermediates (peak C corresponds to
the mono-O-acetate) were accumulated. The data in Figs. 2b and
3b, showing LAS-catalysed hydrolysis of 8 and 11 respectively,
indicated that LAS shows the high hydrolytic activity of O-acetyl
groups in 8 but not in 11. The difference of the activity might be
due to the negative charge on the glucuronic acid moiety in the
molecule of 11.
Fig. 3 HPLC chromatograms of (a) PLE-catalysed hydrolysis of 12, after
4 h; (b) LAS-catalysed hydrolysis of 11 after 5 h; (c) both LAS- and
PLE-catalysed hydrolysis of 8 with DMSO as a co-solvent, after 4 h;
(d) both LAS- and PLE-catalysed hydrolysis of 8 with t-BuOH as a
co-solvent after 4 h. A, 1; B, 5; C, a mono-O-acetyl derivative of 1; D,
11. Conditions: see Experimental.
Recently, selective hydrolysis of methyl and benzyl esters by
microbial enzymes has been reported.44 However, these reactions
required over 24 h, and the enzymes’ chemo-selectivity toward
methyl and benzyl esters in the presence of acetoxy functional
groups was not examined. Additionally, PLE has been also used
for the hydrolysis40 of a methyl ester of 1-b-O-acyl glucuronide
at pH 7.0, but chemo-selectivity toward the substrate was not
high. As shown in Fig. 4, PLE-catalysed hydrolysis of 8 proceeded
smoothly within 4 h, with DF being only slightly formed. Our
methodology using PLE at pH 5.0–6.0 has the advantages of high
chemo-selectivity and a short reaction time.
From these results, the preferred sequential order for enzymatic
treatment was shown to be LAS followed by PLE, namely the
route A shown in Scheme 2. Application of this chemo-enzymatic
method to the synthesis of other 1-b-O-acyl glucuronides, and
screening of more efficient enzymes for the deprotection process,
as well as optimisation of the enzymatic reaction conditions, are
all currently under investigation.
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4.70 (1H, d, J 9.6, C5-H), 4.99 (1H, t, J 8.2, C2-H), 5.01 (1H, t, J
9.6, C4-H), 5.50 (1H, t, J 9.6, C3-H), 6.09 (1H, d, J 8.2, C1-H), 6.22
(1H, d, J 7.9, Ar-H), 6.83 (1H, t, J 7.6, Ar-H), 6.98 (1H, s, NH),
7.05 (1H, t, J 7.6, Ar-H), 7.12–7.23 (2H, m, Ar-H) and 7.52 (2H, d,
J 7.9, Ar-H); dC (67.8 MHz, d6-DMSO) 20.03, 20.18, 20.24, 36.24,
52.58, 68.74, 69.81, 70.80, 71.41, 90.96, 115.83, 120.59, 122.09,
126.11, 127.95, 129.12, 130.80, 131.02, 137.04, 142.86, 166.86,
168.81, 169.25, 169. 39 and 169.62.
Conclusions
A chemo-enzymatic synthesis of 1-b-O-acyl glucuronides was
presented, taking DF 5, MF 6 and NP 7 as examples for
carboxylic acids. In the chemical glucuronidation step, methyl
acetyl derivatives of 1-b-O-acyl glucuronides 8–10 were obtained
exclusively in moderate yields, using neighboring 2-O-acetyl group
participation. In the following enzymatic deprotection step, the
expected free 1-b-O-acyl glucuronides 1–3 were easily obtained
in excellent yields via a sequential LAS- and PLE-catalysed high
chemo-selective hydrolysis.
Methyl 2,3,4-tri-O-acetyl derivative of mefenamic acid 1-b-O-
acyl glucuronide 9. Yield 74%; mp 124–125 ◦C (needles from
EtOH). Found: C, 60.3; H, 5.5; N, 2.45; m/z (FAB, positive)
MNa+, 580.1801. C28H31NO11 requires C, 60.3; H, 5.6; N, 2.5%;
C28H31NO11Na requires m/z 580.1798; mmax (KBr)/cm−1 3340,
1760, 1700, 1610, 1580, 1510, 1460, 1380, 1230, 1100 and 1050;
dH (400 MHz, d6-DMSO) 2.00 (3H, s, OCOCH3), 2.01 (3H, s,
OCOCH3), 2.01 (3H, s, OCOCH3), 2.07 (3H, s, ArCH3), 2.28
(3H, s, ArCH3), 3.61 (3H, s, CO2CH3), 4.77 (1H, d, J 9.8, C5-H),
5.11 (1H, t, J 9.5, C4-H), 5.22 (1H, dd, J 7.8 and 9.5, C2-H), 5.62
(1H, t, J 9.5, C3-H), 6.25 (1H, d, J 7.8, C1-H), 6.60 (1H, d, J 8.6,
Ar-H), 6.72 (1H, m, Ar-H), 7.07–7.16 (3H, m, Ar-H), 7.36 (1H, dt,
J 1.5 and 8.3, Ar-H), 7.78 (1H, dd, J 1.5 and 8.3, Ar-H) and 8.99
(1H, s, NH); dC (100 MHz, d6-DMSO) 13.68, 20.15, 20.21, 20.28,
52.58, 68.80, 69.58, 70.44, 71.38, 90.04, 108.11, 113.42, 116.50,
123.28, 126.16, 127.20, 131.16, 132.10, 135.73, 137.63, 137.98,
149.64, 165.54, 167.00, 169.21, 169.29 and 169.41.
Experimental
Materials
The sodium salt of DF 5 and free acids of MF 6 and NP 7 were
obtained from MP Biomedicals, Lancaster and Wako Pure Chem-
ical Industries, respectively. Methyl 2,3,4-tri-O-acetyl-1-bromo-1-
deoxy-a-D-glucopyranuronate4 was obtained from Sigma-Aldrich
Co. or synthesised according to the reported procedure.45 The
11 enzymes, used in the screening for chemo-selective hydrolytic
activity, were: lipase AS Amano (from Aspergillus niger), lipase
F-AP15 (from Rhizopus oryzae) and lipase G Amano 50 (from
Penicillium camemberti), which were obtained from Wako Pure
Chemical Industries, lipase from wheat germ and esterase from
porcine liver (lyophilized powder), which were obtained from
Sigma-Aldrich Co., lipase MY (from Candida cyclindracea), lipase
OF (from Candida cyclindracea), lipase PL (from Alcaligenes sp.),
lipase QLM (from Alcaligenes sp.), lipase SL (from Pseudomonas
cepacia) and lipase TL (from Pseudmonas stutzeri), which were
kindly gifted from Meito Sangyo Co., Ltd. Amberlite XAD-4 was
obtained from ORGANO Corporation and used after grinding
(80–200 mesh). All other chemicals used were analytical grade
commercial products. 1H- and 13C-NMR were recorded using
either a JNM-GX270 (JEOL) or a JNM-AL400 (JEOL). The
chemical shifts measured in CDCl3, d6-DMSO or CD3OD are
presented by d-values in ppm using the residual solvent peaks as
internal standards relative to TMS.
Methyl 2,3,4-tri-O-acetyl derivative of (S)-naproxen 1-b-O-acyl
glucuronide 10. Yield 46%; mp 182–184 ◦C (needles from EtOH).
Found: C, 59.39; H, 5.59; m/z (FAB, positive) MNa+, 569.1615.
C27H30O12 requires C, 59.34; H, 5.53%; C27H30O12Na requires m/z
569.1625; mmax (KBr)/cm−1 1760, 1630, 1610, 1370, 1230, 1090 and
1040; dH (270 MHz, d6-DMSO) 1.47 (3H, d, J 6.9, CHCH3), 1.86
(3H, s, OCOCH3), 1.95 (3H, s, OCOCH3), 1.97 (3H, s, OCOCH3),
3.59 (3H, s, CO2CH3), 3.86 (3H, s, ArOCH3), 3.97 (1H, q, J 6.9,
CHCH3), 4.66 (1H, d, J 9.9, C5-H), 4.95–5.02 (2H, m, C2-H and
C4-H), 5.50 (1H, t, J 9.6, C3-H), 6.05 (1H, d, J 8.2, C1-H), 7.16
(1H, dd, J 2.6 and 9.2, Ar-H), 7.30 (1H, d, J 2.6, Ar-H), 7.35
(1H, dd, J 1.6 and 8.6, Ar-H), 7.69 (1H, s, Ar-H) and 7.78 (2H,
d, J 8.9, Ar-H); dC (67.5 MHz, d6-DMSO) 17.94, 20.08, 20.14,
20.24, 44.19, 52.56, 55.14, 68.78, 69.71, 70.75, 71.38, 90.93, 105.69,
118.81, 125.88, 126.15, 127.00, 128.29, 129.16, 133.43, 134.27,
157.28, 166.81, 168.86, 169. 21, 169.39 and 172.09.
Glucuronidation of 5–7 using methyl 2,3,4-tri-O-acetyl-1-bromo-
1-deoxy-a-D-glucopyranuronate 4 to provide 8–10
A typical procedure is as follows: a solution of bromide 4 (397 mg,
1.00 mmol) and caesium salt of 5–7 (1.5 mmol) in DMSO (8 cm3)
was stirred at 30 ◦C for 3 h. The reaction mixture was diluted with
EtOAc (100 cm3) and then washed with water (100 cm3), saturated
aqueous NaHCO3 (3 × 40 cm3) and then saturated aq. NaCl. After
drying over Na2SO4, the organic solvent was evaporated to give a
crude product, which was purified by recrystallisation. Analytical
and spectral data of the products 8–10 were as follows.
HPLC analysis
HPLC was performed with a Shimadzu LC-10A equipped with a
C-R8A chromatopac, using a column of Symmetry C18 (5 lm,
4.6 × 150 mm, Waters) for the analysis of the enzymatic
reactions. Mobile phases for the analytical HPLC are given in each
experiment described below. For the final purification of free 1-b-
O-acyl glucuronides 1–3, a semipreparative column of Symmetry
C18 (7 lm, 19 × 150 mm, Waters) was used and the mobile phase
for 1–3 was aqueous CH3CN containing 0.01% (v/v) AcOH at a
flow rate of 3 cm3 min−1. The concentrations of CH3CN were 40,
50 and 30% (v/v) for 1, 2 and 3, respectively.
Methyl 2,3,4-tri-O-acetyl derivative of diclofenac 1-b-O-acyl
glucuronide 8. Yield 79%; mp 155–157 ◦C (needles from EtOH).
Found: C, 53.0; H, 4.5; N, 2.3; Cl, 11.5; m/z (FAB, positive) MNa+,
634.0847. C27H27Cl2NO11 requires C, 52.95; H, 4.4; N, 2.3; Cl,
11.6%; C27H27Cl2NO11Na requires m/z 634.0853; mmax (KBr)/cm−1
3360, 1750, 1510, 1460, 1370, 1230, 1090 and 1040; dH (270 MHz,
d6-DMSO) 1.82 (3H, s, OCOCH3), 1.95 (3H, s, OCOCH3), 1.99
(3H, s, OCOCH3), 3.62 (3H, s, CO2CH3), 3.88 (2H, s, ArCH2),
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Screening of enzymes for the hydrolytic activity of 8 and 12 as
model substrates
LAS-catalysed hydrolysis of 11 (route B); isolation of the
mono-O-acetyl derivative of 1
The incubation was carried out at an initial concentration of
A solution of tri-O-acetyl derivative 11 (20 mg, 0.033 mmol) in
DMSO (3.5 cm3) was added to 14.5 cm3 of 25 mM sodium citrate
buffer (pH 5.0), LAS (270 mg, 15 mg cm−3 incubation mixture)
was dissolved at 40 ◦C and the mixture was incubated for 5 h.
The mobile phase of HPLC analysis for this reaction was 45%
(v/v) CH3CN containing 50 mM NH4OAc (pH 4.5) and 10 mM
TBAB at a flow rate of 0.7 cm3 min−1 with detection at 283 nm.
After incubation for 3 h, the substrate 11 was almost hydrolysed to
give a mixture. The amount of expected free glucuronide 1 for the
5 h incubation was almost same as that for the 3 h incubation (see
Fig. 3b). A product whose retention time was very close to that of 1
was purified by pTLC (CHCl3–MeOH–AcOH = 25 : 9 : 1, v/v) to
give a 3- or 4-mono-O-acetyl derivative of 1 (4.0 mg, 23%), whose
structure was estimated from 1H-NMR; dH (270 MHz, d6-DMSO)
1.92 (3H, s, OCOCH3), 3.28–3.60 (3H, m, sugar-H), 3.84 and 3.92
(each 1H, d, J 15.8, ArCH2), 4.78 (1H, t, J 9.6, C3- or C4-H), 5.43
(1H, d, J 7.6, C1-H), 6.84 (1H, t, J 6.6, Ar-H), 7.07 (2H, t, J 8.9,
Ar-H), 7.16–7.24 (2H, m, Ar-H) and 7.53 (2H, d, J 7.9, Ar-H).
The proton at d 4.78 was assigned to the H-C-OCOCH3 proton
of the sugar moiety due to its resonance at lower field. Its triplet
multiplicity showed the proton to be either C3- or C4-H.
0.5 mM of 8 or 12 in 25 mM sodium citrate buffer (pH 5.0)
containing 20% (v/v) DMSO as a co-solvent at 40
0.1 ◦C
for 30 min and each commercially available enzyme was added
to the incubation mixture at a final concentration of 5 mg cm−3
(except for PLE at 0.4 mg cm−3). For enzyme assays, an aliquot of
the incubation mixture was appropriately diluted with an HPLC
carrier and injected onto a reversed-phase HPLC. The mobile
phase of HPLC analysis for the hydrolysis of 8 was 55% (v/v)
CH3CN containing 50 mM NH4OAc (pH 4.5) and 10 mM tetra-
n-butylammonium bromide (TBAB) at a flow rate of 0.7 cm3 min−1
with detection at 283 nm. For analysis of the formation of 1
from 12, the concentration of CH3CN in the mobile phase was
reduced to 45% (v/v). By adding TBAB (at a final concentration
of 10 mM) to the mobile phase, polar 1-b-O-acyl glucuronides 1–3
could be separated from the other, more polar components derived
from the enzymes, owing to the retardation of the retention times
of 1–3. This is explained by the formation of less polar ion
pairs between the glucuronides and the quaternary ammonium
ion.
Screening of co-solvents for the hydrolysis of 8 or 12
Concurrent usage of LAS and PLE for the hydrolysis of 12
(route C)
In order to optimise the LAS- and PLE-catalysed chemo-selective
hydrolytic conditions, the effect of co-solvents (at the fixed
final concentration of 20% (v/v)) on the enzymatic activity was
examined with substrate 8 for LAS and 12 for PLE.
A solution of 8 in either 20% (v/v) DMSO or 15% (v/v) t-
BuOH was added to 25 mM sodium citrate buffer (pH 5.0), LAS
(15 mg cm−3 incubation mixture) and PLE (0.4 mg cm−3 incubation
mixture) were dissolved at 40 ◦C and the mixture was incubated for
5 h. The mobile phase of HPLC analysis for this reaction was 45%
(v/v) CH3CN containing 50 mM NH4OAc (pH 4.5) and 10 mM
TBAB at a flow rate of 0.7 cm3 min−1 with detection at 283 nm.
As shown in Figs. 3c and 3d, both the enzymatic reactions did
not give good results; yields of the free glucuronide 1 after 4 h
incubation with DMSO and t-BuOH as co-solvents, determined
by HPLC, were ca. 30 and 10%, respectively.
PLE-catalysed hydrolysis of 8 to 11 (route B)
A solution of 8 (77 mg, 0.126 mmol) in CH3CN (37 cm3) was
added to 213 cm3 of 25 mM sodium citrate buffer (pH 5.0)
PLE (135 mg, 0.54 mg cm−3 incubation mixture) was dissolved
at 40 ◦C and the mixture was incubated for 6 h. The mobile
phase of HPLC analysis for this reaction was 55% (v/v) CH3CN
containing 50 mM NH4OAc (pH 4.5) and 10 mM TBAB at a
flow rate of 0.7 cm3 min−1 with detection at 283 nm. After the
reaction mixture was evaporated to remove CH3CN, the residual
aqueous solution was acidified to pH ca. 3 and saturated with
NaCl and then extracted with EtOAc (2 × 100 cm3). After drying
over Na2SO4, the organic solvent was evaporated to give a syrup,
which was purified by silica gel pTLC (EtOAc–acetone–AcOH =
10 : 10 : 0.2, v/v) to provide 11 as a solid (40 mg, 53%); m/z
(FAB, positive) MNa+, 620.0692. C26H25Cl2NO11Na requires m/z
620.0697; dH (400 MHz, CD3OD) 1.76 (3H, s, OCOCH3), 1.96
(3H, s, OCOCH3), 1.99 (3H, s, OCOCH3), 3.83 (1H, d, J 15.1,
ArCH2), 3.88 (1H, d, J 15.1, ArCH2), 4.23 (1H, d, J 9.8, C5-H),
5.11 (1H, t, J 9.3, C4-H), 5.20 (1H, t, J 9.8, C2-H), 5.35 (1H,
t, J 9.5, C3-H), 5.91 (1H, d, J 8.1, C1-H), 6.40 (1H, d, J 8.1,
Ar-H), 6.91 (1H, t, J 7.6, Ar-H), 7.02 (1H, s, NH), 7.06–7.10
(2H, m, Ar-H), 7.19 (1H, d, J 7.6, Ar-H) and 7.40 (2H, d, J 7.9,
Ar-H); dC (67.5 MHz, CD3OD) 20.31, 20.51, 20.59, 38.55, 70.83,
71.70, 73.69, 74.79, 93.09, 118.80, 122.98, 124.85, 126.03, 129.20,
130.06, 131.51, 132.06, 139.10, 144.26, 170.85, 171.15, 171.43 and
171.80.
LAS-catalysed hydrolysis of 8–10 to 12–14 (route A)
Methyl ester of diclofenac 1-b-O-acyl glucuronide 12. Incu-
bation was started by the addition of a solution of 8 (50 mg,
0.082 mmol) in DMSO (32 cm3) to 128 cm3 of 25 mM sodium
citrate buffer (pH 5.0), LAS (1.6 g, 10 mg cm−3 incubation mixture)
was dissolved at 40 ◦C and the mixture stirred magnetically for
3 h. The conversion yield to 12 was 94% by HPLC analysis. The
reaction mixture was then filtered and the filtrate was loaded
on an Amberlite XAD-4 column (5 g), which had been washed
thoroughly with acetone and then equilibrated with water. The
column was washed with water (50 cm3) and then 30% CH3CN
(50 cm3). The hydrolysed product 12 was eluted with 80% CH3CN
(50 cm3) with a recovery yield of 97% (by HPLC analysis).
The solvent was evaporated to provide 12. A portion of the
sample was purified by recrystallisation from CH3CN to confirm
the structure by spectral analyses. m/z (FAB, positive) MNa+,
508.0538. C21H21Cl2NO8Na requires m/z 508.0540; dH (400 MHz,
d6-DMSO) 3.21–3.39 (3H, m, C2-, C3- and C4-H), 3.64 (3H, s,
CO2CH3), 3.82–3.93 (3H, m, ArCH2 and C5-H), 5.28 (1H, d,
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J 4.9, OH, exchangeable with D2O), 5.41 (1H, d, J 5.6, OH,
exchangeable with D2O), 5.43 (1H, d, J 4.9, OH, exchangeable
with D2O), 5.50 (1H, d, J 8.1, C1-H), 6.22 (1H, d, J 8.1, Ar-
H), 6.84 (1H, t, J 7.6, Ar-H), 6.93 (1H, s, NH), 7.06 (1H, t,
J 7.6, Ar-H), 7.21 (2H, t, J 7.8, Ar-H), and 7.52 (2H, d, J
8.3, Ar-H); dC (100 MHz, d6-DMSO) 36.61, 51.96, 71.21, 72.17,
75.18, 75.82, 94.59, 115.65, 120.51, 122.31, 126.15, 127.90, 129.11,
131.01, 131.04, 136.99, 142.90, 168.80 and 170.32.
PLE-catalysed hydrolysis of 12–14 to 1–3 (route A)
Diclofenac 1-b-O-acyl glucuronide 1. The methyl ester 12
(46 mg, 95 lmol) was dissolved in t-BuOH (28 cm3) and the
solution was poured into 123 cm3 of 25 mM sodium citrate buffer
(pH 5.0). To the resultant solution, PLE (76 mg, 0.4 mg cm−3
incubation mixture) dissolved in an aqueous solution (38 cm3) was
◦
added and incubated at 40 C for 4 h. The conversion yield to 1
was 92% by HPLC analysis (the mobile phase is described above).
The reaction mixture was then acidified (to pH ca. 3) with 6 M
HCl and then filtered off. The filtrate was loaded on an Amberlite
XAD-4 column (5 g), which was prepared as described above. The
column was washed with water (100 cm3) and then eluted with
30% CH3CN (50 cm3) and 80% CH3CN (50 cm3). Both aqueous
CH3CN fractions, containing 1, were combined and evaporated
to provide 1 as a white solid in almost quantitative recovery yield.
m/z (ESI, negative) [M − H]−, 470.0409. C20H18Cl2NO8 requires
m/z 470.0409; dH (400 MHz, CD3OD) 3.43–3.76 (4H, m, C2-, C3-,
C4- and C5-H), 3.90 (2H, s, ArCH2), 5.53 (1H, d, J 6.6, C1-H), 6.35
(1H, dd, J 1.0 and 7.9, Ar-H), 6.86 (1H, dt, J 1.0 and 7.6, Ar-H),
7.03 (2H, t, J 7.6, Ar-H), 7.19 (1H, dd, J 1.3 and 7.6, Ar-H) and
7.35 (2H, d, J 8.2, Ar-H); dC (100 MHz, CD3OD) 20.73, 38.60,
72.94, 73.62, 77.17, 77.42, 95.95, 118.79, 122.88, 125.22, 125.86,
129.02, 130.00, 131.48, 132.23, 139.36, 144.53, 172.67 and 175.19.
Methyl ester of mefenamic acid 1-b-O-acyl glucuronide 13.
Incubation was started by the addition of a solution of 9
(40 mg, 0.072 mmol) in DMSO (48 cm3) to 144 cm3 of 25 mM
sodium phosphate buffer (pH 6.0), LAS (3.6 g, 15 mg cm−3
incubation mixture) was dissolved at 40 ◦C and the mixture stirred
magnetically for 5 h. The conversion yield to 13 was 90% by
HPLC analysis; the mobile phase for HPLC analysis was 60%
(v/v) CH3CN containing 50 mM NH4OAc (pH 4.5) and 10 mM
TBAB and the detection was at 283 nm. The reaction mixture was
then filtered and the filtrate was loaded on an Amberlite XAD-4
column (5 g), which was prepared as described for the purification
of 12. The column was washed with water (50 cm3) and then
40% CH3CN (50 cm3). The hydrolysed product 13 was almost
quantitatively recovered by elution with 90% CH3CN (50 cm3).
The solvent was evaporated to provide 13. A portion of the
sample was purified by recrystallisation from CH3CN to confirm
the structure by spectral analyses. dH (270 MHz, CD3OD) 2.13
(3H, s, ArCH3), 2.31 (3H, s, ArCH3), 3.67–3.60 (3H, m, C2-, C3-
and C4-H), 3.76 (3H, s, CO2CH3), 4.04 (1H, d, J 9.6, C5-H), 5.76
(1H, d, J 7.6, C1-H), 6.69–6.59 (2H, m, Ar-H), 7.06–7.11 (3H,
m, Ar-H), 7.27 (1H, t, J 7.3, Ar-H), 8.04 (1H, d, J 8.2, Ar-H),
9.10 (1H, s, NH); dC (67.5 MHz, d6-DMSO) 14.09, 20.60, 52.92,
72.96, 73.66, 77.38, 95.65, 110.62, 114.50, 117.19, 124.78, 127.22,
128.37, 132.93, 133.87, 136.08, 139.45, 151.54, 151.70, 168.33,
170.85.
Mefenamic acid 1-b-O-acyl glucuronide 2. The methyl ester
13 (27 mg, 63 lmol) was dissolved in DMSO (32 cm3) and the
solution was poured into 96 cm3 of 25 mM sodium phosphate
buffer (pH 6.0). To the resultant solution, PLE (65 mg, 0.4 mg cm−3
incubation mixture) dissolved in an aqueous solution (32 cm3) was
◦
added and incubated at 40 C for 1.5 h. The conversion yield to
2 was 99% by HPLC analysis; the mobile phase was 50% (v/v)
CH3CN containing 50 mM NH4OAc (pH 4.5) and 10 mM TBAB
at a flow rate of 0.7 cm3 min−1 with detection at 283 nm. The
reaction mixture was acidified with 6 M HCl (to pH ca. 3) and
then filtered off. The filtrate was loaded on an Amberlite XAD-4
column (5 g), which was prepared as described above. The column
was washed with water (100 cm3) and then with 50% CH3CN
(50 cm3) and eluted with 80% CH3CN (50 cm3). The fraction was
evaporated to give 2 as a white solid in almost quantitative yield.
m/z (FAB, positive) MNa+, 440.1312. C21H23NO8Na requires m/z
440.1317; dH (270 MHz, CD3OD) 2.14 (3H, s, ArCH3), 2.32 (3H, s,
ArCH3), 3.58–3.31 (3H, m, C2-, C3- and C4-H), 3.77 (1H, d, J
9.6, C5-H), 5.77 (1H, d, J 7.6, C1-H), 6.60–6.69 (2H, m, Ar-H),
7.03–7.10 (3H, m, Ar-H), 7.25 (1H, t, J 7.9, Ar-H), 8.07 (1H,
dd, J 1.6 and 8.2, Ar-H) and 9.14 (1H, s, NH); dC (100 MHz,
CD3OD) 14.10, 20.61, 73.47, 73.87, 76.76, 78.01, 95.96, 111.16,
114.45, 117.13, 124.59, 127.16, 128.21, 133.05, 133.76, 135.83,
139.40, 139.69, 151.39, 168.62 and 176.34.
Methyl ester of (S)-naproxen 1-b-O-acyl glucuronide 14. Incu-
bation was started by the addition of a solution of 10 (44 mg,
0.081 mmol) in 2-methoxyethanol (32 cm3) to 128 cm3 of 25 mM
sodium citrate and phosphate buffer (pH 5.5), LAS (2.4 g,
15 mg cm−3 incubation mixture) was dissolved at 40 ◦C and
the mixture stirred magnetically for 2 h. The conversion yield
to 14 was 90% by HPLC analysis; the mobile phase for HPLC
analysis was 50% (v/v) CH3CN containing 50 mM NH4OAc
(pH 4.5) and 10 mM TBAB and the detection was at 271 nm.
The reaction mixture was then filtered and the filtrate was loaded
on an Amberlite XAD-4 column (5 g), which was prepared as
described for the purification of 12. The column was washed with
water (50 cm3) and then 40% CH3CN (50 cm3). The hydrolysed
product 14 was almost quantitatively recovered by elution with
90% CH3CN (50 cm3). The solvent was evaporated to provide
14. A portion of the sample was purified by recrystallisation
from CH3CN to confirm the structure by spectral analyses. dH
(270 MHz, CD3OD) 1.55 (3H, d, J 7.3, CHCH3), 3.39–3.52 (3H,
m, C2-, C3- and C4-H), 3.69 (3H, s, CO2CH3), 3.88–3.98 (5H, m,
C5-H, CHCH3 and ArOCH3), 5.52 (1H, d, J 7.9, C1-H), 7.09 (1H,
dd, J 2.6 and 8.9, Ar-H), 7.18 (1H, d, J 2.0, Ar-H), 7.37 (1H, dd,
J 2.0 and 8.6, Ar-H) and 7.66–7.72 (3H, m, Ar-H); dC (67.5 MHz,
CD3OD) 19.16, 46.39, 52.86, 55.71, 72.88, 73.53, 77.32, 95.84,
106.55, 119.91, 127.13, 127.23, 128.26, 130.25, 130.39, 135.28,
136.49, 159.18, 170.79 and 174.88.
(S)-Naproxen 1-b-O-acyl glucuronide 3. The methyl ester 14
(38 mg, 90 lmol) was dissolved in DMSO (14 cm3) and the solution
was poured into 56 cm3 of 25 mM sodium citrate and phosphate
buffer (pH 5.5) in which PLE was dissolved (28 mg, 0.4 mg cm−3
incubation mixture) and then incubated at 40 ◦C for 3 h. The
conversion yield to 3 was 90% by HPLC analysis; the mobile phase
was 45% (v/v) CH3CN containing 50 mM NH4OAc (pH 4.5) and
10 mM TBAB at a flow rate of 0.7 cm3 min−1 with detection at
271 nm. The reaction mixture was acidified (to pH ca. 3) with
6 M HCl and then filtered off. The filtrate was loaded on an
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Amberlite XAD-4 column (5 g), which was prepared as described
above. The column was washed with water (100 cm3) and then
with 25% CH3CN (50 cm3) and eluted with 80% CH3CN (50 cm3).
The fraction was evaporated to give 3 as a white solid in almost
quantitative recovery yield. m/z (SIMS, positive) MH+, 407.1302.
C20H23NO9 requires m/z 407.1321; dH (400 MHz, CD3OD) 1.56
(3H, d, J 7.2, CHCH3), 3.30–3.50 (3H, m, C2-, C3- and C4-H), 3.84
(1H, d, J 8.8, C5-H), 3.88 (3H, s, ArOCH3), 3.97 (1H, q, J 7.2,
CHCH3), 5.53 (1H, d, J 8.1, C1-H), 7.09 (1H, dd, J 2.4 and 8.9,
Ar-H), 7.18 (1H, s, Ar-H), 7.39 (1H, dd, J 1.7 and 8.5, Ar-H) and
7.67–7.72 (3H, m, Ar-H); dC (100 MHz, CD3OD) 19.23, 46.46,
55.72, 73.02, 73.60, 77.27, 77.60, 95.82, 106.54, 119.88, 127.14,
127.29, 128.26, 130.29, 130.41, 135.27, 136.58, 159.17, 172.87 and
174.92.
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