Development of an automated synthesis system for preparation of glucuronides using a solid-phase extraction column loaded with microsomes

Article abstract of DOI:10.1248/cpb.58.354

An automated synthesis system using a solid-phase extraction (SPE) system and column packed with octadecylsilica (ODS), which was coated with phospholipid and loaded with dog liver microsomes, was developed for synthesis of glucuronides. Preparation of the microsome-immobilized SPE column, glucuronidation of drugs to synthesize the glucuronides and elution of the products were performed by an automated synthesis system. The phospholipid-coated SPE column and then the microsome-immobilized SPE column were readily prepared by allowing a solution containing L-α- dipalmitoylphosphatidylcholine to flow through the SPE column, and then by recycling a buffer solution containing dog liver microsomes through the resulting phospholipid-coated SPE column. The microsome-immobilized SPE column exhibiting the uridine diphosphate (UDP)-glucuronosyltransferase activity catalyzed the glucuronidation of mefenamic acid and estradiol to the corresponding glucuronides in the presence of UDP-glucuronic acid, and three glucuronides of mefenamic acid and estradiol were synthesized using the automated synthesis system, by simply recycling a buffer solution containing UDP-glucuronic acid through the microsome-immobilized SPE column loaded with the substrate. We used β-cyclodextrin as a solubilizing agent for the synthesis of the glucuronides of estradiol that is practically insoluble in aqueous solutions. The productivity of these glucuronides using the microsome-immobilized SPE column was higher than that using the free microsomes (batch method). Furthermore, we developed a fully automated synthesis-isolation system by coupling the automated synthesis system to an automated preparative HPLC system. The automated synthesis system as well as the fully automated synthesis-isolation system should be very useful for synthesizing glucuronides for drug development.

Full text of DOI:10.1248/cpb.58.354

354
Chem. Pharm. Bull. 58(3) 354—358 (2010)
Vol. 58, No. 3
Development of an Automated Synthesis System for Preparation of
Glucuronides Using a Solid-Phase Extraction Column Loaded with
Microsomes
a
Yousuke KASHIMA, ,a Takashi KITADE,b Yuuko KASHIMA,a and Yoshito OKABAYASHI
*
a Developmental Research Laboratories, Shionogi & Co., Ltd.; 5–12–4 Sagisu, Fukushima, Osaka 553–0002, Japan: and
b M&S Instruments Inc.; 2–12–4 Mikuni-honmachi, Yodogawa, Osaka 532–0005, Japan.
Received October 27, 2009; accepted December 14, 2009; published online December 15, 2009
An automated synthesis system using a solid-phase extraction (SPE) system and column packed with oc-
tadecylsilica (ODS), which was coated with phospholipid and loaded with dog liver microsomes, was developed
for synthesis of glucuronides. Preparation of the microsome-immobilized SPE column, glucuronidation of drugs
to synthesize the glucuronides and elution of the products were performed by an automated synthesis system.
The phospholipid-coated SPE column and then the microsome-immobilized SPE column were readily prepared
by allowing a solution containing L-a-dipalmitoylphosphatidylcholine to flow through the SPE column, and then
by recycling a buffer solution containing dog liver microsomes through the resulting phospholipid-coated SPE
column. The microsome-immobilized SPE column exhibiting the uridine diphosphate (UDP)-glucuronosyltrans-
ferase activity catalyzed the glucuronidation of mefenamic acid and estradiol to the corresponding glucuronides
in the presence of UDP-glucuronic acid, and three glucuronides of mefenamic acid and estradiol were synthe-
sized using the automated synthesis system, by simply recycling a buffer solution containing UDP-glucuronic
acid through the microsome-immobilized SPE column loaded with the substrate. We used b-cyclodextrin as a
solubilizing agent for the synthesis of the glucuronides of estradiol that is practically insoluble in aqueous solu-
tions. The productivity of these glucuronides using the microsome-immobilized SPE column was higher than
that using the free microsomes (batch method). Furthermore, we developed a fully automated synthesis-isolation
system by coupling the automated synthesis system to an automated preparative HPLC system. The automated
synthesis system as well as the fully automated synthesis-isolation system should be very useful for synthesizing
glucuronides for drug development.
Key words automated synthesis; glucuronide; microsome: microsome-immobilized solid-phase extraction column
Since glucuronidation, which is catalyzed by uridine water-insoluble substrate such as estradiol, we used b-cyclo-
diphosphate glucuronosyltransferase (UDPGT)1) that is lo- dextrin (b-CD) as a solubilizing agent. The microsome-im-
cated mainly in the liver and other tissues, is one of the main mobilized SPE column and free microsomes were compared
metabolic pathways for drugs in vivo, authentic samples of with respect to their productivity of glucuronides. Further-
glucuronides of drugs are often needed for pharmacokinetics, more, we developed a fully automated synthesis-isolation
pharmacological or toxicological study of drugs during pre- system by coupling the automated synthesis system to an au-
clinical or clinical drug development stages.
tomated preparative HPLC system, isolating the pure product
Glucuronides have been obtained by tedious isolation and from the synthesis mixture, and applied to preparation of
purification of metabolites from biological samples,2,3) by flufenamic acid 1-O-acylglucronide.
limited chemical synthesis,4) by complicated chemo-enzy-
matic synthesis,5) and by inconvenient biosynthesis using
Experimental
free microsomes6,7) or immobilized microsomes.1,8—11)
Materials SPE column (Bond Elut-C18, 500 mg, 3 ml) was obtained
from Varian (Harbor City, CA, U.S.A.). b-CD and L-a-dipalmitoylphos-
phatidylcholine (DPPC) were purchased from Wako (Osaka, Japan). Uridine
Recently, an efficient and useful synthesis of acylglu-
curonides of drugs using immobilized dog liver microsome
5ꢀ-diphosphate-glucuronic acid (UDPGA) was obtained from Nacalai
octadecylsilica (ODS) particles coated with phospholipid
(microsome-immobilized particles) has been reported from
our laboratories.12) However, this synthesis is still inconven-
ient, because preparation of the phospholipids-coated ODS
particles, immobilization of microsomes on the particles, and
glucuronidation of drugs must be conducted by rather
tedious, batch-wise procedures including filtration steps.
Moreover, preparation of glucuronides of water-insoluble
drugs is not practicable by this synthesis.
Tesque (Kyoto, Japan). Mefenamic acid, estradiol and flufenamic acid were
purchased from Sigma (St. Louis, MO, U.S.A.). Acetonitrile, methanol,
water and formic acid were of HPLC grade, and all other chemicals were of
reagent grade.
HPLC and LC/MS Analyses The HPLC system consisted of a Shi-
madzu LC-20AD (Kyoto, Japan) as a pump, a Shimadzu SIL-HTc automatic
injector as an injector, a Shimadzu SPD-20AV spectrophotometer as a UV
detector and a Shimadzu CTO-20AC as a column oven. The chromato-
graphic data were analyzed using a Waters Empower (Milford, MA). The
HPLC column was a Cadenza 5CD-C18 (75ꢁ10 mm i.d., 5 mm, Imtakt,
Kyoto, Japan). The mobile phase was 0.1% formic acid/acetonitrile solution
(70 : 30) or (40 : 60) at a flow rate of 2 ml/min, and the detector was set at a
To overcome these disadvantages, we developed a new au-
tomated process for the synthesis of authentic samples of wavelength of 230 nm or 320 nm. LC/MS experiments were conducted using
a Waters Acquity Ultra Performance LC system coupled to a Waters LCT-
Premier (Milford, MA, U.S.A.).
Preparation of Dog Liver Microsomes Dog liver samples were ob-
tained from female beagle dogs without drug treatment in our laboratories.
glucuronides of drugs, using a solid-phase extraction (SPE)
column loaded with the immobilized dog liver microsomes
ODS coated with phospholipid (microsome-immobilized
SPE column). This automated process was applied to synthe-
sis of glucuronides of mefenamic acid and estradiol. For a
The microsomes were prepared in a conventional manner as described previ-
ously.13) The microsomal pellets were stored in a freezer at ꢂ80 °C until use.
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: yousuke.kashima@shionogi.co.jp

March 2010
355
The microsomal protein concentration was determined by using the Micro HPLC with photodiode array detection confirmed no significant UV spectral
Bicinchoninic Acid Protein Assay Reagent Kit (Pierce, Rockford, IL, shift between each glucuronide and its substrate. The productivity of glu-
U.S.A.) with bovine serum albumin as a standard.14) The microsomal protein curonides by using free microsomes was estimated similarly.
concentration in our study was approximately 25 mg/ml.
Coupling of Automated Preparative HPLC System to Automated
Automated Synthesis System Preparation of the microsome-immobi- Synthesis System to Constitute Fully Automated Synthesis-Isolation
lized SPE column, glucuronidation of drugs to synthesize drug glucuronides
and the elution of products were performed using the automated synthesis
system depicted in Fig. 1a. The automated synthesis system consisted of an
System (Fig. 1b) The automated synthesis system was directly connected
to an automated preparative HPLC system. The Gilson preparative HPLC
system used for the coupling consisted of a model 322-H2 pump and con-
ASPEC XL SPE system (Gilson, Villiers le Bell, France) equipped with a troller, a model 155 UV/Vis detector and a model GX-271 ASPEC auto-
peristaltic pump, two 10-port switching valves (Gilson), two 3-port switch- sampler and fraction collector. Chromatographic data were collected using
ing valves (Takasago Electric, Nagoya, Japan), a thermostatic jacket (M&S Trilution LH software. The preparative column was a Cadenza 5CD-C18
Instruments, Osaka, Japan) and a bath circulator (Thermo Fisher Scientific,
MA, U.S.A.). All steps were programmed with appropriate software. Only water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) at a flow
the SPE column must be renewed in every synthesis. rate of 20 ml/min, and the detector was set at a wavelength of 320 nm. The
(100ꢁ20 mm i.d., 5 mm). The mobile phases used were 0.1% formic acid in
Automated Preparation of Microsome-Immobilized SPE Column and proportion of solvent B was programmed at 10% for 0—3 min, and at 55%
Automated Synthesis of Glucuronides The procedure was conducted at for 3—23 min. The composition of solvent B was then programmed to in-
the ambient temperature of 25ꢃ1 °C. To prepare SPE column coated with crease to 100% in 1 min and this composition was maintained for an addi-
phospholipid (phospholipid-coated SPE column), a solution of 65 mg of
DPPC dissolved in 100 ml of 80% methanol was pumped through an SPE
column with 500 mg of ODS at a flow rate of 6 ml/min for 12.5 min. After
pumping, 30 ml of water was sent through the phospholipid-coated SPE col-
tional 3 min.
NMR Analysis NMR spectra were recorded on a Varian INOVA-500
spectrometer at 500 MHz (1H) and 125 MHz (13C) in CD3OD. 1H and 13C
signals were assigned on the basis of the 1H–1H correlation spectroscopy
umn at a flow rate of 6 ml/min for 5 min to wash the column. The micro- (COSY) and heteronuclear single-quantum correlation (HSQC) and het-
some-immobilized SPE column was prepared by recycling a solution of ap- eronuclear multiple bond correlation (HMBC) spectra, respectively.
proximately 12.3 mg of microsomes in 30 ml of a 50 mM Tris–hydrochloric
Mefenamic Acid 1-O-Acylglucuronide (MFAG) 1H-NMR (500 MHz,
acid buffer (pH 7.4) through the phospholipid-coated SPE column at a flow CD3OD) d: 2.14 (3H, s), 2.32 (3H, s), 3.55 (1H, br t, Jꢄca. 9 Hz), 3.59 (1H,
rate of 3 ml/min for 60 min. To load the substrate, 50 ml of 50 mM Tris–hy-
drochloric acid buffer (pH 7.4) containing 50 mmol of the substrate dis-
solved in 1.5 ml of N,N-dimethylformamide or methanol was pumped 1.0 Hz), 7.05 (1H, m), 7.09 (1H, m), 7.10 (1H, m), 7.27 (1H, ddd, Jꢄ8.6,
br t, Jꢄca. 8 Hz), 3.60 (1H, br t, Jꢄca. 9 Hz), 3.95 (1H, d, Jꢄ9.2 Hz), 5.78
(1H, d, Jꢄ7.9 Hz), 6.63 (1H, dd, Jꢄ8.6, 1.0 Hz), 6.67 (1H, ddd, Jꢄ8.0, 7.1,
through the microsome-immobilized SPE column at a flow rate of 3 ml/min
for 35 min in a continuous circulation. To synthesize the glucuronides, 5 ml
of 50 mM Tris–hydrochloric acid buffer (pH 7.4) containing 50 mM UDPGA
and 10 mM magnesium chloride was pumped through the microsome-immo-
bilized SPE column loaded with the substrate at a flow rate of 0.01 ml/min in
7.1, 1.6 Hz), 8.07 (1H, dd, Jꢄ8.0, 1.6 Hz). 13C-NMR (125 MHz, CD3OD) d:
14.13, 20.65, 73.22, 73.79, 77.79, 77.80, 95.62, 110.89, 114.51, 117.20,
124.76, 127.23, 128.35, 133.02, 133.87, 136.01, 139.46, 139.65, 151.52,
168.45, 173.44. The 13C-NMR data of MFAG prepared in our study were not
fully consistent with those reported by Baba and Yoshioka.5) We believe that
a continuous circulation. The synthesis was achieved at 37 °C. After the re- the MFAG synthesized by Baba and Yoshioka was the carboxylic acid
action, the desired compound was eluted from the microsome-immobilized sodium salt in the glucuronic acid moiety.
SPE column with 10 ml of 70% methanol.
Evaluation of Prepared Microsome-Immobilized SPE Column The
Estradiol 3-O-Glucuronide 1H-NMR (500 MHz, CD3OD) d: 0.77 (3H,
s), 1.20 (1H, m), 1.27 (1H, m), 1.30 (1H, m), 1.35 (1H, m), 1.39 (1H, m),
amount of DPPC coated on the SPE column was calculated from the differ- 1.45 (1H, m), 1.51 (1H, m), 1.70 (1H, m), 1.88 (1H, m), 1.96 (1H, m), 2.03
ence between DPPC concentrations of the solution before and after the (1H, m), 2.16 (1H, m), 2.32 (1H, m), 2.82 (1H, m), 3.47 (1H, m), 3.48 (1H,
preparation, where the DPPC concentrations were determined by HPLC as m), 3.53 (1H, m), 3.66 (1H, t, Jꢄ8.6 Hz), 3.75 (br d, Jꢄca. 9 Hz, 1H), 4.84
described previously.15)
The amount of immobilized microsomes, estimated as the protein content,
on the phospholipid-coated SPE column was calculated from the difference 27.59, 28.49, 30.70, 30.77, 38.08, 40.43, 44.40, 45.51, 51.38, 73.58, 74.83,
(obscured by H2O), 6.81 (1H, d, Jꢄ2.2 Hz), 6.81 (1H, dd, Jꢄ8.6, 2.2 Hz),
7.17 (1H, d, Jꢄ8.6 Hz). 13C-NMR (125 MHz, CD3OD) d: 11.71, 24.07,
in the concentrations of the solution containing microsomes before and after
76.98, 77.83, 82.54, 102.90, 115.59, 118.12, 127.20, 135.37, 138.97,
the preparation, where the concentrations of the solution were determined, as 157.12, 175.66.
described above, using the Micro Bicinchoninic Acid Protein Assay Reagent
Kit.
Estradiol 17-O-Glucuronide 1H-NMR (500 MHz, CD3OD) d: 0.87
(3H, s), 1.22 (1H, m), 1.30 (1H, m), 1.35 (1H, m), 1.37 (1H, m), 1.43 (2H,
Evaluation of Glucuronide Production The productivity of glu- m), 1.68 (1H, m), 1.69 (1H, m), 1.86 (1H, m), 2.06 (1H, m), 2.12 (1H, m),
curonides using the automated synthesis system was calculated from the 2.14 (1H, m), 2.28 (1H, m), 2.76 (1H, m), 3.23 (1H, dd, Jꢄ8.8, 7.8 Hz), 3.38
peak area of the products compared with that of the standard solution of the
substrate injected onto the HPLC system under the same analysis conditions.
(1H, dd, Jꢄ9.2, 8.8 Hz), 3.52 (1H, br t, Jꢄca. 9 Hz), 3.75 (1H, br d, Jꢄca.
10 Hz), 3.78 (1H, t, Jꢄ8.7 Hz), 4.41 (1H, d, Jꢄ7.8 Hz), 6.47 (1H, d,
Fig. 1. Schematic Diagram of Automated Synthesis System (a) and Fully Automated Synthesis-Isolation System (b) for Preparation of Glucuronides of
Drugs
(A) DPPC solution, (B) water, (C) microsomal solution, (D) substrate solution, (E) coenzyme solution (UDPGA, MgCl2), (F) 70% methanol.

356
Vol. 58, No. 3
Jꢄ2.4 Hz), 6.53 (1H, dd, Jꢄ8.4, 2.4 Hz), 7.07 (1H, d, Jꢄ8.4 Hz). 13C-NMR
(125 MHz, CD3OD) d: 12.15, 24.05, 27.66, 28.51, 29.90, 30.76, 38.74,
40.38, 44.65, 45.35, 51.15, 73.32, 75.16, 76.79, 77.73, 90.40, 105.17,
113.78, 116.09, 127.27, 132.65, 138.84, 155.94, 173.00.
Flufenamic Acid 1-O-Acylglucuronide 1H-NMR (500 MHz, CD3OD)
d: 3.57 (2H, m), 3.58 (1H, m), 3.86 (1H, br d, Jꢄca. 8 Hz), 5.77 (1H, d,
Jꢄ7.1 Hz), 6.88 (1H, br t, Jꢄca. 8 Hz), 7.29 (1H, d, Jꢄ8.5 Hz), 7.33 (1H,
br d, Jꢄca. 8 Hz), 7.44 (1H, m), 7.49 (1H, m), 7.50 (1H, m), 7.52 (1H, br t,
Jꢄca. 9 Hz), 8.13 (1H, dd, Jꢄ8.0, 1.4 Hz). 13C-NMR (125 MHz, CD3OD) d:
73.41, 73.80, 77.03, 77.88, 95.78, 114.09, 115.78, 118.62, 119.78, 120.52,
125.56 (q, Jꢄ270.6 Hz), 125.73, 131.47, 132.88 (q, Jꢄ32.8 Hz), 133.39,
135.95, 143.30, 148.18, 168.12, 175.21.
Results and Discussion
Evaluation of Preparation of Microsome-Immobilized
SPE Column The amount of DPPC on the SPE column
and the amount of immobilized microsomes on the phospho-
lipid-coated SPE column were calculated to be approxi-
mately 45 mg and approximately 3 mg as the protein content,
respectively. The microsome-immobilized SPE column could
be readily prepared by allowing a solution containing the de-
sired ligands to flow through the SPE column.
Automated Synthesis of Mefenamic Acid 1-O-Acyl-
glucuronide We synthesized mefenamic acid 1-O-acyl-
glucuronide (MFAG) using the automated synthesis system.
After the reaction, the eluate from the microsome-immobi-
lized SPE column was injected onto the HPLC system. The
peak corresponding to the acylglucuronide was detected
around a retention time of 3 min, while the peak of mefe-
namic acid was detected around a retention time of 11 min
(data not shown). The peaks of mefenamic acid and its acyl-
glucuronide could be completely separated under the present
HPLC conditions. The product obtained from the fraction so-
lution corresponding to the peak of the acylglucuronide was
identified by LC/MS and NMR. Analysis by LC/MS of the
product revealed the presence of molecular ions of m/z 242
[Mꢂ176ꢅH]ꢅ and m/z 418 [MꢅH]ꢅ, corresponding to the
Fig. 2. Chemical Structures of Four Glucuronides Prepared in This Study
Fig. 3. Relationship between Enzymatic Reaction Time and Productivity
of MFAG
molecular weights of mefenamic acid and its acylglu- UDP-glucuronosyltransferase was proven to be reasonably
curonide, respectively. stable during the reactions as the productivity of MFAG in-
1
Figure 2 shows the chemical structure of MFAG. The H- creased proportionally with time up to 32 h. The stability of
and 13C-NMR chemical shifts of synthesized MFAG by the UDP-glucuronosyltransferase was retained due to the immo-
automated synthesis system are given in Experimental. The bilization of microsomes on the phospholipid coating ODS
1H signals of d 5.78 (d), d 3.59 (br t), d 3.55 (br t), d 3.60 particle, as described previously.12)
(br t) and d 3.95 (d) were assigned to H-1ꢆ, H-2ꢆ, H-3ꢆ, H-4ꢆ
Automated Synthesis of Two Estradiol Ether Glu-
b
and H-5ꢆ of the glucuronic acid moiety, respectively. The H- curonides Using -CD as Solubilizing Agent Estradiol is
1ꢆ anomeric proton at 5.78 ppm had a coupling constant of practically insoluble in aqueous solutions. We used b-CD as
7.9 Hz, which showed the characteristic diaxial coupling con- a solubilizing agent for the synthesis of two estradiol ether
stant between H-1ꢆ and H-2ꢆ in the b-glucuronic acid glucuronides. One hundred micromoles of b-CD were added
1
moiety.16,17) Also, a long-range H–13C correlation, which to 50 ml of 50 mM Tris–hydrochloric acid buffer (pH 7.4)
was observed between d 5.78 (H-1ꢆ) and 168.45 (C-1 of the containing 50 mmol of estradiol dissolved in 1.5 ml of
mefenamic acid moiety) on the HMBC spectrum proved that methanol. The amount of estradiol dissolved with b-CD was
C-1 of mefenamic acid was linked to C-1ꢆ of b-glucuronic approximately 37-fold larger than that observed without b-
acid through the oxygen atom.16) As a result, the chemical CD.
structure of MFAG synthesized using the automated synthe-
sis system could be completely identified by using LC/MS some-immobilized SPE column was injected onto the HPLC
and NMR. system, then the peak fractions corresponding to two ether
After the reaction, the eluate obtained from the micro-
Evaluation of MFAG Productivity Figure 3 shows the glucuronides were collected, and the solvent of the fractions
relationship between the enzymatic reaction time and the was evaporated and the residues were lyophilized. The two
productivity of MFAG. The productivity of MFAG increased products were identified by using LC/MS and NMR. The
with increasing the flow time of the coenzyme solution, analysis by LC/MS of the two products revealed the presence
reaching a plateau when the enzymatic reaction time was of the molecular ion of m/z 447 [MꢂH]ꢂ, corresponding to
more than 32 h. As a result, the enzymatic reaction time was the molecular weight of estradiol glucuronides.
set at 32 h for the optimum productivity of glucuronidation.
Figure 2 shows the chemical structure of estradiol 3-O-

March 2010
357
glucuronide (E3G) and estradiol 17-O-glucuronide (E17G).
1
The H- and 13C-NMR chemical shifts of two synthesized
glucuronides using the automated synthesis system are given
1
in Experimental. The H signals of E17G of d 4.41 (d), d
3.23 (dd), d 3.38 (dd), d 3.52 (br t) and d 3.75 (br d) were as-
signed to H-1ꢀ, H-2ꢀ, H-3ꢀ, H-4ꢀ and H-5ꢀ of the glucuronic
acid moiety, respectively. The H-1ꢀ anomeric proton at
4.41 ppm had a coupling constant of 7.8 Hz, which showed
the characteristic diaxial coupling constant between H-1ꢀ and
H-2ꢀ in the b-glucuronic acid moiety.16,17) Also, a long-range
1H–13C correlation which was observed between d 4.41 (H-
1ꢀ) and 90.40 (C-17 of the estradiol moiety) on the HMBC
spectrum proved that the C-17 of estradiol was linked to C-1ꢀ
of b-glucuronic acid through the oxygen atom.16) Similarly, a
long-range 1H–13C correlation of E3G was observed between
d 4.84 (H-1ꢀ) and 157.12 (C-3 of the estradiol moiety)
through the oxygen atom. As a result, the chemical structures
of E3G and E17G synthesized using the automated synthesis
system could be completely identified by using LC/MS and
NMR.
Fig. 4. Relationship between Enzymatic Reaction Time and Productivity
of E3G and E17G
Evaluation of Productivity of E3G and E17G Figure 4
shows the relationship between the enzymatic reaction time
and the productivity of E3G and E17G. The productivity of
E3G and E17G increased with increasing the flow time of the
coenzyme solution. Twenty three micromoles of estradiol
loaded on the microsome-immobilized SPE column almost
completely underwent the reaction, when the enzymatic reac-
tion time was set at 24 h.
Fig. 5. Comparison of the Productivities of MFAG, E3G and E17G by the
Use of Microsome-Immobilized SPE Column and Free Microsomes (Batch
Method)
Microsome-immobilized SPE column: mefenamic acid; substrate loaded on the col-
umn, 38.5 mmol; estradiol; substrate loaded on the column, 23.0 mmol; immobilized
microsomal protein, 3.07 mg. Free microsomes (batch method): mefenamic acid; sub-
strate added to the enzymatic reaction mixture, 38.5 mmol; estradiol; substrate added to
the enzymatic reaction mixture, 23.0 mmol; microsomal protein, 3.07 mg. Enzymatic
reaction mixture: 5 ml of 50 mM Tris–hydrochloric acid buffer (pH 7.4) containing
Comparison of Productivity of Glucuronidation with
Free Microsomes The productivities of glucuronides of
mefenamic acid and estradiol using the microsome-immobi- 50 mM UDPGA and 10 mM magnesium chloride; reaction conditions, 37 °C, 24 h.
lized SPE column were compared to those observed with free
microsomes (batch method) under the same reaction condi- eluate obtained from the microsome-immobilized SPE col-
tions. Figure 5 shows that the productivities of MFAG, E3G umn was automatically injected onto the automated prepara-
and E17G using the microsome-immobilized SPE column tive HPLC system, and the peak fraction corresponding to
were higher than those observed with the free microsomes. It the acylglucuronide was collected using a UV trigger. The
was previously assumed that the difference of the glu- solvent of the fraction was evaporated and the residue was
curonide productivity between the microsome-immobilized lyophilized. The product was identified by using LC/MS and
particles and the free microsomes would be caused by the NMR. The analysis by LC/MS of the product revealed the
difference of the concentration of substrate in the enzymatic presence of the molecular ion of m/z 458 [MꢅH]ꢅ, corre-
reaction mixture.12) In the synthesis of glucuronides with free sponding to the molecular weight of FFAG.
1
microsomes, the concentration of substrate in the mixture
Figure 2 shows the chemical structure of FFAG. The H-
should be lower in order to keep the enzyme activity higher. and 13C-NMR chemical shifts of FFAG synthesized by the
On the other hand, in the case of the microsome-immobilized fully automated synthesis-isolation system are given in Ex-
1
SPE column, the glucuronidation could be efficiently ef- perimental. The H signals of d 5.77 (d), d 3.58 (m), d 3.57
fected with excessive substrate, because it was loaded on the (m), d 3.57 (m) and d 3.86 (br d) were assigned to H-1ꢆ, H-
SPE column without inhibiting the enzyme activity. As a re- 2ꢆ, H-3ꢆ, H-4ꢆ and H-5ꢆ of the glucuronic acid moiety, re-
sult, the productivities of MFAG, E3G and E17G with the spectively. The H-1ꢆ anomeric proton at 5.77 ppm had a cou-
microsome-immobilized SPE column were higher as com- pling constant of 7.1 Hz, which showed the characteristic di-
pared to those obtained with the same protein content of free axial coupling constant between H-1ꢆ and H-2ꢆ in the b-glu-
1
microsomes. These findings indicate that the immobilized curonic acid moiety.16,17) Also, a long-range H–13C correla-
microsomes on the microsome-immobilized SPE column tion which was observed between d 5.77 (H-1ꢆ) and 168.12
could efficiently achieve the synthesis of glucuronides, even (C-1 of the flufenamic acid moiety) on the HMBC spectrum
when there was an excessive substrate.
proved that C-1 of flufenamic acid was linked to C-1ꢆ of b-
Fully Automated Synthesis-Isolation of Flufenamic glucuronic acid through the oxygen atom.16) As a result, the
Acid 1-O-Acylglucuronide A schematic diagram of the chemical structure of FFAG synthesized using the fully auto-
fully automated synthesis-isolation system is depicted in Fig. mated synthesis-isolation system could be completely identi-
1b. The automated synthesis system was directly connected fied by using LC/MS and NMR.
to an automated preparative HPLC system. We prepared the
flufenamic acid 1-O-acylglucuronide (FFAG) using this fully using the fully automated synthesis-isolation system (in the
The productivity of FFAG was approximately 8.8 mmol
automated synthesis-isolation system. After the reaction, the presence of 50 mM UDPGA, at 37 °C for 24 h).

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Conclusion
We have developed an automated synthesis system for ob-
taining authentic samples of glucuronides, using a micro-
some-immobilized SPE column and SPE system. By using
the automated synthesis system, the acylglucuronide of
mefenamic acid and two ether glucuronides of estradiol
could be easily synthesized. Furthermore, estimation of the
glucuronidation productivity showed an advantage of the
method using the microsome-immobilized SPE column over
that using free microsomes. We used b-CD as a solubilizing
agent for the synthesis of the glucuronides of estradiol that is
practically insoluble in aqueous solutions. The usage of b-
CD means that this system could accept a water-insoluble
substrate. Furthermore, we coupled the automated synthesis
system to an automated preparative HPLC system, an on-line
system, to constitute a fully automated system. This allows
easy operation to obtain the pure product from the synthetic
mixture. We have concluded that the automated synthesis
system as well as the fully automated synthesis-isolation sys-
tem would be helpful in drug development stages for obtain-
ing authentic samples of acylglucuronides, ether glu-
curonides or other metabolites of drugs that are slightly
soluble in aqueous solutions or those hardly synthesized by
chemical methods.
Acknowledgement We thank Dr. Yukiko Kan for many helpful discus-
sions.
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