Development of a rapid and detailed structural identification system with an on-line immobilized enzyme reactor integrated into LC-NMR

Article abstract of DOI:10.1248/cpb.58.423

Immobilized enzyme reactors (IMERs) integrated into an LC-NMR system were developed for rapid and detailed structural identification of enzymatic reaction products. An on-column enzymatic reaction was achieved for immobilized cytochrome-c and dog microsomes. After the reaction, these products were analyzed by LC-NMR without any work-up processes. The immobilized cytochrome-c column integrated into LC-NMR was used to characterize the reaction product formed on N-demethylation by measurement of 1H-NMR. In the case of reaction taking place on the microsome column, a glucuronidation product was identified by 1H-NMR and 1H-1H correlated spectroscopy. The chemical structures of the enzymatic reaction products could be elucidated by IMER-LC-NMR without the need for authentic samples or isolation processes.

Full text of DOI:10.1248/cpb.58.423

March 2010
Notes
Chem. Pharm. Bull. 58(3) 423—425 (2010)
423
Development of a Rapid and Detailed Structural Identification System
with an On-Line Immobilized Enzyme Reactor Integrated into LC-NMR
Yousuke KASHIMA* and Yoshito OKABAYASHI
Developmental Research Laboratories, Shionogi & Co., Ltd.; 5–12–4 Sagisu, Fukushima, Osaka 553–0002, Japan.
Received November 2, 2009; accepted November 30, 2009; published online December 2, 2009
Immobilized enzyme reactors (IMERs) integrated into an LC-NMR system were developed for rapid and
detailed structural identification of enzymatic reaction products. An on-column enzymatic reaction was achieved
for immobilized cytochrome-c and dog microsomes. After the reaction, these products were analyzed by LC-
NMR without any work-up processes. The immobilized cytochrome-c column integrated into LC-NMR was used
1
to characterize the reaction product formed on N-demethylation by measurement of H-NMR. In the case of re-
1
1
1
action taking place on the microsome column, a glucuronidation product was identified by H-NMR and H– H
correlated spectroscopy. The chemical structures of the enzymatic reaction products could be elucidated by
IMER-LC-NMR without the need for authentic samples or isolation processes.
Key words immobilized enzyme reactor; LC-NMR; microsome; cytochrome-c
Immobilized enzyme reactors (IMERs) have been devel- tem. Next, we used an immobilized dog microsome column
oped for use as high-performance liquid chromatographic for the system to identify glucuronide as a reaction product.
(HPLC) columns and have been used as pre- or post-columns Glucuronidation is one of the main metabolic pathways for
in HPLC systems for on-line analysis of biological sub- drugs in vivo. Authentic samples of drug glucuronides can
1,2)
stances. IMERs have been used to study the enzymatic not be easily obtained by chemical synthesis or biosynthesis
3
)
12)
enantioselective recognition of racemic compounds and the using free microsomes. The results of this study demon-
4,5)
on-line synthesis and analysis of drug metabolites.
We strate that an IMER-LC-NMR system can be used for on-line
have also previously prepared immobilized enzyme phospho- synthesis and characterization of the glucuronide. Drug de-
lipid columns with biomimetic characteristics for HPLC velopment worked can be aided by being able to identify
columns and examined their properties as enzyme reactors compounds which may be glucuronidated and to synthesize
6)
and their usage for drug development. IMERs have been and characterize the resulting products.
utilized in various applications combined with HPLC sys-
tems. However, such IMER-HPLC systems could not be used Experimental
Materials Deuterium oxide was purchased from Sigma-Aldrich
Dorset, U.K.). Cyt-c and uridine 5ꢀ-diphosphate-glucuronic acid (UDPGA)
were purchased from Nacalai Tesque (Kyoto, Japan). N-Methylaniline (MA),
-nitrophenol (4NP) and L-a-dipalmitoylphosphatidylcholine (DPPC) were
to identify reaction products without the use of authentic
samples nor could they elucidate the detailed chemical struc-
tures of the products. Recently, IMERs have been combined
(
4
with LC-MS for drug metabolism and inhibition studies. purchased from Wako (Osaka, Japan). Acetonitrile, methanol, water and tri-
7)
fluoroacetic acid were of HPLC grade and all other chemicals were of
reagent grade. The HPLC column used for this study was Develosil ODS-
UG-5 (35ꢁ4.6 mm i.d., 5 mm, Nomura Chemicals, Aichi, Japan).
Nicoli et al. incorporated two cytochrome P450-based im-
mobilized enzyme reactors into an LC-MS system to per-
form automated on-line phase I drug metabolism studies.
Apparatus The HPLC system consisted of a Shimadzu LC-20AD
LC-MS has been often used for the identification of small (Kyoto, Japan) as a pump, a Shimadzu SIL-HTc automatic injector, a Shi-
amounts of drug metabolites and impurities in pharmaceuti- madzu SPD-20AV spectrophotometer as a UV detector and a Shimadzu
8)
CTO-20AC as a column oven. The chromatographic data was analyzed
using a Waters Empower software (Milford, MA, U.S.A.). The LC-NMR
system consisted of a Varian INOVA 600-MHz spectrometer (Varian NMR
cals. However, LC-MS by itself usually does not provide
sufficient structural information for unambiguous identifica-
tion. Therefore, time-consuming preparative chromato-
1
13 15
Instruments, Palo Alto, CA, U.S.A.) equipped with a H{ C/ N} pulse field
graphic isolation is often necessary to obtain pure com- gradient triple resonance 5 mm cryogenic probe with a flow cell (active vol-
pounds for NMR spectroscopic analysis, if authentic com- ume: 60 ml) and a Varian HPLC system (a Prostar 230 pump and a Proster
3
35 photdiode array UV detector). This was operated in the loop-collection
pounds for comparison are not available commercially.
Recently, LC-NMR has been increasingly applied for the
structural determination of drug metabolites, impurities and
modes using Varian VNMRJ software. The field frequency was locked on
the H resonance of D O. Suppression resonances from HDO resonance
were attained by using water elimination through a transverse gradients
2
2
13)
1
degradation products in pharmaceuticals to obtain detailed (WET) technique. The H-NMR spectra were obtained using an acquisi-
structural information on compounds in mixtures without tion time of 3 s, a delay between the successive pulses of 1 s, and a spectral
9,10)
width of 9592 Hz. A total of 256 scans (measuring time ca. 17 min) were ac-
using isolation processes.
Also, NMR analysis allows
1
1
cumulated to obtain an appropriate signal-to-noise ratio. The H– H corre-
lated spectroscopy (COSY) spectrum was acquired with a spectral width of
identification of compounds with small molecular weights or
poor ionization characteristics, which are likely to be difficult
5940 Hz into 2048 data points in t , with 256 increments in the t dimension.
2
1
11)
for MS detection.
Number of transients per increment was 24, and the measuring time was ca.
h. The cryogenic probe was used for increasing NMR sensitivity and re-
2
In this study, we developed an IMER-LC-NMR system for
on-line enzymatic reaction and structural identification of the
resulting products and evaluated the system. First, we devel-
ducing the measuring time.
Preparation of Dog Liver Microsomes Dog liver samples were ob-
tained from female beagle dogs without drug treatment in our laboratory and
12)
oped a system using an immobilized cytochrome-c (Cyt-c) the microsomes were prepared as described previously. The microsomal
column as a model case to assess the performance of the sys- pellets obtained were stored in a freezer at ꢂ80 °C until use. The microso-
∗
To whom correspondence should be addressed. e-mail: yousuke.kashima@shionogi.co.jp
© 2010 Pharmaceutical Society of Japan

4
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Vol. 58, No. 3
mal protein concentration was determined by using the Micro Bicinchoninic umn, 35ꢁ4.6 mm i.d.), respectively. The amounts of immobi-
Acid Protein Assay Reagent Kit (Pierce, Rockford, IL, U.S.A.) with bovine
serum albumin as standard. The microsomal protein concentration in this
study was approximately 20 mg/ml.
lized enzyme on the phospholipid columns were 16.3 mg
14)
(Cyt-c column) and 27.6 mg (microsome column), respec-
tively. Thus, the immobilized enzyme phospholipid columns
Preparation of Immobilized Enzyme Columns The phospholipid col-
umn used to prepare the immobilized enzyme column was a Develosil ODS- could readily be prepared by allowing a solution containing
UG-5 (35ꢁ4.6 mm i.d.) coated with DPPC. The phospholipid column was the desired ligands to flow through the HPLC column.
15)
prepared by a dynamic coating technique developed in our laboratory. The
procedure was done under an ambient temperature of 25ꢃ1 °C. An immobi-
lized Cyt-c column was prepared by recycling a solution of 20 mg of Cyt-c
Development and Evaluation of IMER-LC-NMR Cyt-
c is one of the electron transport hemoproteins located on
the biomembrane surface and usually does not catalyze the
dissolved in 50 ml of a 10 mM sodium phosphate buffer in D O (pD 7.1) at a
2
flow rate of 1 ml/min through the phospholipid column for up to 240 ml of oxidation reaction. However, Cyt-c is known to show N-
1
6)
total delivery. Similarly, an immobilized microsome column was prepared demethylase activity on addition of an oxygenating agent.
by recycling a solution of 31 mg of microsome added to 100 ml of a 10 mM
We tried various methods of adding the oxygenating agent
during the separation. First, a solution of N-methylaniline
MA) as the substrate and hydrogen peroxide as the oxy-
sodium phosphate buffer in D O (pD 7.4) at a flow rate of 0.5 ml/min
2
through the phospholipid column for total delivery of up to 360 ml. The im-
mobilized enzyme columns were washed with a 10 mM sodium phosphate
(
buffer in D O (pD 7.0) at 1 ml/min for 30 min. The amounts of DPPC coat- genating agent were simultaneously injected onto the immo-
2
ing on the octadecyl silica (ODS) columns and immobilized enzyme on the
phospholipid column were calculated as described previously. The activi-
ties of immobilized enzymes were estimated by N-demethylation of MA
bilized Cyt-c column with a mobile phase of 10 mM sodium
13)
phosphate buffer in D O (pD 7.1). Since the yield of the oxi-
2
dation product of MA was low, time-consuming measure-
(Cyt-c) and glucuronidation of 4NP (microsomes).
1
ment of H-NMR was necessary to identify the product.
IMER-LC-NMR Analyses On-line enzyme reaction, analytic separa-
tion and identification of products were performed using the IMER-LC- Next, we tried to inject the hydrogen peroxide solution onto
NMR system depicted in Fig. 1. The mobile phase was 10 mM sodium phos-
the column after injection of the substrate. The yield of the
phate buffer in D O. NMR measurements were performed in the loop-col-
2
N-demethylation product was sufficient to easily obtain the
lection mode. The loop-collection mode trapped chromatographic peaks of
interest in nine loops according to UV detection, and the loop contents were
1
H-NMR spectrum. N-Demethylation was efficiently induced
transferred to an NMR flow cell for NMR measurements.
by successive injection of the oxygenating agent when hydro-
gen peroxide solution passed through the previously injected
substrate band in the column. Figure 2 shows the chro-
Results and Discussion
Evaluation of Preparation of Immobilized Enzyme matogram obtained when 16 mmol/50 ml hydrogen peroxide
Phospholipid Columns The amount of DPPC coating on solution as the oxygenating agent was injected onto the col-
the ODS columns and that of immobilized enzyme on the umn after injection of 3 mmol MA as the substrate. Both the
phospholipid columns were calculated. The amounts of substrate and the product were simultaneously separated on
DPPC coating on the ODS columns were 0.072 mmol (Cyt-c the on-line column and detected after N-demethylation. The
1
column, 35ꢁ4.6 mm i.d.) and 0.072 mmol (microsome col-
H-NMR spectra of peak 2 (MA) and the peak 1 (product)
1
are presented in Fig. 3. The H-NMR spectrum of the prod-
uct showed no singlet at 2.73 ppm (compared with MA),
indicating the product to be aniline. As a result, the IMER-
LC-NMR system could be successfully developed and ap-
plied to identify the enzymatic reaction product.
Fig. 1. Schematic Diagram of On-Line IMER Integrated into LC-NMR
System
Next, we applied this system to the identification of glu-
Fig. 2. UV Chromatogram of N-Demethylation of MA on Immobilized Cyt-c Column
Flow rate: 0.6 ml/min; detection: 280 nm; column temperature: 25 °C. Peaks: 1, aniline; 2, MA.
1
Fig. 3. H-NMR Spectra of (a) MA and (b) Aniline

March 2010
425
Fig. 4. UV Chromatogram of Glucuronidation of 4NP on Immobilized Microsome Column
Mobile phase: 10 mM sodium phosphate buffer in D O (pD 7.4) containing 10 mM magnesium chloride; flow rate: 0.5 ml/min; detection: 320 nm; column temperature: 37 °C.
2
Peaks: 1, 4NP glucuronide; 2, 4NP.
1
Fig. 5. H-NMR Spectra of (a) 4NP and (b) 4NP Glucuronide
curonide formed from immobilized dog microsomes. Two enzymatic reaction and the subsequent identification of the
micromoles of UDPGA as a coenzyme was injected onto the products using simple compounds. The IMER-LC-NMR sys-
column after injection of 1 mmol 4-nitrophenol (4NP) as sub- tem should be applicable for the structural identification of
strate at the mobile phase rate of 0.5 ml/min, and the flow various drug metabolites by selecting the appropriate column
was stopped after 55 s, enabling the substrate and UDPGA to and enzyme.
be in contact with the microsome immobilized within the
Acknowledgement We thank Takashi Kitade of M&S Instruments for
many helpful discussions.
column. After 10 min, the mobile phase flow was restarted at
1
0
.5 ml/min (Fig. 4). For identification, analyses of the H-
NMR of 4NP (peak 2) and 4NP glucuronide (peak 1) and References
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1
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2
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2
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