Study on the metabolic mechanism of chiral inversion of S-Mandelic acid in vitro

Article abstract of DOI:10.1002/chir.21031

Mandelic acid (MA) is generally used as a biological indicator of occupational exposure to styrene, which is classified as a class of hazardous environmental pollutants. It was found to undergo one-directional chiral inversion (S-MA to R-MA) in Wistar and Sprague-Dawley rats in vivo. This study was aimed to explore the metabolic mechanism of chiral inversion of S-MA in vitro. S-MA was converted to R-MA in rat hepatocytes, whereas MA enantiomers remained unchanged in acidic and neutral phosphate buffers, HepG2 cells, and intestinal flora. In addition, the synthesized S-MA-CoA thioester was rapidly racemized and hydrolyzed to R-MA by rat liver homogenate and S9, cytosolic and mitochondrial fractions. The data suggest that chiral inversion of S-MA may involve the hydrolysis of S-MA-CoA, and its metabolic mechanism could be the same as that of 2-arylpropionic acid (2-APA) drugs.

Full text of DOI:10.1002/chir.21031

CHIRALITY 24:86–95 (2012)
Study on the Metabolic Mechanism of Chiral Inversion of
S-Mandelic Acid In vitro
LING-BO GAO,1 JIN-ZHAO WANG,2 TONG-WEI YAO,1 AND SU ZENG
1
*
1Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China
2Department of Quality Control, Zhejiang Huahai Pharmaceutical Ltd, Taizhou, China
ABSTRACT
Mandelic acid (MA) is generally used as a biological indicator of occupational
exposure to styrene, which is classified as a class of hazardous environmental pollutants. It was
found to undergo one-directional chiral inversion (S-MA to R-MA) in Wistar and Sprague-
Dawley rats in vivo. This study was aimed to explore the metabolic mechanism of chiral inver-
sion of S-MA in vitro. S-MA was converted to R-MA in rat hepatocytes, whereas MA enantiom-
ers remained unchanged in acidic and neutral phosphate buffers, HepG2 cells, and intestinal
flora. In addition, the synthesized S-MA-CoA thioester was rapidly racemized and hydrolyzed to
R-MA by rat liver homogenate and S9, cytosolic and mitochondrial fractions. The data suggest
that chiral inversion of S-MA may involve the hydrolysis of S-MA-CoA, and its metabolic mecha-
nism could be the same as that of 2-arylpropionic acid (2-APA) drugs. Chirality 24:86–95,
C
VV
2012.
2011 Wiley Periodicals, Inc.
KEY WORDS: mandelic acid; chiral inversion; intestinal bacteria; chemical synthesis; ibuprofen
INTRODUCTION
types of pseudomonas bacteria, a stereospecific particulate S-
Mandelic acid dehydrogenase is responsible for the S-MA me-
tabolism, whereas R-MA is metabolized only after its isomeriza-
tion to S-MA by mandelate racemase.12,13
To date, the mechanism of one-directional chiral inversion
of MA in mammals remains unknown. Our present study
was undertaken to further characterize the potential meta-
bolic mechanism of chiral inversion of S-MA by several in
vitro models.
Styrene is widely used in plastic industry and has been
implicated as reproductive toxicant, neurotoxicant, or possi-
ble carcinogen.1,2 Occupational and environmental exposure
to styrene occurs predominately via inhalation.3 Mandelic
acid (MA, structure of its two enantiomers see Fig. 1) is one
of the major urinary metabolites of styrene in both rodents
and humans.4,5 It can be further metabolized to phenylglyox-
ylic acid (PGA). Therefore, MA and PGA are generally used
as biological indicators of occupational exposure to styrene.6
Though MA was deemed low toxic in the past, a recent study
indicated that it may contribute to the depletion of dopamine
in rabbit and the adverse effects on the peripheral nervous
system in rats.7,8 In recent years, there has been an
increased awareness of the effects of stereochemistry on
drug metabolism and toxicity. Thus the investigation of
mechanism of the chiral inversion of MA would be meaning-
ful to evaluate toxicity and stereo-metabolism of styrene and
also helpful to assess the hazard of styrene to organisms.
Besides monitoring workers exposed to styrene vapor,
MA is also used as an intermediate for the synthesis of target
molecules, such as urinary tract bactericide hexamine man-
delate, peripheral vasodilator hacosan, eyedrops hydroben-
zole, and spasmolytic agent. It has also been used effectively
as a urinary antiseptic, particularly as a bladder irrigant dur-
ing urological procedures.9 Lately, MA has gained popularity
as a topical skin care treatment for adult acne.
Early investigation on MA metabolism showed that it can
partly dehydrogenate to PGA in human and dogs. In 1990s,
besides stereoselectively metabolized to PGA, MA was also
found could undergo one-directional chiral inversion (S-MA
to R-MA) in Wistar rats by Drummond et al for the first
time.10 Our laboratory study also indicated that an obvious
phenomenon of S-MA inverted to R-MA had occurred in SD
rats after a single oral administration of 100 mg/kg S-MA.5
The first information regarding the bi-directional chiral inver-
sion of MA enantiomers in some bacterias was published by
Kenyon and Hegeman in 1970.11 Later, it was found in some
MATERIALS AND METHODS
Chemicals and Reagents
S-MA and R-MA (purity >99.5%) were kindly presented by Yiming
Fine Chemicals Ltd. (Taixing, China). (S)-(2)-alpha-(1-naphthyl) ethyla-
mine (S-NEA) was purchased from Sigma Chemical Company
(St. Louis). 1-Hydroxy-benzotriazole (HOBT) and 1-(3-dimetthylamino-
propyl)-3-ethylcarbodiimide HCl (EDC) were purchased from Acros
Organics. (NJ). Phenylglyoxal was purchased from Alfa Aesar. 2, 6-di-
methyl pyridine, magnesium acetate tetrahydrate, magnesium chloride,
hydroquinone, calcium chloride, epsom salt, dipotassium hydrogen
phosphate, potassium dihydrogen phosphate, sodium bicarbonate, so-
dium chloride, dimethylformamide, and acetic ether were supplied by
Sinopharm Chemical Reagent (Beijing, China). Tryptone, yeast extract,
cysteine hydrochlorate were obtained from BBI (Canada). Coenzyme A
was kindly presented by Biochemical Pharmaceutical. Jinan Weier
Abbreviations: 2-APA, 2-Arylpropionic acid; CoA, coenzyme A; DMF, di-
methyl formamide; EDC, 1-(3-dimetthylaminopropyl)-3-ethylcarbodiimide;
ESI, Electrospray ionization; HOBT, 1-Hydroxy-benzotriazole; HPLC, high-
performance liquid chromatography; MA, mandelic acid; MeCN, methyl
cyanide; PGA, phenylglyoxylic acid; S-NEA, (S)-(2)-alpha-(1-naphthyl)
ethylamine.
Contract grant sponsor: National Program on Key Basic Research Project;
Contract grant number: 2011CB710800.
Contract grant sponsor: Natural Science Foundation of China; Contract grant
number: 81072691.
*Correspondence to: Su Zeng, Department of Pharmaceutical Analysis and
Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou 310058, China. E-mail: zengsu@zju.edu.cn
Received for publication 21 August 2010; Accepted 10 August 2011
DOI: 10.1002/chir.21031
Published online 2 December 2011 in Wiley Online Library
(wileyonlinelibrary.com).
C
VV
2011 Wiley Periodicals, Inc.

MOLECULAR MECHANISM OF CHIRAL INVERSION OF S-MA TO R-MA
87
Kang. Deionized water was purified using a Milli-Q water system (Milli-
pore, Bedford, MA). HPLC grade methanol and ethanol were obtained
from Merck (Darmstadt, Germany), other chemicals used were of ana-
lytical grade.
Chiral Derivatization and HPLC Analysis
The enantiomers R- and S-MA were determined by HPLC using S-
NEA as a chiral derivatization reagent. In brief, MA enantiomers were
extracted with ethyl acetate from reaction mixture at acidic pH. After
centrifugation at 10,000g for 5 min, the upper organic layer was removed
Fig. 1. Structure of S- and R-MA. (A) S-MA and (B) R-MA.
Fig. 2. The synthesis procedure from phenylglyoxal to S-MA-CoA thioester using Mg(OAc)2l4H2O and 2.6-dimethylpyridine as catalyst.
Fig. 3. Chromatograms of MA enantiomers coincubated with intestinal bacteria. (A) Blank intestinal bacteria incubate spiked with MA enantiomers and inter-
nal standard; (B) Blank intestinal bacteria incubate for 24 h; (C) S-MA coincubated with intestinal bacteria for 24 h; (D) R-MA coincubated with intestinal bacteria
for 24 h. 1: S-MA-naphthylethylamide derivative; 2: R-MA-naphthylethylamide derivative; 3: I.S-naphthylethylamide derivative.
Chirality DOI 10.1002/chir

88
GAO ET AL.
Fig. 4. Chromatograms of MA incubated with rat hepatocytes for 2 h. (A) blank, (B) S-MA incubate, (C) R-MA incubate. 1: S-MA-naphthylethylamide deriva-
tive; 2: R-MA-naphthylethylamide derivative.
and evaporated to dryness under a gentle stream of nitrogen and then
derivatized with S-NEA. EDC and HOBT were used as coupling agents.
The chiral derivatization was performed as described previously.5
Chromatography was performed on a ZORBAX SB-C18 column (250 3
4.6 mm2, I.D., 5 lm, Agilent) coupled with a C18 guard column (20 3 4.6
mm2, I. D., 5 lm). The mobile phase was composed of methanol-10 mM
phosphate buffer (pH 2.5) [65:35, (v/v) at a flow-rate of 0.8 ml/min
(flow-rate of 1.0 ml/min for S-MA-CoA thioester incubation]. Detection
was set at UV wavelength of 254 nm. The column temperature was main-
tained at room temperature. The injected volume was 20 ll.
was added and incubated for 0, 0.5, 1, and 2 h to analyze the chiral inver-
sion of MA.
Chemical Synthesis, Racemization and Hydrolysis of
S-MAndelic Acid-CoA Thioester
Synthesis of S-MA-CoA thioester. S-MA-CoA thioester was synthe-
sized in a flask by the method of Stan S. Hall14 used for the synthesis of
a-hydroxythiol esters, with some modifications (Fig. 2). The method
involved the following:
To a solution of 0.38 g (0.5 mM) of phenylglyoxal and 0.03 g (0.25
mM) of Mg(OAc)2 4H2O in 3 ml of DMF that had stirred for 10 min, a
solution of 0.38 g (1 mM) of CoA and 0.009 g (0.016 mM) of hydroqui-
none in 6 ml of DMF was added. After reaction for 2 h at 258C, 0.9 mg
(0.25 mM) of 2, 6-dimethylpyridine was added to the reaction mixture.
After an additional 4 h of stirring, some cold water was added into the
reaction mixture, then extracted with acetic ether for three times.
Nonenzyme Metabolism of MA Enantiomers In Vitro
MA enantiomers (20 lg/ml) were incubated with 0.1M phosphate
buffer (pH 1.5 or 7.4) at 378C in triplicate, respectively. After 0, 1, 2, 4, 8,
12, and 24 h of incubation, samples were treated and analyzed as Chiral
derivatization and HPLC analysis. Phenylacetic acid was used as internal
standard.
Purification and identification of S-MA-CoA thioester. The reac-
tion mixture was extracted with 3 3 10 ml ethyl acetate. Then the ethyl
acetate layer was concentrated by vacuum centrifugal concentrator. S-
MA-CoA thioester was purified from the mixture by preparative HPLC. It
was purified on a semi-preparative column Hypersil ODS2 (5 lm, 10 3
250 mm2) column with a flow of 3.0 ml/min using a gradient elution
(100% A 20 min, 20.01–35.00 min 0–50% MeCN). The mobile phase A
was composed of H2O:MeCN:1M ammonium acetate 95:5:1 (v:v:v). Frac-
Incubation of MA Enantiomers with Intestinal Bacteria
Preparation of intestinal bacteria in vitro. Male Sprague-Dawley
rats (150–180 g) obtained from Laboratory Animal Center of Zhejiang
University (Hangzhou, China), were raised under laboratory condition to
collect fresh feces. Two volumes of sterile physiological saline were
added to the feces, filtered. Then the filtrate was added with nine vol-
umes of anaerobic medium, cultivated overnight in anaerobic incubator.
The anaerobic medium was modified PY medium contained 20 mg/ml
tryptone, 10 mg/ml yeast extract, 0.5 mg/ml cysteine hydrochlorate, 40
ml/l VPI salt solution, pH 7.2. The VPI salt solution was consisted of 0.2
mg/ml calcium chloride, 0.2 mg/ml epsom salt, 1 mg/ml dipotassium
hydrogen phosphate, 1 mg/ml potassium dihydrogen phosphate, 10
mg/ml sodium bicarbonate, 2 mg/ml sodium chloride.
Incubation of MA enantiomers in SD rats intestinal bacteria. MA
enantiomers (final concentration 50 lg/ml) were added to the overnight
cultivated intestinal bacteria, misce bene, cultivated in anaerobic incuba-
tor for 0, 2, 4, 8, 12, and 24 h. Incubation in the absence of intestinal bac-
teria was used as negative control. Samples were treated and analyzed
as Chiral derivatization and HPLC analysis. Phenylacetic acid was used
as internal standard.
Chiral Inversion of MA Enantiomers in Hepatocyte
Rat hepatocytes were isolated by the two-step collagenase perfusion
method with a minor modification. The isolated cells were resuspended
in pH 7.4 Krebs-Henseleit buffer (containing 12.5 mM HEPES and 0.1%
bovine serum albumin) and seeded in six-well plates (Costar Corning) at
a density of 1 3 106 cells/well. After seeded for 4 h, the medium was dis-
carded, and new culture medium containing 20 lg/ml MA enantiomers
Fig. 5. Time profile of R-MA produced by different concentrations of S-
MA incubated in hepatocytes suspension.
Chirality DOI 10.1002/chir

MOLECULAR MECHANISM OF CHIRAL INVERSION OF S-MA TO R-MA
89
Fig. 6. Mass spectra of MA-CoA thioester and CoA. (A) The fragmentation of synthesized MA-CoA thioester; (B) Daughters of CoA; (C) Daughters of MA-
CoA thioester. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
tions containing S-MA-CoA were collected, pooled, lyophilized to dry-
ness, and stored at 2208C until use.
weighted and homogenized in four volumes of 20 mM Tris-HCl buffer
(pH 7.4), containing 0.25 M sucrose. The homogenate was centrifuged
for 15 min at 2000g and the supernatant, designed as the whole homoge-
nate fraction. The S9, mitochondrial, cytosolic, and microsomal fractions
were prepared by successive centrifugation at 9000g for 20 min, 19,000g
for 20 min, and then at 105,000g for 60 min. The mitochondrial and
microsomal fractions were washed twice with the same buffer. Protein
concentration was determined using BCA Protein Assay Reagent with
bovine serum albumin as the standard.
The identity of the synthesized and purified S-MA-CoA thioester was
determined by LC-MS and LC-MS/MS with ESI using the infusion
mode. The mass spectrometer (UPLC-TQD-MS, Waters Company) was
operated in negative ion MS scan mode, with the following settings:
dwell time, 0.5 s; source temperature, 1008C; desolvent gas temperature,
3008C; Capillary voltage, 3600 V. The collision gas was argon gas; colli-
sion energy was 20 eV. Masslynx V4.1 (Waters) was used for instrument
control, data acquisition and data processing.
In vitro incubation of S-MA-CoA thioester with rat liver homoge-
nate and subcellular fractions. The reaction mixture was composed
of 50 mM Tris-HCl buffer (pH 7.4, containing 150 mM KCl and 15 mM
MgCl2), S-MA-CoA thioester and either rat liver homogenate, S9, cyto-
solic fractions (final concentration of 5 mg/ml) or mitochondrial fraction
(final concentration of 4 mg/ml). These samples were all placed in a
shaking water bath maintained at 378C. All incubates were monitored for
Preparation of rat liver homogenate, S9, cytosolic and mitochon-
dria fractions. Male Sprague-Dawley rats obtained from Laboratory
Animal Center of Zhejiang University (Hangzhou, China), weighing 180
6 10 g, were fasted overnight before treatment. Tissue processing was
performed at 48C. Briefly, freshly obtained rat liver was cut into small
pieces and washed with physiological saline. The samples were
Chirality DOI 10.1002/chir

90
GAO ET AL.
Fig. 7. Schizolysis spectrum of MA-CoA thioester and CoA.
2 h, acidified with 20 ll of 5M HCl, and treated as Chiral derivatization
and HPLC analysis to assay for S- and R-MA.
Chiral Inversion of MA Enantiomers in SD Rat Hepatocyte
The in vitro results showed that chiral inversion of S-MA
to R-MA was observed in rat hepatocyte (Fig. 4). The
concentrations of R-MA inverted from S-MA at different
times were shown in Figure 5.
In addition, the time-response curve and concentration-response curve
of S-MA-CoA thioester in rat liver homogenate and subcellular fractions
were also investigated. The incubation mixtures were the same with
above. To perform the tests for time-response curve, the incubation mix-
tures were incubated for different times, 5, 15, 30, 45, and 60 min. To
perform the tests for concentration-response curve, the final enzyme
concentrations were set at 1, 2, 5, and 10 mg/ml (mitochondrial was 1,
2, and 4 mg/ml) and the incubation time was set for 45 min. Samples
were treated as described in the previous section. Chiral derivatization
and HPLC analysis to determine the R-MA.
Chemical Synthesis, Racemization and Hydrolysis of
S-MAndelic Acid-CoA Thioester
Synthesis and identification of S-MA-CoA thioester. The syn-
thesized and purified S-MA-CoA thioester was lyophilized to
a yellow powder solid. It was identified to have the parent
ion fragmentation of m/z 900 ([MA-CoA-H]2) with MS (Fig.
6A). Among all the individual daughter ions of parent ion
fragmentation m/z 900 obtained by MS/MS, we could see
that except the fragmentation of m/z 766 ([CoA-H]2), the
other daughter ions m/z 686, 408, 338, 159, and 134 were
the same with that of CoA. In other words, the spectra
obtained were in accordance with the fragmentation patterns
of CoA (Fig. 6B, 6C, and 7). So it was confirmed that the syn-
thesized and purified compound was the target molecule.
RESULTS
Nonenzyme Metabolism of MA Enantiomers In Vitro
Chiral inversion did not occur between MA enantiomers
after 24 h incubation in pH 7.4 and 1.5 phosphate buffers. It
indicated that MA configuration is stable and S-MA and R-
MA cannot be inverted to each other in gastro-intestine with-
out enzymes.
Incubation of S-MA-CoA thioester with rat liver homogenate and
subcellular fractions. S-MA-CoA thioester was rapidly hydro-
lyzed by rat liver homogenate with formation of R-MA (Fig.
8). The same phenomenon was also observed in rat S9, cyto-
solic, and mitochondrial fractions (Fig. 9). It indicated the
Chiral Inversion of MA Enantiomers in Intestinal Bacteria
Chiral inversion of S-MA to R-MA was not observed after
incubated for 24 h with SD rats intestinal bacteria in vitro
(Fig. 3).
Chirality DOI 10.1002/chir

MOLECULAR MECHANISM OF CHIRAL INVERSION OF S-MA TO R-MA
91
Fig. 8. Chromatograms of S-MA-CoA thioester incubated with SD rats liver homogenate after derivatization. (A) 0 h incubation; (B) S-MA-CoA incubated for
2 h; (C) R-MA standard; (D) S-MA standard. 1: R-MA; 2: S-MA. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
racemization of S-MA-CoA thioester to R-MA-CoA thioester
and subsequent release of R-MA, which suggests that one of
the mechanisms of chiral inversion of MA is probably similar
with that of 2-arypropionic acids.
a function of enzyme concentrations. From 1 to 5 mg/mL pro-
tein concentration, the curve had better linearity.
DISCUSSION
Up to now, the mechanism of one-directional chiral inver-
sion of MA in mammal has never been able to explain, and
the corresponding report is rather rare. Generally speaking,
the change of chiral drug configuration may be mainly
caused by three mechanisms.
One is nonenzymatic mechanism, the chiral drug is unsta-
ble and will undergo isomerization in the certain environ-
ment (acidic or basic or highlight). For instance, stiripentol
in the acidic environment and thalidomide in aqueous media
The Time-Response Curve and Concentration-Response
Curve of S-MA-CoA Thioester in Rat Liver Homogenate
and Subcellular Fractions
The time-response curve and concentration-response curve
for rat liver homogenate and subcellular fractions were shown
in Figure 10. For the time-response curve, one could see that
the reaction rate was almost linear until 45 min. For the
concentration-response curve, the metabolic rate increased as
Chirality DOI 10.1002/chir

92
GAO ET AL.
Fig. 9. Chromatograms of S-MA-CoA thioester incubated with SD rat liver S9, cytosol protein and mitochondria protein after derivatization. (A) S9; (B) cytosol
protein; (C) mitochondria protein; (D) R-MA Standard 1: R-MA-naphthylethylamide derivative. The three curves from top to the bottom respectively mean chromato-
gram after 2 h incubation, chromatogram before incubation and chromatogram of blank system. [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
can undergo chemical racemization.15,16 In this study, we
simulated the physiological environment of stomach and
intestine to investigate the chiral inversion of MA. The result
showed MA enantiomers were stable under the environment
of pH 1.5 and 7.4, which confirmed the idea of that the chiral
inversion of MA was catalyzed by the enzymes in SD rat.
The second is bacteria mechanism. Extensive metabolic
inversion of MA mediated by mandelate racemase was
observed in bacteria.12,13 Intestinal bacteria are also able to
contribute to inversion of one enantiomer to its antipode in
the organism. The potential contribution of intestinal bacteria
has been demonstrated to chiral inversion of flosequinan.17
Chiral inversion at sulfoxide position of flosequinan enan-
tiomers occurred in conventional rats but not in either germ-
free rats or rats treated with antibiotics. Several strains of in-
testinal bacteria stereoselectively reduced flosequinan to
give sulfoxide, followed by oxidation of the sulfoxide in the
body to produce the antipode of flosequinan. Here according
to our research, rat intestinal bacteria have nothing to do
with the chiral inversion of MA.
The last is enzymatic mechanism, which based on two op-
posite metabolic processes or the reversible metabolic path-
way and result in chiral inversion. According to some litera-
tures, MA was thought to be stereoselectively metabolized
by rat alcohol dehydrogenase.18 Hence, we hypothesized
that the chiral inversion of MA may mediate via oxido-reduc-
Chirality DOI 10.1002/chir

MOLECULAR MECHANISM OF CHIRAL INVERSION OF S-MA TO R-MA
93
Fig. 10. The reactive curve (A) and concentration curve (B) of S-MA-CoA thioester metabolism by rat liver homogenate and subcellular fractions (n 5 3).
tion of keto-alcohol. We expressed the recombinant proteins
of rat alcohol dehydrogenase 1 and aldo-keto reductase 1A1
by pET Escherichia coli system and these recombinant pro-
teins were incubated with MA or PGA to investigate whether
the chiral inversion was mediated by alcohol dehydrogenase
and aldo-keto reductase.19 The result suggested that chiral
inversion of S-MA in rat may not be mediated through reac-
tions of oxidoreduction by alcohol dehydrogenase 1 and
aldo-keto reductase 1A1. Whether the other isoforms of alco-
hol dehydrogenase and aldo-keto reductase were involved in
this process needs much more in-depth investigation.
In parallel with the previous result of our laboratory,20 we
also found S-MA could be biotransformed to R-MA in SD rat
hepatocytes, which showed rat liver is the right organ
where the chiral inversion takes place. It has been reported
that ketoprofen could undergo chiral inversion in rat
hepatocytes.21
: at first, thioesterification of R-enantiomer to R-ibuprofen
CoA via an adenosine monophosphate intermediate catalyzed
by the microsomal long-chain acyl-CoA synthetase; second,
enzymatic epimerization of R-ibuprofenoyl-CoA thioester cat-
alyzed by the cytosolic and mitochondrial 2-aryl-CoA-epimer-
ase to S-ibuprofenoyl-CoA via an enolate intermediate; at last,
nonstereoselective hydrolysis of the acyl-CoA thioesters by
one or more hydrolases to yield free S-ibuprofen.24
The chiral structure of MA is similar with 2-Arylpropionic
acid drugs. The difference is that in MA a-methyl group is
changed to hydroxyl group. The polarity of hydroxyl group
is much higher than that of methyl group so the chemical
properties between the two drugs should have some diver-
sity. In this article, we also investigated the chemical synthe-
sis, racemization and hydrolysis of S-MA-CoA thioester to
clarification whether the mechanism of the chiral inversion
of MA is similar with ibuprofen.
To date, 2-Arylpropionic acid derivatives are probably the
most frequently cited drugs exhibiting the phenomenon that
is best known as chiral inversion. Ibuprofen is the most
extensively studied example of unidirectional chiral inversion
in different animal species and man.22,23 The molecular
mechanism of chiral inversion of profens involves three steps
The direct synthesis of S-MA-CoA thioester was tried
using several procedures according to literatures about
2-APA-CoA thioester synthesis. At first, we tried Ulrik Side-
nius method,25 1,10-Carbonyldiimidazole was used to react
with the carboxylic acid at room temperature in a nitrogen
atmosphere, then to the reaction mixture was added CoA to
Fig. 11. The probable mechanism of chiral inversion of S-MA according to our investigation.
Chirality DOI 10.1002/chir

94
GAO ET AL.
4. Manini P, Andreoli R, Poli D, Palma GD, Mutti A, Niessen WMA. Liquid
chromatography/electrospray tandem mass spectrometry characteriza-
tion of styrene metabolism in man and in rat. Rapid Commun Mass Spec-
trom 2002;16:2239–2248.
form the acyl-CoA. The acyl-CoA thioesters of eight carbox-
ylic acids [ibuprofen, clofibric acid, indomethacin, fenbufen,
tolmetin, salicylic acid, 2-phenoxypropionic acid and
(4-chloro-2-methyl-phenoxy) acetic acid (MCPA)] were suc-
cessfully synthesized by this method, but it did not work for
S-MA-CoA thioester synthesis.
5. Wang JZ, Wang XJ, Tang YH, Shen SJ, Jin YX, Zeng S. Simultaneous
determination of mandelic acid enantiomers and phenylglyoxylic acid in
urine by high-performance liquid chromatography with precolumn
derivatization. J Chromatogr B Analyt Technol Biomed Life Sci 2006;
840:50–55.
Then we tried Carabaza et al.’s method26: oxalyl chloride
was added to a solution of MA in methylene chloride, the
solution was stirred for 1 h under an argon atmosphere and
then evaporated to an oily residue with a stream of argon.
The residue was added dropwise into a stirred solution of
sodium Coenzyme A in H2O adjusted to pH 8.9 with 0.5M
NaOH. Mart´ın et al.’s method27 was performed also: trifluoro-
acetic acid, anhydride, and thioglycollic acid were used to
react with ibuprofen formed ibuprofen-thioglycollate and
finally react with CoA to form ibuprofen-CoA. Unfortunately,
none of them was successful for MA-CoA thioester synthesis.
In the end, we decided to try the Hall’s method14 to syn-
thesis S-MA-CoA thioester indirectly. A series of a-hydroxy-
thiol esters were successfully prepared by this method. First,
equilibration of the glyoxal with the thiol was carried out in
the presence of Mg21 (0.5 equiv) in DMF solvent. The
resulting a-ketohemithiol acetal, which is presumably che-
lated to the Mg21, is subsequently converted to the corre-
sponding a-hydroxythiol ester by the addition of 2,6-dime-
thylpyridine. Increased isolated yields were realized by the
presence of trace amounts of hydroquinone that retarded oxi-
dation of the thiols to dimmers and by the use of the steri-
cally hindered base 2,6-dimethylpyridine, rather than pyri-
dine or triethylamine, which catalyzes the hydrolysis of the
a-hydroxythiol esters during work-up.
6. Mingyue M, Tomohiro U, Yuko M, Yingyan G, Yasuaki S, Fumihiro S,
Toshio K, Reiko K. Influence of genetic polymorphisms of styrene-metab-
olizingenzymes and smoking habits on levels of urinary metabolitesafter
occupational exposure to styrene. Toxicol Lett 2005;160:84–91.
7. Gagnaire F, Chalansonnet M, Carabin N, Micillino JC. Effects of sub-
chronic exposure to styrene on the extracellular and tissue levels of dopa-
mine, serotonin and their metabolites in rat brain. Arch Toxico 2006;
80:703–712.
8. Junichi M, Megumi N, Zhao WY, Kazuo A. Neurophysiological changes
in rats subchronically treated with styrene or its metabolite. J Occup
Health 2001;42:328–335.
9. Herold BC, Scordi BI, Cheshenko N, Marcellino D, Dzuzelewski M,
Francois F. Mandelic acid condensation polymer: novel candidate
microbicide for prevention of human immunodeficiency virus and herpes
simplex virus entry. J Virol 2002;76:11236–11244.
10. Drummond L, Caldwell J, Wilson HK. The stereoselectivity of 1, 2-
phenylethanediol and mandelic acid metabolism and disposition in the
rat. Xenobiotica 1990;20:159–168.
11. Kenyon GL, Hegeman GD. Mandelic acid racemase from Pseudomonas
putida. Evidence favoring a carbanion intermediate in the mechanism of
action. Biochem 1970;9:4036–4043.
12. Maurice SM, Bearne SL. 2 Kinetics and thermodynamics of mandelate
racemase catalysis. Biochemistry 2004;1:4048–4058.
13. Hegeman GD, Rosenberg EY, Kenyon GL. Mandelic acid racemase from
Pseudomonas putida. Purification and properties of the enzyme. Biochem-
istry 1970;9:4029–4036.
14. Hall SS, Doweyko LM, Doweyko AM, Zilenovski JS. Synthesis and evalu-
ation of alpha-hydroxythiol esters as antitumor agents and glyoxalase I
inhibitors. J Med Chem 1977;20:1239–1242.
S-MA-CoA thioester was racemized and hydrolyzed to
R-MA in rat liver homogenate. Thus it gives us a hint that
one of the mechanisms of MA chiral inversion might be simi-
lar with that of 2-Arylpropionic acid drugs. Thioesterification
of S-MA to S-MA-CoA thioester was taken place in priority,
and then isomerized to R-MA-CoA thioester via an enolate
intermediatetic, at last nonstereoselective hydrolyzed of the
R-MA-CoA thioesters to yield free R-MA (Fig. 11).22,23,27,28
S-MA-CoA thioester was also racemized and hydrolyzed to
R-MA in rat S9, cytosolic and mitochondrial fractions. It is
paralleled with the report of that R-ibuprofen-CoA thioester
hydrolyzed to S-ibuprofen in rat microsome and mitochon-
drial fractions as same as in liver homogenate.29 Either of
the experimental result demonstrates the chiral inversion of
MA could happen in rat liver homogenate, mitochondrial
fractions and so on. There is no location restriction for the
chiral inversion. According to literature, there are a number
of hydrolases which could possibly catalyze the hydrolysis
reaction of thioesters. Therefore, whether the hydrolases cat-
alyze the hydrolysis reaction of S-MA-CoA thioester and R-
ibuprofen-CoA thioester are similar still remains unknown
and needs more investigation.
15. Zhang K, Tang C, Rashed M, Cui D, Tombret F, Botte H, Lepage F, Levy
RH, Baillie TA. Metabolic chiral inversion of stiripentol in the rat. I.
Mechanistic studies. Drug Metab Dispos 1994;22:544–553.
16. Reist M, Carrupt PA, Francotte E, Testa B. Chiral inversion and hydroly-
sis of thalidomide: mechanisms and catalysis by bases and serum albu-
min, and chiral stability of teratogenic metabolites. Chem Res Toxico
1998;11:1521–1528.
17. Kashiyama E, Yokoi T, Todaka T, Odomi M, Kamataki T. Chiral inver-
sion of drug: role of intestinal bacteria in the stereoselective sulphoxide
reduction of flosequinan. Biochem Pharmaco 1994;48:237–243.
18. Ma M, Umemura T, Mori Y, Gong YY, Saijo Y, Sata F, Kawai T, Kishi R.
Influence of genetic polymorphisms of styrene-metabolizing enzymes
and smoking habits on levels of urinary metabolites after occupational
exposure to styrene. Toxicol Lett 2005;160:84–91.
19. Gao LB, Wang JZ, Zeng S. Cloning, expression and the application of
human, rat alcohol dehydrogenase and aldo-keto reductase. Acta Pharma
Sin 2009;44:778–784.
20. Gao LB, Wang JZ, Yao TW, Zeng S. Stereoselective metabolism of man-
delic acid in rat, mouse and rabbit tissue preparations. Chin J Pharmacol
Toxicol 2009;23:351–355.
21. Soglowek MS, Geisslinger G, Brune K. Metabolic chiral inversion of 2-
arylpropionates in different tumor cell lines. Agents Actions Suppl
1993;44:23–29.
22. Sanins SM, Adams WJ, Kaiser DG, Halstead GW, Hosley J, Barnes H,
Baillie TA. Mechanistic studies on the metabolic chiral inversion of R-ibu-
profen in the rat. Drug Metab Dispos 1991;19:405–410.
LITERATURE CITED
1. Filser JG, Kessler W, Csana´dy GA. Estimation of a possible tumorigenic
risk of styrene from daily intake via food and ambient air. Toxicol Lett
2002;126:1–18.
23. Baillie TA, Adams WJ, Kaiser DG, Olanoff LS, Halstead GW, Harpootlian
H, Van Giessen GJ. Mechanistic studies of the metabolic chiral inversion
of (R)-ibuprofen in humans. J Pharmacol Exp Ther 1989;249:517–523.
2. Speita G, Henderson L. Review of the in vivo genotoxicity tests per-
formed with styrene. Mutat Res 2005;589:67–79.
24. Wsol V, Skalova L, Szotakova B. Chiral inversion of drugs, coincidence
or principle? Curr Drug Metab 2004;5:517–533.
3. Limasset JC, Simon P, Poirot P, Subra I, Grzebyk M. Estimation of the
percutaneous absorption of styrene in an industrial situation. Int Arch
Occup Environ Health 1999;72:46–51.
25. Sidenius U, Skonberg C, Olsen J, Hansen SH. In vitro reactivity of
carboxylic acid-CoA thioesters with glutathione. Chem Res Toxicol 2004;
17:75–81.
Chirality DOI 10.1002/chir

MOLECULAR MECHANISM OF CHIRAL INVERSION OF S-MA TO R-MA
95
26. Carabaza A, Suesa N, Tost D, Pascual J, Gomez M, Gutierrez M, Ortega
E, Montserrat X, Garcia AM, Mis R, Cabre F, Mauleon D, Carganico G.
Stereoselective metabolic pathways of ketoprofen in the rat: incorporation
into triacylglycerols and enantiomeric inversion. Chirality 1996;8:163–172.
28. Nakamura Y, Yamaguchi T, Takahashi S, Hashimoto S, Iwatani K,
Nakagawa Y. Optical isomerization mechanism of R(2)hydratropic acid
and derivatives. J Pharmacobiodyn 1981;4:s–l.
29. Knihinicki RD, Day RO, Williams KM. Chiral inversion of 2-arylpropionic
acid non-steroidal anti-inflammatory drugs-II. Racemization and hydro-
lysis of (R)- and (S)-ibuprofen-CoA thioesters. Biochem Pharmacol
1991;42:1905–1911.
27. San Mart´ın MF, Soraci A, Fogel F, Tapia O, Islas S. Chiral Inversion of
(R)-(2)-fenoprofen in guinea-pigs pretreated with clofibrate. Vet Res
Commun 2002;26:323–332.
Chirality DOI 10.1002/chir