Focusing Review

Chromatography, Vol.24 No.1 (2003)

Focusing Review

Highly Selective Molecular Recognition of Biologically Active
Substances Using Liquid Phase Separation
Nariyasu Mano1＊, Naoki Asakawa2 and Junichi Goto1,3
1

Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Sendai 980−8578, Japan
2

3

Tsukuba Research Laboratories, Eisai Co., Ltd., 5−1−3 Tokodai, Tsukuba, Ibaraki 300−2635, Japan

Department of Pharmaceutical Sciences, Tohoku University Hospital, 1−1 Seiryo−machi, Aoba−ku, Sendai 980−8574, Japan
Received January 7, 2003 Revised manscript received February 5, 2003 Accepted February 5, 2003

Abstract
The development of new chiral stationary phases has been very important in the accurate analysis of drug enantiomers and their metabolites inbiological samples during drug discovery and development. New chiral stationary phases have been developed using conalbumin and flavoprotein from chicken egg whites, which have been applied to a broad range of drug enantiomers. The application and characterization of thesetwo chiral columns for high−performance liquid chromatography have been documented. Both specific and non−specific interactions, basedon the silica gel surface and linker moiety, influenced retention and chiral separation of solutes. Interactions between drug enantiomers andproteins, as a pseudo chiral stationary phase, were investigated with affinity capillary electrophoresis, in order to avoid the effects of non−specific interactions. The chiral discrimination region for ketoprofen on the flavoprotein surface was concluded to consist of an α−helix structure.
Studies with chemically modified flavoprotein indicated that two types of interactions at the chiral discrimination region were required forchiral separation: a π−π interaction between a tryptophan residue and the aromatic ring of ketoprofen, and an ionic interaction between the
carboxyl group of ketoprofen and an amino and carboxyl group of the protein.
In the body, drugs and biologically active substances having a carboxyl group have been known to transform various metabolites such as acyl
glucuronide. The acyl adenylate has also been noted as a chemically active intermediate of coenzyme A ligation. Both the acyl adenylate andthe acyl glucuronide produced protein adducts by reacting with nucleophilic groups such as amino groups on protein molecules. To characterize both active intermediates and protein adducts, analytical techniques conferring highly selective molecular recognition, such as high−performance liquid chromatography and mass spectrometry, were required.
Keywords: protein−conjugated chiral stationary phase, affinity capillary electrophoresis, chiral discrimination, acyl glucuornide, acyl
adenylate, bile acid, protein−bound adductreceptors can therefore distinguish a specific ligand from others.

1. Introduction
Molecular recognition by biomolecules such as proteins is

In liquid chromatography, four basic mechanisms underlie re-complicated because many intermolecular interactions, including

tention and separation of solutes: adsorption chromatography, par-ionic interactions, hydrogen bonding, hydrophobic interactions,tition chromatography, ion−exchange chromatography, and sizeand coordinate bonding, function between binding sites on the pro-exclusion chromatography. Except for size exclusion chromatogra-

tein surface and the ligand. Biopolymers possess the ability tophy, each of these depends on hydrogen bonding, hydrophobic in-clearly discriminate between different binding molecules at theteractions, van der Waal’s forces and ionic interactions, respec-

binding site. The affinity of binding depends on the sum total of

tively. For example, the retention of solutes to the stationary phasethese interactions, and proteins such as enzymes, antibodies, andcan mainly be explained by hydrophobic interactions in the re-

TEL: +81−22−217−6819, FAX: +81−22−217−6816*E−mail: n−mano@mail.pharm.tohoku.ac.jp―１９―Chromatography, Vol.24 No.1 (2003)

Figure 1. Structures of drugs and bile acids used in this review.

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Nariyasu Mano, Naoki Asakawa and Junichi Gotoversed−phase partition liquid chromatography. Such hydrophobic

conducted on optical resolution by high−performance liquid chro-interactions play a key role in anchoring molecules to binding sites

matography (HPLC), and the direct resolution of racemic com-on biopolymers. Chromatographic analysis of retention and separa-pounds has been achieved by use of many chiral stationary phasestion mechanisms of solutes can be performed, and understanding(CSPs). The usefulness of protein−conjugated columns was firstthese mechanisms may lead to a more complete understanding ofdemonstrated by Allenmark et al. [1−3] and Hermansson [4]. Al-intermolecular interactions between biologically active substanceslenmark et al. successfully resolved acidic compounds using a bo-

and biopolymers.

vine serum albumin−conjugated CSP, and Hermansson resolved
racemic amines using α1−acid glycoprotein−conjugated CSP.

In this review, the mechanism for molecular recognition isanalyzed for the interaction between drug enantiomers and proteins

Miwa et al. have developed a highly effective CSP using ovomu-

as chiral selectors. Affinity capillary electrophoresis (CE) is effec-

coid, an acid glycoprotein found in chicken egg whites [5]. Antive for this purpose. The combination of chemical modifications

ovomucoid column can achieve chiral resolution within a broadof the protein surface with spectroscopic analysis is also effective

range, and is quite resistant to variations in pH, heat, and organic

for analyzing the mechanism of interaction. On the other hand,solvents [6,7]. An ovomucoid column alone, used as a protein−highly specific molecular recognition and capturing of target mole-conjugated CSP, however, is not sufficient for separation of a great

cules are available for analyzing post−translational modifications

variety of drug enantiomers, and thus, the development of newof proteins. Accurate chromatographic separation is also important

CSPs is necessary.

for reliable analysis of unstable metabolites in biological fluids.
2.1. Development of protein−conjugated chiral stationary
2. Separation of drug enantiomers by protein−conjugatedphaseschiral stationary phases

There are two methods for immobilization of protein onto a

Chiral discrimination has been an issue in the development

support, immobilization by non−covalent conjugation and by cova-

and use of pharmaceutical drugs, because drug enantiomers canlent conjugation. Covalent conjugation of protein as a CSP onto ahave different pharmacokinetic properties and produce different

support provides superior reproducibility of immobilization, pro-physiological responses. For this reason, many studies have been

ducing reproducible retention and high stability. Haginaka et al.

Figure 2. Typical Chromatograms of each compound on a flavoprotein−conjugated column: (A) ketoprofen;(B) ibuprofen; (C) flurbiprofen; (D) α, ε−dibenzoyllysine; (E) warfarin; (F) benzoin.
Conditions: mobile phase, (A), (B), (C), (E) 50 mM KH 2 PO4 /ethanol (9:1), (D) 50 mM potassium phosphate buffer (pH 4.0) /ethanol (94:6), (F) 50 mM potassium phosphate buffer (pH 5.6) /tert.−butanol (96:4);
detection, UV at 230 nm; flow rate, 1.0 mL/min; column temperature, room temperature.

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compared the efficiency of immobilization of the epoxide and ac-

Wainer and co−workers reported that k/ (k+1) showed a linear rela-tive ester method, and found that the latter is more efficient as antion to protein−binding capacity when human serum albumin was

immobilization method [8].

employed as a chiral selector and allosteric effects were demonstrated [13−15]. Therefore, protein binding is essential for retention

Conalbumin is an egg−white protein that binds iron, copper,and chiral separation on protein−CSPs [16−18].

manganese, and zinc at pH 6 or above, and acts to block the growthof bacteria. Its molecular weight is approximately 70000−78000,

The retention and chiral separation properties of protein−CSPs

and its pI value is 6.1−6.6. Conalbumin was immobilized onto an

have been investigated in detail, and the results indicated that theaminopropyl silica gel support activated with N, N−disuccinimidylhydrophobic and ionic interactions between enantiomers and acarbonate [9], and this new CSP achieved chiral separation for achiral recognition moiety were important for chiral separation withbasic compound, azelastine, and was also able to separate drug

each CSP. In addition, protein binding of enantiomers of native

enantiomers in a plasma sample with high sensitivity, using col-proteins was examined, and enantiomers, which displayed signifi-

umn−switching liquid chromatography/frit−fast atom bombard-

cant protein binding ability, showed strong retention in chromatog-

ment mass spectrometry [10].

raphy [19]. Also, each CSP had a similar property in that enanti-

Flavoprotein is a glycoprotein made up of 14% carbohydrate,omers, which showed significant differences in protein binding

consisting of mannose, galactose, and glucosaminide. Its pI value

ability, were well resolved by chromatography. The results, how-

is 3.9−4.1, which is similar to that of ovomucoid (3.9−4.3), and its

ever, also suggested that retention of solutes on protein−CSPs de-molecular mass is 32000−36000. Interestingly, it also has the abil-pended on different contributions from non−specific interactions.

ity to bind riboflavin at a 1:1 ratio at pH 4.0 or above. This protein

Enzymes can efficiently discriminate between drug enanti-

plays an important role in the transfer of riboflavin from blood toomers, contributing to differences in metabolism and biological ac-

egg whites. It is very stable to heat, retaining its riboflavin−binding

tivity between enantiomers. Since chiral discrimination by proteinscapacity after heating to 100℃ at pH 7.0 for 15 min. The flavopro-is due to differences in the affinity of drug enantiomers for bindingtein−conjugated CSP performed chiral separation of acidic, weakly

sites, chiral separation on protein CSPs can be displayed by a gen-acidic, and neutral compounds as shown in Fig. 2 [11]. Racemic 2−

eral equation including the association constants [20]. The interac-arylpropionates are extensively used in clinical medicine as anti−

tions between solutes and protein CSPs require consideration of theinflammatory drugs. These profens, ketoprofen, ibuprofen and flur-fact that the solute interacts not only with specific binding sites on

biprofen, were separated using 50 mM KH 2 PO4 (pH 4.6)/ethanola protein molecule, but also with non−specific regions, such as the(9:1, v/v) as a mobile phase, whereas ibuprofen and flurbiprofen

surface of the silica support, aminopropyl spacer, and linkage re-were not appreciably resolved, despite their similar structures. α, εgion (Fig. 3). The theoretical equations for calculating the retention−Dibenzoyllysine, an amino acid derivative and another carboxylic

factor (k) and enantioselectivity (α) of protein CSPs were createdacid used in this experiment, also did not give good resolution with
50 mM potassium phosphate buffer (pH 4.0) /ethanol (94:6, v/v) asa mobile phase. On the other hand, both warfarin and benzoin,
which are weakly acidic and neutral compounds, respectively, gave
good results for chiral separation using 50 mM KH 2 PO4 (pH 4.6) /ethanol (9:1, v/v) and 50 mM potassium phosphate buffer (pH 5.6)
/tert.−butanol (96:4, v/v), respectively.
2.2. Affinity of drug−protein for chiral discrimination
HPLC, using a protein−conjugated CSP (protein−CSP) and an
aqueous mobile phase, can separate a broad range of enantiomers,
and is effective for pharmacokinetic and toxicokinetic studies during drug discovery and development. Many kinds of protein−CSPs
have been reported [1−5, 9, 11, 12]. The mechanism for chiral discrimination and retention with protein−CSP, however, has not yet
been clarified in detail, because proteins are complex biopolymers
consisting of l−amino acid residues. They are capable of numerous
interactions with small molecules, such as ionic interactions, hy-

Figure 3. Schematic illustration of the interaction of solutes
with a protein−CSP.

drophobic interactions, hydrogen bonding, and π−π interactions.

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Nariyasu Mano, Naoki Asakawa and Junichi Goto

protein−solute complex, Vpro and Vnsp are the volume of the pro-as follows: tein as a CSP and that of the non−specific regions in the column,



kcsp

Kp [P] Vpro VnspVm

Kp2 [P] Vpro Knsp Vnsp
Kp1 [P] Vpro Knsp Vnsp

Vm is the mobile phase volume, and Knsp is the distribution constant of solute molecules in the non−specific phase. These equa-     

tions show that the k and α values are influenced by the association
constant, because [P], Vpro, Vnsp, Vm, and Vnsp are the eigenvalues for a column.

where the association constant (Kp) between solute and protein

The validity of the above theory was verified by experiments

molecules is [PS] / ([S]•[P]), [P] is the molar concentration of pro-using a flavoprotein−CSP, in combination with variable numbers

tein molecules in the mobile phase, [S] is the molar concentrationof non−specific stationary phases in series. The results indicatedof solutes in the mobile phase, [PS] is the molar concentration ofthat specific and non−specific interactions contribute differently tothe retention of ketoprofen enantiomers at pH 4.0 and 5.0 as shownin Fig. 4 and Table 1. At pH 4.0, the specific interaction with theprotein has a greater effect than the non−specific interaction in theretention of both enantiomers. On the other hand, the ratio of thenon−specific interaction at pH 5.0 is greater than that at pH 4.0.
The ionic interaction between the carboxyl group in the ketoprofen
molecule may be the predominant factor in the non−specific interaction. Overall, the results support the validity of the complex interactions. Thus, to understand chiral separation on protein−CSP,
all interactions, including non−specific interactions must be taken
into consideration. That is to say, chiral discrimination observedwith native proteins is not necessarily reflected in chromatographic
separation, because of effects of non−specific interactions.

Table 1. Effect of non−specific interaction on k and α values.
1. Experimental values

2. Caluculated values
Figure 4. Chromatograms obtained in minicolumn experiments.
A flavoprotein−CSP minicolumn (4.0 mm I.D. x 10mm) was used in combination with various numbers(n = 0−4) of non−specific stationary phase minicolumns.
Conditions: mobile phase, 20 mM potassium phosphate buffer (pH 4.0 or 5.0); flow rate, 0.5 mL/min;detection, UV at 254 nm; column temperature, 25℃;
injection amount, 40 ng/5 µL as racemate.

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Chromatography, Vol.24 No.1 (2003)of the carboxyl group in ketoprofen and a component of the spe-

2.3. Analysis of the molecular recognition mechanism using affinity capillary electrophoresis

cific binding site was reduced by the methanol−induced conforma-

Kaliszan et al. investigated the relationship between chiraltional change in flavoprotein, whereas hydrophobic interactions

separation and physicochemical properties of solutes such as hy-

were unaffected. In addition, the decreased binding capacity at

drophobicity, molecular size, and excess electron charge on the ni-

more than 25% methanol may be due to weakening of the interac-

trogen atom [21−23]. They concluded that solutes bind to the chiraltion between the carboxyl group of ketoprofen and an amino groupdiscrimination region through hydrophobic interactions and that ain the binding site.

nearby ionic region is also related to the chiral separation. Pinker-

In order to investigate the nature of the amino acid residue inton et al. found that only the third domain of turkey ovomucoid has

the chiral discrimination region that interacts with ketoprofen, thechiral discrimination capacity, and they considered that ionic inter-molecular ellipticity of flavoprotein was measured with various

actions, hydrophobic interactions, and hydrogen bonding all con-concentrations of ketoprofen solution. Flavoprotein has a weakly

tribute to chiral discrimination, on the basis of NMR and computa-

negative Cotton effect at 245 nm in methanol−acetic acid buffer

tional studies [24]. Protein−CSPs not only show specific binding,(pH 5.0, I=0.05) mixed solution (5:95) as shown in Fig. 6 A. Whenbut also non−specific interactions, e.g., at the silica gel surface, at
unreacted aminopropyl groups, and in the linkage region. It is very
Table 2. Effect of methanol content in running buffer on k and α
values in affinity CE.

difficult to investigate only the specific interaction involved inchiral discrimination by using HPLC [20]. Capillary electrophoresis (CE) has very high separation efficiency and many drug enantiomers have been separated by using various chiral selectors. Chiralseparation by CE, using protein as a pseudo stationary phase, hasbeen reported [25,26]. In CE, the protein used as a chiral selector isin a nearly native conformation in solution, so retention of the solute may reflect quite well the specific interactions between protein
and solute in solution. Lloyd et al. compared retention on HPLC
and CE with human serum albumin as a chiral selector, and foundthat the k value of benzoin on HPLC and CE showed a linear relationship in running buffer, containing various amounts of 1−propanol [27].
The content of organic modifiers greatly affected retention
and chiral separation in chiral protein HPLC, implying that hydrophobic interactions between solute and protein binding sites arevery important [28]. Table 2 shows the effect of the methanol content of the running buffer on binding and chiral separation in affinity CE. The k values increased slightly up to 20% methanol content, and then decreased at more than 25% methanol. Furthermore,they decreased with increasing ethanol content, in the range of 4−
12%, in HPLC. On the other hand, the α values decreased with increasing of methanol content, and the chiral separation of ketoprofen was not achieved at 25% or more methanol. Fig. 5 shows thechange in molecular ellipticity of flavoprotein, in relation to themethanol content of the running buffer. Increases in methanol content up to 20% caused a marked change of molecular ellipticity at

Figure 5. Effect of methanol content on the molar ellipticity at
245 nm of flavoprotein.
Conditions: sample concentration, 5 µM; solvent,acetate buffer (pH 5.0, I = 0.05) /methanol mixture;cell length, 1 mm; scan range, 200−350 nm; scan
speed, 50 mm/min; resolution, 0.1 nm; band width,
1.0 nm; accumulation, 5.

208 nm, reflecting conformational change of the secondary structure (α−helix) of flavoprotein, which coincides with a change of α
value in affinity CE. The decrease in chiral separation of ketoprofen with increasing methanol content seems to be closely related toa conformational change in flavoprotein. The behavior of the k values indicated that electrostatic repulsion between negative charges―２４―Chromatography, Vol.24 No.1 (2003)

Nariyasu Mano, Naoki Asakawa and Junichi Goto

Figure 6. CD spectra of (A) flavoprotein induced by binding of ketoprofen and (B) modified flavoproteins.
Conditions: sample concentration, 50 µM; solvent, acetate buffer (pH 5.0, I = 0.05) /methanol (95:5); cell length, 10 mm;
scan range, 240−350 nm; scan speed, 50 mm/min; resolution, 0.1 nm; band width, 1.0 nm; accumulation, 5.

Figure 7. Electropherograms of ketoprofen by affinity CE using (A) native, tryptophan− and tyrosine−modified flavoproteins and (B)native, carboxyl and amino group modified flavoproteins as chiral selectors.
(A−1, B−1) native flavoprotein, (A−2) HNBB flavoprotein, (A−3) O −acetyl flavoprotein, (B−2) amino−modified flavoprotein, (B−3) carboxy−modified flavoprotein.
Conditions: capillary, µSIL (linear polyacrylamide coated capillary, 375 µm O.D., 50 µm I.D.); total length, 40 cm; effective length, 25 cm; electrophoretic buffer, acetate buffer (pH 5.0, I =0.05) /methanol (95:5) containing 200 µM flavoprotein
or modified flavoprotein; applied voltage, 10 kV; detection, UV at 254 nm; sample amount, 1 mM.

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Chromatography, Vol.24 No.1 (2003)

the molar ratio of ketoprofen and flavoprotein was 1:1, the Cotton

electrophoretic mobility of the protein based on the change in na-

effect at 245 nm was enhanced, while it was decreased at a molar

tive charge. These results indicated that ionic interactions between

ratio of more than 2. Moreover, the addition of 100 µM of ketopro-a carboxyl group in ketoprofen and an amino group in the chiralfen removed this Cotton effect. These results suggest a strong inter-

discrimination region are important for chiral separation. A repul-

action between ketoprofen and the amino acid side chain, whichsive interaction between the carboxyl group of ketoprofen and acontributed to the Cotton effect at 245 nm.

carboxyl group of the protein, however, destabilizes the interaction.

Tryptophan, tyrosine, phenylalanine, and cysteine residues in-

These studies have established that the chiral recognition re-fluence the long−wavelength CD of proteins. Among them, trypto-

gion of flavoprotein for ketoprofen in affinity CE consists of an α−phan can be easily and specifically modified with 2−hydroxy−5−

helix structure, and the critical groups involved are a tryptophan

nitrobenzylbromide (HNBB) under aqueous conditions and tyro-

residue, an amino group, and a carboxyl group of the protein. At

sine can be specifically O−acetylated by N−acetylimidazole. Fig. 6

more than 25% methanol, the putative π−π interaction between the

B shows the CD spectra of native, HNBB−modified and O−acetyl−

aromatic rings of ketoprofen and the tryptophan residue is retained,

modified flavoprotein. The CD spectrum of O−acetyl−modified

so that binding capacity is largely maintained, although the α−heli-flavoprotein was similar in shape to that of native flavoprotein. In

cal structure is considered to be substantially denatured, thereby

contrast, HNBB−modified flavoprotein decreased both the native

abrogating the ionic interaction, and consequently, chiral discrimi-

Cotton effect at 245 nm and the positive Cotton effect at aroundnation.

280 nm. These results suggested that a tryptophan residue on the
2.4. Quantitative determination of drug enantiomers and theirflavoprotein surface is related to the flavoprotein−ketoprofen inter-metabolites

action. Fig. 7 A shows electropherograms of ketoprofen obtainedwith native, HNBB−modified, and O−acetyl−modified flavoprotein

E 3810 is an antiulcer agent which inhibits gastric acid secre-

as chiral selectors. With O−acetyl−modified flavoprotein, the mi-tion as a consequence of blocking an H+, K+−ATPase. The region

gration and chiral separation of ketoprofen were almost the same asaround the sulfur atom and the side chain on the pyridine ring of Ewith the native flavoprotein. The HNBB−modified flavoprotein,3810 is mainly metabolized. To confirm the utility of the flavopro-

however, showed diminished chiral discrimination, though the mi-tein column, E 3810 and its metabolites were applied with a col-gration time was almost unchanged. These results indicated that aumn−switching technique using an avidin−conjugated column for

tryptophan residue is involved in chiral discrimination of ketopro-

deproteinization, as a pretreatment column made it possible to per-fen by flavoprotein, and a π−π interaction between the aromatic

form automatic in−line analysis [29]. Typical chromatograms of aring of ketoprofen and the indole side chain of the tryptophan resi-

blank plasma sample and a sample spiked with E 3810 and its me-

due presumably plays an important part in chiral separation of ke-tabolites are shown in Fig. 8, A and B. A chromatogram of a

toprofen.

plasma sample obtained following the intravenous administration

Chiral discrimination by flavoprotein also appears to be influ-

of racemic E 3810 is shown in Fig. 8 C. (S)−(−)−E 3810 and (R)−enced by ionic interactions between the carboxyl group in ketopro-(+)−E 3810 were separated from one another on the flavoprotein

fen and ionic group (s) at the binding site. Therefore, carboxyl

column, in that order of elution, and M 3 enantiomers were alsogroups of glutamic acid and aspartic acid residues on the flavopro-

separated. All peaks of interest were clearly separated with chiraltein, whose pI values are 3.9−4.1, were modified by coupling withseparation by this column−switching system using the flavoprotein

glycine ethyl ester in the presence of 1−ethyl−3 (3−dimethylamino-column, and there was no interference at the retention time of any

propyl) carbodiimide, and amino groups of lysine and arginineof the eluted compounds of interest.

residues were modified by acylation using sulfosuccinimidyl ace-

A column−switching high−performance liquid chroma-tate. Fig. 7 B shows electropherograms of ketoprofen obtained with

tographic method using a flavoprotein column for simultaneous de-native, amino−modified, and carboxy−modified flavoprotein as

termination of drug enantiomers and their metabolites has beenchiral selectors. The modification of amino groups quenched chiraldemonstrated. This method employs an avidin column as a pre-separation capacity, and the peak shape was sharper than that withtreatment column for in−line deproteinization and concentration,

native flavoprotein, though the migration time was almost theallowing the direct injection of a large volume of plasma. This typesame. This phenomenon is considered to be due to loss of ionic in-of on−line automatic system using a protein−CSP allows simple,

teractions between the carboxyl group in ketoprofen and an amino

rapid, accurate, and precise determination of drug enantiomers andgroup in the chiral discrimination region. On the other hand, thetheir metabolites in biological samples, and should be applicable tocarboxyl−modified flavoprotein achieved chiral separation of keto-enantioselective pharmacokinetic studies of various drugs.

profen, although the migration time was longer due to reduced―２
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Nariyasu Mano, Naoki Asakawa and Junichi Goto

water−soluble forms, plays a significant role in the metabolism of
drugs and endogenous compounds. For a long time, this conjugation process, which is catalyzed by the hepatic glucuronosyltransferase, has been considered an important detoxification mechanism. Recent observations indicate, however, that compounds
which act as substrates for the transferase become more toxic upon
glucuronidation. Acyl glucuronides of carboxylic acid derivatives
are chemically active and irreversibly bind to proteins to produce
protein−bound adducts [30−32], which may result in hypersensitivity reactions to acidic compounds [33,34]. Therefore, the formationof acyl glucuronides by the hepatic glucuronosyltransferase requires further characterization.
In contrast, it has been proposed that during the activation of
the carboxyl group of biological interest to the acyl coenzyme A(CoA) thioester, which is known to be the intermediate for the formation of amino acid conjugates, the acyl adenylate is first formed
and acyl CoA synthetase may be responsible for its formation [35−37]. This acyl adenylate, which is a mixed anhydride consisting ofa carboxylic acid derivative and an adenosine monophosphate,shows high reactivity towards nucleophiles such as amino groups
on proteins. Most bile acids are metabolized to amino acid conjugates in the hepatocyte through the carboxyl group at position C−
24. Therefore, acyl adenylates can be produced as active intermediates during amino acid conjugations of bile acids, and both the biosynthesis of bile acid acyl adenylates and their reactivity to aminogroups on protein molecules requires further investigation.
3.1. Acyl glucuronide in the human body
Nonsteroidal anti−inflammatory drugs (NSAIDs), α−aryl-

Figure 8. Typical chromatograms of (A) blank beagle dog
plasma, (B) plasma spiked with 10 µg/mL of E 3810
enantiomers and 1 µg/mL of their metabolites and(C) plasma from a beagle dog 30 min after intravenous administration of racemic E 3810 (3 mg/kg).
Conditions: analytical column, flavoprotein column(4.6 mm I.D. x 250 mm); trapping column, avidin
column (4.0 mm I.D. x 10 mm); mobile phase fortrapping, 0.1 M potassium phosphate buffer (pH 7.5)
at a flow rate of 1.0 mL/min; mobile phase foranalysis, A) 20 mM potassium phosphate buffer (pH5.5) /acetonitrile (99:1), B) 20 mM potassium phosphate buffer (pH 5.5) /acetonitrile (1:1); gradient
program, 0% of solvent B for 30 min and 0 to 40%
of solvent B over 35 min at a flow rate of 1.0 mL/min; detection, UV at 290 nm.

propionic acid derivatives having an asymmetric center at the α−
position of the carboxyl group, are commonly used in racemate
form. Almost all NSAIDs are subjected to a chiral inversion of the
R −enantiomer into its counterpart, the S −enantiomer, through theacyl CoA thioester. Only the R −enantiomer is a substrate for hepatic acyl CoA thioester ligases. On the other hand, the enantiomerof flurbiprofen does not act as a substrate for acyl CoA thioester ligases [38], and therefore, there is no chiral inversion of flurbiprofen in humans [39], which suggests that producing amino acid conjugates of flurbiprofen by phase II metabolic reactions would be
difficult.
Glucuronidation is one of the major phase II metabolic pathways for endogenous compounds, drugs, and other xenobiotics.
The stereoselectivity of acyl glucuronosyltransferases towards
NSAIDs has been investigated. R−Flurbiprofen metabolism into its
acyl glucuronide was found to occur at a rate 2−fold higher thanthat of the S −enantiomer [40]. Furthermore, based on a kinetic

3. Analysis of low molecular weight compounds−protein ad-

study with rat liver microsomes, Magdalou et al. found a 5−foldductshigher rate of formation of flurbiprofen acyl glucuronide than

Glucuronidation, which converts lipophilic substances into―２７―

Chromatography, Vol.24 No.1 (2003)

ibuprofen acyl glucuronide [41]. Therefore, acyl glucuronidation,

target compounds [42]. This LC/ESI−MS method was effective forwhere a stereoselective reaction may take place, is regarded to be

the simultaneous resolution of S − and R −flurbiprofen glucuronides

the most important phase II reaction for flurbiprofen.

and an internal standard (IS), possessing a fine base−line separation

The elimination of coexisting substances, such as protein andwith a detection limit of 7.4 pg (17.6 fmol) /injection of the S −flur-inorganic salts in biological fluids, is a common prerequisite for the

biprofen glucuronide, at a signal−to−noise ratio of 10 under a se-separation and determination of trace compounds. Because acyl

lected ion monitoring mode.

glucuronides are chemically unstable due to the active ester bond,This method was applied to assay 11 human urine samples

biological samples must be rapidly stabilized or quenched by expo-

from healthy volunteers, who received a 40−mg tablet of flurbipro-

sure to acidic conditions to prevent degradation of biosynthetic

fen orally. Urine specimens were collected 3 hours after drug ad-

acyl glucuronides during sample handling and storage. Therefore,

ministration and subjected to the LC/ESI−MS analysis, with a se-urine specimens were immediately acidified with 10% trichlo-

lected ion−monitoring mode utilizing corresponding deprotonatedroacetic acid upon their collection. An LC/ESI−MS system with amolecules (m/z 419 for glucuronides and m/z 314 for IS). The typi-

simple column−switching technique was employed for an on−line

cal mass spectrum and chromatogram of urinary flurbiprofen glu-

pretreatment procedure, enabling the direct injection analysis ofcuronides are illustrated in Fig. 9 A and Fig. 9 B, respectively. The

Figure 9. (A) Electrospray negative ion mass spectrum of urinary (R)−flurbiprofen glucuronide and (B) typical selected ion recording of a urine sample.
Conditions: electrospray voltage, −2.5 kV; orifice voltage, 0 V; ring lens voltage, −60 V; orifice temperature, 150℃; desolvating plate temperature, 250℃; analytical column, TSKgel ODS−80 Ts (2.0 mm I.D. x
150 mm); trapping column, Inertsil ODS−3 (4.0 mm I.D. x 10 mm); mobile phase for trapping, 100 mM
ammonium acetate buffer (pH 4.0) at a flow rate of 1.0 mL/min; mobile phase for analysis, 20 mM ammonium acetate buffer (pH 5.6)/acetonitrile/ethanol (20:7:2) at a flow rate of 200 µL/min.

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Chromatography, Vol.24 No.1 (2003)

Nariyasu Mano, Naoki Asakawa and Junichi Goto

Table 3. Concentration of the flurbiprofen glucuronides in humanurine 3 hours after oral administration of 40 mg flurbiprofen tablet.

3.2. Production of protein bound adduct through acyl glucuronides
Bile acids are synthesized from cholesterol in hepatocytes andexcreted as glycine or taurine conjugates into the intestine via thebile duct. In the intestinal lumen, bile acids undergo deconjugationand dehydroxylation at the 7 α−hydroxy group by intestinal bacteria. These bile acids are then re−adsorbed from the ileum−proximal
colon and returned to the liver via the portal vein. Conjugation withsulfuric acid and glucuronic acid also takes place through the 3 α−hydroxy group of bile acids. The levels of sulfates in urine significantly increase in patients with hepatobiliary disease. Although the
conjugation of bile acids with glucuronic acid involves the hydroxyl group at the C−3 position on the steroid nucleus, the levelsof bile acid 3−glucuronides in human urine are very low [43]. Acylhigh−resolution mass values corresponding to deprotonated mole-

glucuronides of bile acids, conjugated through the carboxyl groupcules were 419.1130 and 419.1171 for the peaks corresponding toat C−24, have also been demonstrated to be present in human

the S − and R −flurbiprofen glucuronides (theoretical exact massurine. Bile acid acyl glucuronides were also detected at low levels

value: 419.1142) on the chromatogram, with mass errors of onlyin human urine obtained from both healthy subjects and from pa-

1.2 and 2.9 mMU, respectively. As shown in Table 3, the concen-

tients with hepatobiliary disease [44]. These acyl glucuronides

trations of R − and S −flurbiprofen glucuronides in human urine

were also preferentially biosynthesized following incubation withfrom healthy subjects were 6.8~29.4 µg/mL and 3.9~18.0 µg/mL,

rat hepatic microsomal fractions [45]. It has been reported that therespectively, suggesting that a slightly higher value of the acyl glu-

acyl glucuronide of a drug having a carboxyl group is capable ofcuronide exists for the R −enantiomer.

reacting with protein, such as human serum albumin, to form a covalent drug−protein adduct [32].

Figure 10. MALDI−TOF mass spectra of (A) the incubation mixture of LCA 24−G with dynorphin A and (B) the incubation
mixture of LCA 24−G and lysozyme.
Conditions: mass spectrometer, Voyager RP with a 337 nm pulsed nitrogen laser (Perseptive Biosystems); matrix,(A) α−cyano−4−hydroxycinnamic acid, (B) 3,5−dimethoxy−4−hydroxycinnamic acid; accelerating voltage, 25.0
kV; grid voltage, 14.0 kV; guide wire voltage, 25 V.

―２
９―Chromatography, Vol.24 No.1 (2003)

Table 4. Observed and calculated peptide fragment of covalent adduct produced by incubation of LCA 24−G and lysozyme.

LCA 24−G, which was effectively biosynthesized by the ac-compared with those obtained for the authentic lysozyme. As a re-tion of hepatic glucuronosyltransferase, was incubated with dynor-sult, the covalent LCA− and LCA 24−G−lysozyme adducts boundphin A, and the mass spectrum of the reaction mixture was ob-

through Lys−1 and Lys−97 of the protein were definitely con-tained using MALDI−TOFMS. In addition to the protonated form

firmed (Table 4).

of dynorphin A at m/z 1611.5, intestine ions were newly observed
3.3. Inhibition of acyl glucuronidation by bile acids

at m/z 1969.4 and 2145.7 along with the corresponding potassium
adduct ions (Fig. 10 A). The modified peptide A was the covalentAcyl glucuronides are hydrolyzed into aglycones under alka-binding product of LCA with the peptide through the Arg−1 or theline conditions and, in the presence of alcohol, easily generate theε−amino function of Lys−8 of dynorphin A, and the modified pep-corresponding esters of aglycones under neutral conditions. To

tide B was the peptide covalently bound to LCA 24−G.

characterize the rat hepatic bile acid glucuronosyltransferase, a reli-

The next lysozyme as a model protein was incubated withable method for determining such unstable 24−Gs was needed. LC/

LCA 24−G, and MALDI−TOFMS analysis gave only three ions at

ESI−MS in the negative ion detection mode was therefore em-

m/z 14306.5, 14665.5, and 14840.2, corresponding to lysozyme,

ployed, due to its high specificity and selectivity. Various amounts

protein−LCA adduct, and protein−LCA 24−G adduct, respectivelyof bile acids were also incubated with the enzyme preparation [47].

(Fig. 10 B). The lyophilized powder was then subjected to reduc-A saturation curve following the Michaelis theory was not ob-

tion and S−carboxymethylation. After protein digestion with ly-

served for LCA as a substrate, and the initial velocity of LCA glu-

sylendopeptidase, the obtained peptide fragment mixture was sub-curonidation decreased with increasing concentrations of substrates

jected to HPLC separation. The peak of interest was collected and(data not shown). This result strongly suggested that LCA had in-subjected to MALDI−TOFMS analysis. The mass values obtained

hibitory activity towards bile acid acyl glucuronidation. In humanfor the lysylendopeptidase digests of the modified lysozyme werebody fluids, most bile acids exist as glycine and taurine conjugates,

Figure 11. Effect of (A) amino acid conjugated CDCA, (B) unconjugated and amino acid conjugated UDCA and (C) bile
acid acyl glucuronides on the formation of CDCA acyl glucuronide.
Twenty µM of CDCA was incubated with microsomal preparations (400 µg of protein/mL) in the presence ofvarious amounts of bile acid derivatives as inhibitors at 37℃ for 10 min.

―３０―Chromatography, Vol.24 No.1 (2003)

Nariyasu Mano, Naoki Asakawa and Junichi Gotowhich are the analogues of substrates. The inhibitory effects of gly-

placing the AMP moiety with CoA. The carboxylic acid deriva-

cine and taurine conjugated CDCA on the formation of CDCA 24−

tives may also be condensed by AMP during the biosynthesis of

G were therefore investigated. Both glycine and taurine conjugates

those acyl−CoAs.

inhibited the metabolism of CDCA into its 24−G by 20−25%, as il-

To confirm the preferential formation of CA−AMP, preceding

lustrated in Fig. 11 A. Glycine and taurine conjugated UDCA also

the production of the CA−CoA, LC/ESI−MS was applied to theinhibited CDCA 24−G formation by 20−25%, and unconjugatedseparation and characterization of cholyl−adenylate in an incuba-

UDCA inhibited the glucuronidation of CDCA 2−fold more po-tion mixture with a rat liver microsomal fraction [48]. The potas-

tently than conjugated UDCAs (Fig. 11 B).

sium salt of cholic acid was incubated with a hepatic microsomal

The enzyme reaction product, the bile acid acyl glucuronide,

fraction from a male Wistar rat, and then the reaction mixture wasis a kind of derivatives of the substrate. LCA 24−G, as an inhibitor,

subjected to solid−phase extraction and a portion of the extract was

was therefore added into the incubation mixture. Addition of an

subjected to LC/MS analysis. Typical selected ion recordings areequal molar amount of LCA 24−G into the incubation mixture re-illustrated in Fig. 12 A. In the presence of ATP in the incubation

sulted in a 40% inhibition of acyl glucuronidation of CDCA (Fig.

mixture, the peak corresponding to cholyl−adenylate was detected,

11 C). In addition, the formation of CDCA 24−G was reduced bywhereas no peak of cholyl−adenylate on the chromatogram was ob-

25% in the presence of a 5−fold molar excess of LCA 24−G.

served in the absence of ATP in the incubation medium.

UDCA 24−G also inhibited the acyl glucuronidation of CDCA andCholic acid was then incubated in the presence of ATP withits degree of inhibition was almost identical to that of glycine andCoA. As shown in Fig. 12 B, peaks of not only cholyl−adenylatetaurine conjugated UDCA. CA 24−G in the incubation mixture re-but also of cholyl−CoA were detected. The amount of cholyl−

sulted in increased formation of the CDCA 24−G. Since the acyl

adenylate was increased by the addition of excess cholic acid, in

glucuronides seem to be an activated form of glucuronic acid, thecontrast to increasing relative amounts of cholyl−CoA by using

excess amounts of CA 24−G may act as a glucuronic acid moiety

low concentration of cholic acid as a substrate. These results may

donor, such as UDPGA.

indicate that enzymatic formation of cholyl−adenylate is preferredto the biotransformation of cholyl−CoA.
Since acyl−adenylates correspond to the activated form of car-3.4. Enzymatic formation of acyl adenylate
Prior to conjugation with glycine and taurine, the carboxyl

boxylic acids, it seems that cholyl−adenylate reacts with com-groups of bile acids must be activated by hepatic bile acid acyl−

pounds having an amino group. Therefore, the nonenzymatic con-

CoA synthetases, transforming them into the corresponding CoA

densation of cholyl−adenylate with taurine to produce taurocholate

thioester. This enzyme system, existing in a distinct compartment

was performed. The cholyl−adenylate was incubated with or with-from fatty acid−CoA synthetases, is localized to hepatic microso-

out taurine and a portion of the incubation mixture was subjected tomal fractions. The reaction mechanism governing the biosynthesis

an LC/MS analysis monitored with a deprotonated molecule hav-of fatty acyl−CoA proceeds via a ping−pong mechanism. The firsting an m/z 736 for cholyl−adenylate and 514 for taurocholate. As

step is the transfer of an adenyl group to form an acyl−adenylate

depicted in Fig. 12 C, cholyl−adenylate was transformed into tauro-(AMP) and pyrophosphate. The acyl−CoA is then formed by dis-cholate in the presence of taurine, exhibiting the nonenzymatic

Figure 12. Selected ion recordings of (A) an incubation mixture of cholic acid in the presence of ATP without CoAor (B) with CoA using male Wistar rat liver microsomal preparations (250 µg of protein) and (C) an incubation mixture of cholyl adenylate in the presence of taurine without any enzymes.

―３１―

Chromatography, Vol.24 No.1 (2003)

body is effective for the group separation of modified and unmodi-amidation of bile acids.

fied peptides as an efficient pretreatment procedure. Substance P(SP), a lysine−containing undecapeptide amide formed by the post-

3.5. Production of protein bound adducts through acyl
adenylatetranslational processing of preprotachykinin, was used as a modelTransacylation mechanisms to produce drug−protein adducts

peptide. The extraction of the adduct chemically synthesized SP

operate via nucleophilic substitution through the ε−amino group of

bound (S)−ibuprofen was performed using immobilized anti−(S)−lysine in proteins. The structural analysis of such drug−protein ad-

ibuprofen antibody, and can clearly distinguish the S−form fromducts is needed, and mass spectrometry is a powerful tool for thethe R−form [49]. This enrichment procedure is very useful for se-

structural characterization of proteins with low sample levels. This

lection of targets of post−translationally modified peptides frommethod can also characterize the binding sites of low molecularcomplex peptide fragment mixtures.

weight compounds on a covalently modified protein. Immunoaffin-

The deoxycholyl adenylate was incubated with an equimolar

ity extraction with a highly specific and selective immobilized anti-

amount of SP, and the reaction was monitored for 72 h by analyz-

Figure 13. (A) MALDI−TOF mass spectrum and (B) HPLC chromatogram of the reaction mixture resulting fromincubation of deoxycholyl adenylate with substance P.
Conditions: mass spectrometer, Voyager RP with a 337 nm pulsed nitrogen laser (Perseptive Biosystems); matrix, α−cyano−4−hydroxycinnamic acid; accelerating voltage, 25.0 kV; grid voltage, 14.0 kV;
guide wire voltage, 25 V; column, PRODIGY 5 u C 8 (4.6 mm I.D. x 150 mm); mobile phase, A, water/acetonitrile (9:1) containing 0.08% TFA, B, acetonitrile containing 0.08% TFA; gradient program, 1 to
50% of solvent B over 60 min; detection, UV at 215 nm.

Table 5. Observed and calculated m/z values of Lys−C digests of covalent adducts produced by incubation of CA−AMP
and lysozyme.

―３２―Chromatography, Vol.24 No.1 (2003)

Nariyasu Mano, Naoki Asakawa and Junichi Gotoacid residues or surface functions was also useful for analyzing in-ing a portion of the mixture by MALDI−TOFMS [50]. As illus+

trated in Fig. 13 A, in addition to [M+H] for SP at m/z 1348.8, ad-teractions between solutes and chiral discrimination regions. The

ditional abundant ions were detected at m/z 1723.4 and 2097.7.

interactions functioning on chromatography are the same as those

These values were shifted by 374 and 748 Da (∆M) from that ofinteractions between solutes and their binding sites on the protein

the unmodified SP, indicating the addition of one and two mole-surface, and they contribute a main part of binding to the active site

cules of DCA, respectively. Liquid chromatographic separation of

on enzymes. Acyl glucuronides and acyl adenylates produced by

the reaction mixture was carried out. The SP, deoxycholyl

the action of enzymes are chemically active compounds, which

adenylate, and DCA−SP adducts, represented as adducts A, B, andproduces protein adducts displaying immunogenicity in the body.

C, were effectively resolved (Fig. 13 B). The fractions correspond-

The enzymatic formation of these active chemicals and their reac-

ing to peaks A, B, and C were collected, and the components intivity towards amino groups on proteins has been demonstrated. To

these fractions were confirmed as the modified peptides by obser-

characterize both active intermediates and protein adducts, analyti-

vation of the [M+H]+ ions at m/z 1722.3, 1722.7, and 2096.7, re-cal techniques including highly selective molecular recognition

spectively, in the MALDI−TOF mass spectra. PSD analyses ofsuch as high−performance liquid chromatography and mass spec-these modified peptides were then carried out in order to establish

trometry are required.

the binding site of DCA. In the PSD mode, an amino acid sequence
Acknowledgmentsof a modified peptide can be established by comparison of the

The authors are very grateful to Honorary Professor T. Nam-product ion pattern of a modified peptide with that of an unmodi-

bara (Tohoku University), Professor S. Ikegawa (Kinki Univer-fied peptide.
Next, the ten−fold molar amount of cholyl adenylate was in-sity), and Professor N. Kobayashi (Kobe Pharmaceutical Univer-cubated with lysozyme as a model protein. The mass spectrum in-sity). The authors also thank many co−workers for their assistancedicated an abundant ion at m/z 15079.9, and a moderately−abun-and discussions; Dr. Y. Oda, Dr. Y. Ishihama and Dr. H. Katayama+

dant ion at m/z 15428.0, along with the [M+H] ion of unmodified

in Eisai Co., and the members of Professor Goto’s laboratory, in-lysozyme. These m/z values correspond to lysozyme modified withcluding graduates.

two and three molecules of CA, where the discrepancies between
the theoretical and observed mass values were 5 (0.03% error) and

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