Enantioselective hydrolysis of some 2-aryloxyalkanoic acid methyl esters and isosteric analogues using a penicillin G acylase-based HPLC monolithic silica column

Article abstract of DOI:10.1021/ac0204193

A technique based on liquid chromatography has been developed to facilitate studies of enantioselectivity in penicillin G acylase (PGA)-catalyzed hydrolysis of some 2-aryloxyalkanoic acid methyl esters and isosteric analogues. PGA was covalently immobilized on an aminopropyl monolithic silica support to create an immobilized HPLC-enzyme reactor. Two sets of experimental data were drawn to calculate the enantioselectivity (E) of the kinetically controlled enantiomer-differentiating reaction, the degree of substrate conversion and the enantiomeric excess of the product. The developed enzymatic reactor was coupled through a switching valve to an achiral analytical column for separation and quantitation of the hydrolysis products. The enantiomeric excess was determined off-line on a PGA-chiral stationary phase. In this way, highly precise E values were determined. A computational study related to the hydrolysis of the considered racemic esters was also carried out in order to unambiguously clarify both the substrate specificity and the enantioselectivity displayed by PGA.

Full text of DOI:10.1021/ac0204193

Anal. Chem. 2003, 75, 535-542
Enantioselective Hydrolysis of Some
2-Aryloxyalkanoic Acid Methyl Esters and Isosteric
Analogues Using a Penicillin G Acylase-Based
HPLC Monolithic Silica Column
|
Gabriella Massolini,*,† Enrica Calleri,† Antonio Lavecchia,‡ Fulvio Loiodice,§ Dieter Lubda,
Caterina Temporini,† Giuseppe Fracchiolla,§ Paolo Tortorella, Ettore Novellino,‡ and
Gabriele Caccialanza†
Dipartimento di Chimica Farmaceutica, Universita’ di Pavia, Via Taramelli 12, I-27100 Pavia, Italy,
Dipartimento di Chimica Farmaceutica e Tossicologica, Universita’ di Napoli “Federico II”, Via Domenico Montesano,
49, I-80131 Napoli, Italy, Dipartimento Farmaco-Chimico, Via Orabona 4, I-70126 Bari, Italy, LSP R&D MDA,
Merck KGaA, Frankfurter Str 250, D-64293 Darmstadt, Germany, and Dipartimento di Scienze del Farmaco,
Universita’ “G.D’Annunzio”, Via dei Vestini, I-66100 Chieti, Italy
strong affinity for the phenyl-acetyl moiety and a high tolerance
as far as the rest of the molecule is concerned. This capacity has
been used advantageously to achieve moderate-to-excellent ster-
eochemical discrimination between corresponding enantiomers
in the hydrolytic cleavage of the phenyl-acetyl group from
R-aminoalkylphosphoric acids, R-, â-, and γ-amino carboxylic acids,
sugar, amines, peptides, and esters of phenylacetic acid. However,
a detailed understanding of the structural factors affecting the rate
and stereochemistry of these reactions is still lacking, and a deeper
knowledge of the enzyme’s specificity could facilitate the engi-
neering of this catalyst, enhancing its potential for chemical and
industrial applications.
Recently, the X-ray structures of PGA complexed with a series
of phenylacetic acid analogues6 and with penicillin G/ penicillin
G sulfoxide ligands7 have been solved, shedding light on the
functional properties of PGA and on its catalytic mechanism. PGA
belongs to the newly recognized structural superfamily of N-
terminal nucleophile (Ntn) amidohydrolases.8 Other Ntn hydro-
lases identified so far are aspartylglucosaminidase (AGA),9 pro-
teasome (PRO),10 and glutamine-PRPP-amidotransferase (GAT).11
The enzymes of the superfamily share similar three-dimensional
folds and function analogously. The architecture of their active
sites is also similar with corresponding catalytic elements arranged
in analogous ways.8 On the basis of the X-ray structures and
biochemical studies, the well-known serine protease-like catalytic
A technique based on liquid chromatography has been
developed to facilitate studies of enantioselectivity in
penicillin G acylase (P GA)-catalyzed hydrolysis of some
2 -aryloxyalkanoic acid methyl esters and isosteric ana-
logues. P GA was covalently immobilized on an aminopro-
pyl monolithic silica support to create an immobilized
HP LC-enzyme reactor. Two sets of experimental data were
drawn to calculate the enantioselectivity (E) of the kineti-
cally controlled enantiomer-differentiating reaction, the
degree of substrate conversion and the enantiomeric
excess of the product. The developed enzymatic reactor
was coupled through a switching valve to an achiral
analytical column for separation and quantitation of the
hydrolysis products. The enantiomeric excess was deter-
mined off-lin e on a P GA-chiral stationary phase. In this
way, highly precise E values were determined. A compu-
tational study related to the hydrolysis of the considered
racemic esters was also carried out in order to unambigu-
ously clarify both the substrate specificity and the enan-
tioselectivity displayed by P GA.
Penicillin G acylase (PGA) is best known for the regioselective
hydrolysis of the side chains of penicillin G and penicillin V in
the commercial synthesis of 6-aminopenicillanic acid.1 PGA
selectivity toward substrates other than penicillin G has attracted
the interest of researchers since it has been demonstrated that
this enzyme is able to hydrolyze a large variety of amides and
esters containing aromatic acyl moieties.2-5 The enzyme shows a
(4) Rossi, D.; Romeo, A.; Lucente, G. J. Org. Chem. 1 9 7 8 , 43, 2576-2581.
(5) Baldaro, E.; D’Arrigo, P.; Pedrocchi-Fantoni, G.; Rosell, C. M.; Servi, S.;
Tagliani, A.; Terreni, M. Tetrahedron Asymmetry 1 9 9 3 , 4, 1031-1034.
(6) Done, S. H.; Brannigan, J. A.; Moody, P. C. E.; Hubbard, R. E. J. Mol. Biol.
1 9 9 8 , 284, 463-475.
* Corresponding author: (fax) +39 382 422975; (e-mail) g.massolini@unipv.it.
† Universita’ di Pavia.
(7) McVey, C. E.; Walsh, M. A.; Dodson, G. G.; Wilson, K. S.; Brannigan, J. A.
‡ Universita’ di Napoli “Federico II”.
J. Mol. Biol. 2 0 0 1 , 313, 139-150.
§ Dipartimento Farmaco-Chimico.
(8) Brannigan, J. A.; Dodson, G.; Duggleby, H. J.; Moody, P. C. E.; Smith, J. L.;
Tomchick, D. R.; Murzin, A. G. Nature 1 9 9 5 , 378, 416-419.
(9) Mononen, I.; Fischer, K. J.; Kaartinen, V.; Aronson, N. N. FASEB J. 1 9 9 3 ,
7, 1247-1256.
| Merck KGaA.
Universita’ “G.D’Annunzio”.
(1) Duggleby, H. J.; Tolley, S. P.; Hill, C. P.; Dodson, E. J.; Moody, P. C. E.
Nature 1 9 9 5 , 373, 264-268.
(10) Lo¨ we, J.; Stock, D.; Jap, B.; Zwickl, P.; Baumeister, W.; Huber, R. Science
(2) Fuganti, C.; Rosell, C. M.; Servi, S.; Tagliani, A.; Terreni, M. Tetrahedron
Asymmetry 1 9 9 2 , 3, 383-386.
(3) Rossi, D.; Calcagni, A.; Romeo, A. J. Org. Chem. 1 9 7 9 , 44, 2222-2225.
1 9 9 5 , 268, 533-539.
(11) Smith, J. L.; Zaluzec, E. J.; Wery, J. P.; Niu, L.; Switzer, R. L.; Zalkin, H.;
Satow, Y. Science 1 9 9 4 , 264, 1427-1433.
10.1021/ac0204193 CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/21/2002
Analytical Chemistry, Vol. 75, No. 3, February 1, 2003 535

mechanism11-16 involving Ntn amidohydrolases1,10 has been dem-
onstrated.
they have the same fast mass transfer as 3-µm particulate materials
(as shown by comparing the H/ U behavior of comparable
columns), but at the same time they offer a low-pressure drop as
∼15-µm particles. Both properties play a crucial role in chroma-
tography performance and speed.
The developed enzymatically active monolithic reactor was
coupled through a switching valve to an analytical column for on-
line separation and quantitation of the hydrolysis product to
calculate the total ester hydrolysis. The enantiomeric excess of
the product was calculated off-line on a CSP utilizing immobilized
penicillin G acylase (PGA-CSP).
With the aim of shedding light on the experimentally observed
substrate specificity, a computational study was also undertaken.
The recognition process between PGA and its substrates was
simulated through the docking and molecular dynamics of the
studied compounds inside the active site of the enzyme.
A novel feature of the catalytic machineries of the Ntn
amidohydrolases is that the nucleophile, which attacks the
carbonyl carbon of the scissile amide or ester bond, and the base,
which facilitates the attack by accepting the proton from the
nucleophile, are located in the same N-terminal amino acid. In
the case of PGA, the nucleophilic residue is the N-terminal serine,
in the case of AGA and PRO, threonine, and in the case of GAT,
the N-terminal cysteine. The neutral R-amino group of the
N-terminal amino acid, corresponding to histidine of the serine
proteases, is thought to act as a base in the catalytic mechanisms
of Ntn amidohydrolases. This is supported by the fact that the
optimum pH of the rate of the catalytic reactions is between 7
and 9 for these enzymes.1,17,18 The oxyanion binding site (i.e.,
oxyanion hole) has also been identified from the structures of
Ntn amidohydrolases.
In the present paper, a series of nine racemic 2-aryloxyal-
kanoic acid methyl esters and isosteric analogues were con-
sidered in order to study the structural effects on the rate and
enantioselectivity of the esterolytic reactions catalyzed by im-
mobilized PGA.
EXPERIMENTAL SECTION
Chemicals. Penicillin G acylase crude extract from Escherichia
coli ATCC 11105 (EC 3.5.1.11) was kindly donated by Recordati
(Milan, Italy). N,N′-Disuccinimidyl carbonate (DSC), Bradford
reagent, penicillin G potassium salt, phenylacetic acid (PAA), and
6-aminopenicillanic acid (6-APA) were purchased from Sigma-
Aldrich (Milan, Italy). Potassium dihydrogen phosphate and
dipotassium hydrogen phosphate used for the preparation of the
mobile phases were of analytical grade and purchased from Merck
(Darmstadt, Germany). Acetonitrile, methanol, and 1-propanol
were from Carlo Erba (Milan, Italy). Water was deionized by
passing through a Direct-Q (Millipore) system (Millipore, Bedford,
MA). Chromolith Performance NH2 (2-µm macropores, mesopore
size 13 nm), (10 cm × 0.46 (i.d.) cm) research sample was from
Merck. Zorbax RX-C8 column (15 cm × 0.46 (i.d.) cm) was
purchased from Agilent Technologies.
Studies of such hydrolytic reactions are usually carried out
“in batch” and these procedures are often followed by additional
steps involving substrate and product extraction and off-line
quantitation of the hydrolysis products. This multistep process is
time-consuming and may yield unreproducible results; therefore,
an immobilized PGA reactor for on-line conversion of substrates
was applied. This approach allows the direct evaluation of the
unreacted substrate as well as the two product enantiomers.
In this work, PGA was immobilized on monolithic chromato-
graphic support. These innovative materials are based on a
development of Nakanishi and Soga,19 who used a new sol-gel
process for the preparation of monolithic silica columns with a
bimodal pore structure (i.e., with throughpores and mesopores).
Tanaka et al.20,21 and Cabrera et al.22 demonstrated that this
method allows the preparation of chromatographic columns with
high efficiencies and low column back pressures. Due to their
properties concerning the fast mass transfer between the sub-
stance within the eluent and the active sites inside the skeleton
of the monolithic silica support, these materials seem to be an
ideal material for the immobilization of enzymes and the fast
conversion of substrates. Monoliths differ from the conventional
columns as regards their hydrodynamic properties especially since
Racemic acids were prepared according to our previous
papers23,24 and quantitatively converted to methyl esters 1-9 using
a solution of diazomethane (caution!! diazomethane is explosive,
toxic, and carcinogenic) in diethyl ether. Mass and NMR spectra
were consistent with the identity and purity of the so obtained
compounds. The methyl esters’ chemical structures are listed in
Table 1. The pure (+)-enantiomers of the considered acid
racemates were prepared25 and used for the determination of the
enantiomeric elution order on PGA-chiral stationary phase.26
Apparatus. Titration. Titration was performed by means of
718 STAT Titrino from Metrohm Italiana (Saronno, VA, Italy).
Chromatography. Chromatographic experiments were performed
with three modular systems. The systems were connected to
HPLC ChemStation, (Revision A.04.01). All the chromatographic
experiments were carried out at 25 °C. System 1. System 1
consisted of a Hewlett-Packard HP1050 chromatographic pump
(Palo Alto, CA), a Rheodyne sample valve (20-µL loop), and the
enzyme reactor. System 2. System 2 consisted of a Hewlett-Packard
(12) Warshel, A.; Naray-Szabo, G.; Sussman, F.; Hwang, J.-K. Biochemistry 1 9 8 9 ,
28, 3629-3637.
(13) Daggett, V.; Schro¨ der, S.; Kollman, P. J. J. Am. Chem. Soc. 1991, 113, 8926-
8935.
(14) Warshel, A.; Russell, S. J. Am. Chem. Soc. 1 9 8 6 , 108, 6569-6579.
(15) Nakagawa, S.; Yu, H.-A.; Karplus, M.; Umeyama, H. Proteins 1993 , 16, 172-
194.
(16) Kraut, J. Annu. Rev. Biochem. 1 9 7 7 , 46, 331-358.
(17) Kaartinen, V.; Williams, J. C.; Tomich, J.; Yates, J. R.; Hood, L. E.; Mononen,
I. J. Biol. Chem. 1 9 9 1 , 266, 5860-5869.
(18) Seemu¨ ller, E.; Lupas, A.; Zu¨ hl, F.; Zwickl, P.; Baumeister, W. FEBS Lett.
(23) Romstedt, K. J.; Lei, L-P.; Feller, D. R.; Witiak, D. T.; Loiodice, F.; Tortorella,
V. Farmaco 1 9 9 6 , 51, 107-114.
1 9 9 5 , 359, 173-178.
(19) Nakanishi, K.; Soga, N. J. Non Cryst. Solids 1 9 9 2 , 139, 1-13, 14-24.
(20) Tanaka, N.; Kobayashi, H.; Ishizuka, N.; Minakuchi, H.; Nakanishi, K.;
Hosoya, K.; Ikegami, T. J. Chromatogr., A 2 0 0 2 , 965, 35-49.
(21) Minakuchi, H.; Nakanishi, K.; Soga, N.; Ishizuka, N.; Tanaka, N. Anal. Chem.
1 9 9 6 , 68, 3498-3501.
(24) Bettoni, G.; Ferorelli, S.; Loiodice, F.; Tangari, N.; Tortorella, V.; Gasparrini,
F.; Misiti, D.; Villani, C. Chirality 1 9 9 2 , 4, 193-203.
(25) Ferorelli, S.; Loiodice, F.; Tortorella, V.; Amoroso, R.; Bettoni, G.; Conte-
Camerino, D.; De Luca, A. Farmaco 1 9 9 7 , 52, 367-374.
(26) Calleri, E.; Massolini, G.; Loiodice, F.; Fracchiolla, G.; Temporini, C.;
Felix, G.; Tortorella, P.; Caccialanza, G. J. Chromatogr., A 2 0 0 2 , 958, 131-
140.
(22) Cabrera, K.; Lubda, D.; Eggenweiler, H-M.; Minakuchi, H.; Nakanishi, K.
J. High Resolut. Chromatogr. 2 0 0 0 , 23, 99-104.
536 Analytical Chemistry, Vol. 75, No. 3, February 1, 2003

tion of 5 mM. The substrate solutions were injected into the
coupled LC system. The rates of substrate conversion (C) were
calculated from the peak areas of unreacted esters. Standard
curves for the substrates were linear over the concentration range
of 0.1-5 mM corresponding to 2 and 100% of conversion.
The product of the enzymatic hydrolysis was collected from
the analytical column in a 1-mL volumetric flask and directly
injected into system 3 for chiral resolution and determination of
the product enantiomeric excess (eep). For the compound with
the highest enantioselectivity (compound 3 ) the lowest level of
the R enantiomer was determined. The limit of quantitation (LOQ)
for the R enantiomer was ∼0.05% (w/ w) at a signal-to-noise ratio
of 10. A typical chromatogram of the S enantiomer of the acid
corresponding to ester 3 spiked with 0.05% R enantiomer is
presented in Figure 3.
Table 1. Chemical Structures of the Substrates Used
in the Study
racemate
A
B
R
1
2
3
4
5
6
7
8
9
Cl
O
O
O
S
NH
CH2
O
O
O
CH3
C2H5
C6H5
CH3
CH3
CH3
CH3
CH3
CH3
Cl
Cl
Cl
Cl
Cl
Br
F
CH3
PGA Immobilization. PGA was covalently immobilized on a
Chromolith-NH2 column. The immobilization of the enzyme via
amino groups was carried out according to a previously described
procedure following the in situ method.27,28 A total of 2.24 g of
DSC was dissolved in 100 mL of acetonitrile, and the resulting
solution was continuously circulated for 18 h at 0.5 mL/ min
through the column previously equilibrated with the same solvent.
The column was then washed at the same flow rate, first with 60
mL of acetonitrile and then with water and with 60 mL of 1 mM
phosphate buffer (pH 7.0). A total of 300 mg of PGA was dissolved
in 100 mL of 1 mM phosphate buffer (pH 7.0) and the enzyme
solution continuously circulated at 0.5 mL/ min for 24 h. The flow
was inverted every 15 min in the first hour and then every 30
min in the following 3 h. After 24 h, the column was washed with
200 mL of water, 200 mL of a 0.5 M solution of NaCl, and finally
with 100 mL of a 0.2 M glycine solution to block the remaining
activated groups and equilibrated with a 50 mM solution of
phosphate buffer (pH 7.0).
The amount of immobilized units was calculated from the
difference between the initial and the final enzymatic activity of
the enzymatic solution. The hydrolysis of penicillin G potassium
salt has been used as a standard assay for the determination of
the catalytic activity of the enzyme. The hydrolysis was carried
out in 20 mL of a 100 mM penicillin G solution in 50 mM
phosphate buffer (pH 7.0) and the PAA produced was titrated with
0.1 M NaOH with automatic pH correction. The experiments were
carried out at room temperature. The enzymatic activity was
measured as international units (U), equivalent to micromoles of
penicillin G hydrolyzed per minute. The amount of immobilized
units were found to be 1406 U, corresponding to 58.58 mg of
enzyme as determined with Bradford reagent. When not in use,
the columns were stored at 4 °C in a solution of 0.01% sodium
azide (w/ v).
HP 1100 liquid chromatograph with a Rheodyne sample valve
(20-µL loop) equipped with a Hewlett-Packard HP 1100 vari-
able-wavelength detector, a HP 1100 thermostat, and the Zorbax
RX-C8 column. System 1-2. Systems 1 and 2 could be used
independently or the eluent from system 1 could be directed onto
system 2 through a HP six-port switching valve as shown in Figure
1. The UV detector could be connected to system 1 or to system
2 through a second switching valve (detector selector valve).
System 3. System 3 consisted of a Hewlett-Packard HP 1100 liquid
chromatograph with a Rheodyne sample valve (20-µL loop)
equipped with a Hewlett-Packard HP 1100 diode-array detector,
a HP 1100 thermostat, and a PGA-epoxide chiral stationary phase
(15 cm × 0.46 (i.d.) cm). The mobile phase was 120 mM
phosphate buffer (pH 7.0). The flow rate was maintained at 0.8
mL/ min and the detection was performed at 225 nm.
Methods. Column-Switching Setup. A schematic drawing of
the column-switching system (system 1-2) is given in Figure 1.
Step 1 (0-1.2 min, valve 1 in position A): the substrates were
loaded on the enzymatic column (system 1); 50 mM phosphate
buffer (pH 7.0) at a flow rate of 0.8 mL/ min was used as eluent
(pump 1).
Step 2 (1.2-14.0 min, valve 1 in position B): valve 1 was
switched to position B and the analytes (products and unreacted
substrate) were flushed and focused directly to the reversed-phase
analytical column (pump 1) using 50 mM phosphate buffer (pH
7.0) as mobile phase at a flow rate of 0.8 mL/ min.
Step 3 (14.0-39.0 min, valve 1 position A): the valve was then
switched back to its original position for separation of products
and unreacted substrate on Zorbax RX-C8 (system 2). The
following mobile-phase gradient was applied (pump 2): 50 mM
phosphate buffer (pH 7.0)-methanol (60:40) for the first 10 min,
step gradient to 50 mM phosphate buffer (pH 7.0)-methanol (40:
60) at 10.01 min, and return to the starting condition at 25 min.
The flow rate was 1.0 mL/ min, and the detection was performed
at 225 nm.
A representative chromatogram obtained from the chroma-
tography of ester 6 on the coupled PGA-column/ analytical column
is presented in Figure 2.
Analytical Procedure. Stock solutions of each racemic substrate
were prepared in 1-propanol at a concentration of 10 mM and then
diluted with 50 mM phosphate buffer (pH 7.0) to a final concentra-
PGA Column Activity Determination. The hydrolysis of penicil-
lin G potassium salt has been used as a standard assay for the
determination of the catalytic activity of the enzyme in the
immobilized form using chromatographic system 1.29 The enzyme
column was equilibrated for 30 min with 50 mM phosphate buffer
(pH 7.0). A 20-µL aliquot of penicillin G diluted in the same buffer
(27) Massolini, G.; Calleri, E.; De Lorenzi, E.; Pregnolato, M.; Terreni, M.; Felix,
G.; Gandini, C. J. Chromatogr., A 2 0 0 1 , 921, 147-160.
(28) Felix, G.; Descorps, V. Chromatographia 1 9 9 9 , 49, 595-605.
(29) Andrisano, V.; Bartolini, M.; Gotti, R.; Cavrini, V.; Felix, G. J. Chromatogr.,
B 2 0 0 1 , 753, 375-383.
Analytical Chemistry, Vol. 75, No. 3, February 1, 2003 537

Figure 1. Chromatographic system coupling the enzyme column with the reversed-phase analytical column. The substrate is loaded onto the
enzyme column using position A; the product and the unreacted substrate are switched to the analytical column using position B; the conversion
percentage is measured on the analytical column using position A. 1, switching valve; 2, detector selector valve.
Figure 2. Chromatogram showing the separation of the product from the unreacted substrate racemate 6 obtained on-line with the coupled
system. See text for experimental conditions. Peak 1, acidic product; peak 2, unreacted substrate.
solution, concentration range between 0.1 and 4 M, was injected
onto the HPLC system with a flow rate of 0.5 mL/ min and UV
detection at 225 nm. Each concentration was injected in duplicate.
The species eluted from the PGA column were contained in the
first 3-mL fraction of each eluate (6 min). Each fraction was
collected in a 10-mL volumetric flask (dilution factor, df ) 10/ 3)
adjusting the volume with 50 mM phosphate buffer (pH 7.0). Each
solution was injected off-line in system 2. The mobile phase
employed was 50 mM phosphate buffer (pH 7.0)-methanol (70:
30). Flow rate was 0.8 mL/ min, and the detector was set at 225
nm. The chromatographic retention factors were 0.17 for 6-APA,
0.60 for phenylacetic acid, and 2.55 for penicillin G. The amount
of PAA from PGA-catalyzed hydrolysis was found to be dependent
on the concentration of injected penicillin G. The Michaelis-
538 Analytical Chemistry, Vol. 75, No. 3, February 1, 2003

Figure 3. Typical chromatogram of the S enantiomer of the acid corresponding to ester 3 containing 0.05% R enantiomer.
Menten trend was found by plotting the rate of enzymatic reaction
against the substrate concentration [S].
The rate of the enzymatic reaction V expressed as (∆area PAA/
min) was calculated by
analyte and to is the retention time of an unretained compound;
in this study, to was calculated from the first disturbance of the
baseline after injection. The void volume was calculated from the
first disturbance of the baseline after injection.
The product enantiomeric excess was calculated as follows:
V (∆area/ min) ) area (PAA) × df/ time (min) )
area (PAA) × (10/ 3)/ 6
eep ) (x1 - x2)/ x1 + x2
(1)
To obtain estimate of the Vmax, Lineweaver and Burk reciprocal
plots of 1/ V versus 1/ [S] were constructed.
To calculate the units (U) of immobilized enzyme, the following
equation was used:
where x1 and x2 denote the concentrations of the two acidic
enantiomers separated on the chiral stationary phase.
The enantioselectivity E, for an irreversible enantiomer-
differentiating enzyme-catalyzed hydrolysis, is expressed as
U (µmol/ min) ) [(∆area/ min)max/ extintion coefficient] ×
column void volume (mL) × 103
E ) ln[1 - C(1 + eep)]/ ln[1 - C(1 - eep)]
(2)
where C is the degree of substrate conversion, or conversion
factor.30
where one unit (U) is the amount of enzyme that hydrolyses 1
µmol of penicillin G in 1 min.
Modeling of Transition-State Analogues in P GA. All model-
ing was done with the Sybyl software package,31 running on a
Silicon Graphics R10000 workstation. Energy minimizations and
molecular dynamics calculations were done using the Tripos force
field32 including the electrostatic contribution. Atom-centered
partial charges were calculated according to the Gasteiger-Hu¨ ckel
method.33,34
We used a distance-dependent dielectric constant of 4.0 and
scaled the 1-4 van der Waals interactions by 50%. The distance-
dependent dielectric constant damps long-range electrostatic
interactions and thus compensates for the lack of explicit solva-
To determine the phenylacetic acid extinction coefficient for
concentration assessment (area of a 1 M concentration of PAA),
increasing concentrations of phenylacetic acid were injected onto
the enzyme column. Four calibration solutions in the range be-
tween 0.1 and 4 M were prepared, and each solution was injected
twice. For each injected concentration, the PAA area was deter-
mined by collecting 10 mL of each eluate from the enzyme column
in a volumetric flask; each solution was then injected off-line on
the RX-C8 Zorbax, measuring the area at 225 nm and plotting
against the corresponding PAA concentration. A linear correlation
(r2 ) 0.999) was found, providing the extinction coefficient value.
The amount of active immobilized enzyme was found to be
1420.09 ( 29.5 units, this result is comparable to the value of
immobilized units (1406 U); therefore, all the immobilized enzyme
was found to be active after the immobilization procedure.
Calculations. The retention factor (k) was calculated using
the equation k ) (tr/ to) - 1, where tr is the retention time of the
(30) Chen, C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. Chem. Soc. 1 9 8 2 ,
104, 7294-7299.
(31) Sybyl Molecular Modelling System (version 6.8); Tripos Inc.: St. Louis, MO.
(32) Vinter, J. G.; Davis, A.; Saunders, M. R. J. Comput.-Aided Mol. Des. 1 9 8 7 ,
1, 31-55.
(33) Gasteiger, J.; Marsili, M. Tetrahedron 1 9 8 0 , 36, 3219-3228.
(34) Purcel, V. P.; Singer, J. A. J. Chem. Eng. Data 1 9 6 7 , 12, 235-246.
Analytical Chemistry, Vol. 75, No. 3, February 1, 2003 539

Figure 4. Intermediates and their analogues in the PGA-catalyzed hydrolysis of esters. Amino acids correspond to those in PGA. (a) In the
accepted mechanism for hydrolysis of esters, the catalytic serine (SerB1) attacks the ester once it binds in the active site. This attack forms the
tetrahedral intermediate marked 1. Hydrogen bonds from two amide N-H’s stabilize the oxyanion in this intermediate. Collapse of tetrahedral
intermediate 1 releases the alcohol and forms an acyl enzyme intermediate. Attack of water on the acyl enzyme yields tetrahedral intermediate
2. Collapse releases the acid and regenerates the free enzyme. (b) A phosphonate mimics the transition states for formation and collapse of
tetrahedral intermediate 1. Four hydrogen bonds, marked a-d, stabilize the charges. To be judged catalytically competent, the minimized structures
must contain all four hydrogen bonds.
tion.35 The crystal structure of the p-nitrophenylacetic acid/ PGA
complex solved by Done et al. was retrieved from the Brookhaven
Protein Data Bank36 (entry code 1ajn).
Table 2. Influence of Solute Structure on Hydrolysis
Rate and Enantioselectivitya
substrate
C (%)
RSD (%)
eeb (%)
E
Using the Biopolymer module of Sybyl, hydrogen atoms were
added to the unfilled valences of the amino acids. Histidines were
uncharged, aspartates and glutamates were negatively charged,
and arginines and lysines were positively charged. The inhibitor
found in the crystal structure was considered as a template to
select a trial conformation of the phosphonates (R)-1 , (S)-1 , (R)-
3 , and (S)-3 . Docking of phosphonates (R)-1 , (S)-1 , (R)-3 , and
(S)-3 into the PGA active site was carried out in two steps. First,
we linked the phosphorus covalently to the Oγ of SerB1. Then,
we identified a low-energy conformation of these compounds
superimposable on the experimentally determined bioactive
conformation of the PGA inhibitor p-nitrophenylacetic acid by
manual adjustment of the torsion angles about the P-C1, C1-B2
and B2-C3 rotatable bonds (see Figures 4 for atom labeling). The
overlay on p-nitrophenylacetic acid was performed by minimizing
the root-mean-square distance between the centroids of aromatic
rings and the COO-/ POO-moieties. The resulting complexes
were geometry-optimized by keeping fixed the coordinates of the
protein backbone. Energy minimization proceeded in two stages:
first, 1000 iterations of steepest descent algorithm and second,
1000 iterations of conjugate gradients algorithm until the rms
deviation reached 0.005 Å mol-1. Water molecules and the
substrate were not constrained through any of the minimization
cycles.
1
2
3
4
5
6
7
8
9
50.6
5.4
47.9
19.7
6.7
10.5
46.7
28.2
22.7
2.97
0.46
2.75
4.8
81.1 (S)
39.0 (S)
>99.9 (S)
4.3 (S)
66.4 (S)
38.2 (S)
82.9 (S)
17.1 (S)
85.3 (S)
23
2.3
>200
1.1
2.2
5.3
2.3
23
1.5
15
0.64
0.73
0.42
0.25
a
Reactor B. C, conversion (%); ee, enantiomeric excess (%); E,
enantioselectivity; RSD, experimental error (n )2). See text for
experimental details. b Fast reacted enantiomer.
tion, during which the protein backbone atoms were constrained
as done in the previous step. A time step of 1 fs and a nonbonded
pair list updated every 25 fs were used for the molecular dynamics
simulations. The temperature was mantained at 300 K using the
Berendsen algorithm with a 0.2-ps coupling constant. An average
structure was calculated from the last 100-ps trajectory and energy-
minimized using the steepest descent and conjugate gradient
methods as specified above.
RESULTS AND DISCUSSION
A chromatographic bioreactor based on PGA immobilized on
monolithic support was developed in order to investigate the
hydrolytic activity of PGA toward a series of 2-aryloxyalkanoic acid
methyl esters and isosteric analogues, whose chemical structures
are reported in Table 1. We decided to use monolithic supports
because they are characterized by an almost complete lack of
diffusion resistance during mass transfer; thus, they represent
The geometry-optimized structures were then used as the
starting point for subsequent 200-ps molecular dynamics simula-
(35) Weiner, S. J.; Kollman, P. A.; Case, D. A.; Singh, U. C.; Ghio, C.; Alagona,
G.; Profeta, S., Jr.; Weiner, P. J. Am. Chem. Soc. 1 9 8 4 , 106, 765-784.
(36) Bernstein, F. C.; Koetzle, T. F.; Williams, G. J. B.; Meyer, E. F., Jr.; Brice,
M. D.; Rodgers, J. R.; Kennard, O.; Shimanouchi, T.; Tasumi, T. J. Mol.
Biol. 1 9 7 7 , 112, 535-542.
540 Analytical Chemistry, Vol. 75, No. 3, February 1, 2003

ideal materials for the immobilization of enzymes and fast
conversion of substrates. Moreover, the large throughpores of
monolithic materials allow high-speed analysis and low back
pressures and consequently enable the coupling of the enzymatic
column with the analytical column by means of a switching valve.
The column-switching device was used to calculate the rate of
substrate conversion (C) and to collect the product whose
enantiomeric excess (ee) was calculated off-line for the determi-
nation of enantioselectivity (E). The C and ee values together
with E, calculated from the chromatographic data, are reported
in Table 2.
High reaction rates (C >40%) and enantioselectivity (ee >80%)
were obtained for 1 , 3 , and 7 ; 1 and 7 are characterized by a
methyl group in the R-position while 3 presents a second aromatic
ring on the asymmetric carbon. For this compound, a cleavage
greater than 99.9% of the (S)-ester, corresponding to 47.9%
conversion of the racemic ester, was observed. It is interesting to
remark that a change of the substituent on the R-position from a
methyl (analyte 1 ) to an ethyl (analyte 2 ) group led to a dramatic
reduction in the conversion rate and E.
The substituent on the aromatic ring (see 1 and 7 -9 ) seems
to play a role in the reaction rate and enantioselectivity. The
compounds with the most hydrophobic and electron-withdrawing
substituent (1 and 7, respectively) are the most rapidly hydrolyzed
with a high ee value. The presence of the oxygen in the R-position
is also important for the catalytic activity, all the isosters (4 -6 )
present a low conversion rate, a low ee value, or both in
comparison with 1 .
To explain the results of PGA enantioselectivity toward the
aryloxyalkanoic acid methyl esters 1 -9 , we used computer
modeling of enzyme-transition-state analogue complexes. The
binding site of PGA has been found to consist of three major
regions that are responsible for the hydrolysis reaction of the
substrates by the enzyme:1,6,7 the catalytic residue SerB1, the
oxyanion hole (stabilizing the negative charge present on one of
the oxygens of the tetrahedral intermediates by hydrogen bond-
ing) formed by GlnB23, AlaB69, AsnB241, and a hydrophobic
pocket that is able to accommodate hydrophobic groups. The first
chemical step in PGA-catalyzed ester hydrolysis is the attack of
the active site serine (SerB1) on the ester carbonyl forming a
tetrahedral intermediate. Collapse of this tetrahedral intermediate
releases the alcohol, Figure 4a. We assume that the transition
state involved in the formation or collapse of this first tetrahedral
intermediate defines the selectivity of PGA toward aryloxyalkanoic
acid methyl esters 1 -9 . To mimic this transition state, we used
the phosphonates linked to PGA shown in Figure 4b. Indeed,
researchers have shown that phosphonates mimic the transition
state for ester hydrolysis well.37,38
Figure 5. (a) Superimposition of the phosphonate transition-state
analogues of (R)-1 (red) and (S)-1 (green), located at their proper
respective three-dimensional position inside the active site of PGA.
(b) Catalytically competent orientation of the phosphonate transition-
state analogue of (S)-3 (yellow) in the active site of PGA. Only amino
acids located within 3 Å of the intermediates are displayed for the
sake of clarity. Hydrogen-bonding interactions are depicted as dashed
lines. Intermediates maintain all the catalytically essential hydrogen
bonds identified in Figure 4b.
The docking geometries of compounds (R)-1 and (S)-1 gave
their respective tetrahedral intermediates without losing favorable
van der Waals and electrostatic interactions inside the active site
of PGA. As depicted in Figure 5a, where the superimposition of
the two intermediates is shown, the oxygen anion of both the
tetrahedral intermediates gave the frame of four hydrogen bond
interactions with the oxyanion hole residues GlnB23, AlaB69, and
AsnB241. Moreover, in both intermediates, the p-chlorophenyl
ring was located inside a hydrophobic pocket of the active site
formed by the side chains of residues PheB24, ValB56, PheB57,
AlaB69, SerB67, MetB142, TrpB154, and IleB177. In this pocket,
the PheB24 side chain appeared in a suitable orientation for a
π-stacking charge-transfer interaction with the p-chlorophenyl of
the intermediates. Finally, it should be pointed out that in both
cases the relevant intramolecular hydrogen bond interactions of
PGA were not disrupted. However, a different scenario appeared
(37) Jacobs, J.; Schultz, P. G.; Sugasawara, R.; Powell, M. J. Am. Chem. Soc. 1987,
109, 2174-2176.
(38) Hanson, J. E.; Kaplan, A. P.; Bartlett, P. A. Biochemistry 1 9 8 9 , 28, 6294-
6305.
Analytical Chemistry, Vol. 75, No. 3, February 1, 2003 541

when the two structures were analyzed according to the position
of the methyl substituent on the stereogenic center.
the aromatic ring, atom in the R-position to the stereogenic center,
and type of substituent on the asymmetric carbon dramatically
changed the conversion rate or the enantioselectivity of the
reaction involving the selected substrates. The molecular modeling
study supports the experimental observations on structure-
property relationships of esters 1 -9 processed by PGA. The
reaction occurs well when a relatively small group such as methyl
is present on the stereogenic center, preferentially in the S
configuration. In the case of larger substituents, such as ethyl,
the hydrolysis reaction is prevented, possibly due to steric
hindrance within the active site. However, when a planar system
is attached to the stereogenic center, the enzymatic hydrolysis
reaction occurs again, since the complex is stabilized by favorable
stacking interactions between the planar ring and PheB71 and
PheB146 side chains.
As regards substituents on the aromatic ring, hydrophobic and
electronic properties seem to play an important role in the
enzymatic process control. The bromo derivative 7 , in fact, has a
behavior similar to the chloro derivative 1 , as expected; the
analogue 8 shows low conversion rate and enantioselectivity,
possibly due to the poor hydrophobicity of the fluorine substituent.
In the case of analogue 9 , on the contrary, a low conversion rate
would be the result of the methyl group electron-donor properties
that would weaken the π-stacking interaction formed between 9
and PheB24 aromatic rings. The isosteric replacement of the
oxygen atom in the position R to the stereogenic center (analogues
4 -6 ) affects the enzymatic hydrolysis too, producing lower
conversion rate and enantioselectivity. One may speculate that
the detrimental effect of the O/ S/ NH replacement originates from
a less good accommodation of the p-chlorophenyl ring into the
hydrophobic pocket of the enzymatic cavity due to the different
stereoelectronic features of the isosteric substituents.
For the tetrahedral intermediate of (R)-1 , the methyl group
was located in a shallow pocket between PheB24, GlnB23, and
ProB22, where bulkier groups could hardly be allocated. In
contrast, the methyl group of (S)-1 enantiomer was projected
toward a hydrophobic pocket made up by the side chains of Phe71,
Phe146, and Ala69, which leads to the opening of the active site.
This pocket is medium-sized so that the methyl group fits properly
in this configuration; however, it may preferentially accommodate
planar π-electron systems to make favorable stacking and hydro-
phobic interactions. Accordingly, the phenyl group attached to
the stereogenic center of (S)-3 , showing an high reaction rate
and enantioselectivity, was found to point toward this hydrophobic
cavity at the opening of the active site and to contact PheB71 and
PheB146 side chains favorably via a π-stacking and a T-shaped
interaction, respectively (Figure 5b). Burley and Petsko have, in
fact, shown that the aromatic-aromatic interactions are significant
contributors to stabilization of both protein structure and ligand-
protein complexes.39,40 These interactions operate at centroid-to-
centroid distances of 4.5-7.0 Å between interacting rings. The
angle between the normal vectors of interacting aromatic rings
is typically between 30° and 90°, producing a “T-shaped” or “edge-
to-face” arrangement of interacting rings. Such interactions are
reported to have energies between 1.0 and 2.0 kcal/ mol.39
CONCLUSIONS
PGA was successfully immobilized on a monolithic support,
and the developed reactor combined with a switching valve was
used as a highly efficient tool to study the enantioselectivity of
ester hydrolyses catalyzed by PGA. The advantages of this
chromatographic setup are the complete elimination of the
downstream process, involving separation of product from the
remaining substrate, the large decrease in enzyme and sample
amount needed, and the precision by which C and ee can be
calculated for the determination of E. Therefore, the described
chromatographic system can be used to investigate the optimum
reaction conditions, temperature, ionic strength, and pH, that
influence enzyme enantioselectivity.
This information could expand the application of immobilized
PGA as a chiral biocatalyst for the considered compounds and
new processes. It could also be used to optimize the steric purity
of products in the catalyzed transformation of chiral substrates.
ACKNOWLEDGMENT
This work is supported by MURST (progetto: Analytical pro-
file of drugs: advanced methodologies MM03104549•002) and
Galileo project.
The hydrolysis of a series of 2-aryloxyalkanoic acid methyl
esters and isosteric analogues was considered, and it has been
observed that small structural modification, e.g., substituent in
Received for review June 26, 2002. Accepted November 4,
2002.
(39) Burely, S. K.; Petsko, G. A. Science 1 9 8 5 , 229, 23-28.
(40) Burely, S. K.; Petsko, G. A. Adv. Protein Chem. 1 9 8 8 , 39, 125-189.
AC0204193
542 Analytical Chemistry, Vol. 75, No. 3, February 1, 2003