Enantioselective hydrolysis of naproxen ethyl ester catalyzed by monoclonal antibodies

Article abstract of DOI:10.1016/S0968-0896(02)00074-3

This report described that a hapten of racemic phosphonate 3 designed as the mimic of the transition state of hydrolysis of naproxen ethyl ester was successfully synthesized from easily available 2-acetyl-6-methoxy-naphthalene 5. Then BALB/C mice were immunized and one of the monoclonal catalytic antibodies, N116-27, which enantioselectively accelerated the hydrolysis of the R-(-)-naproxen ethyl ester was given. The Michaelis-Menton parameter for the catalyzed reaction was K_M=6.67 mM and k_cat/k_uncat=5.8×10⁴. This enantioselective result was explained by the fact that the R-isomer of rac-hapten was more immunogenic than the S-isomer.

Full text of DOI:10.1016/S0968-0896(02)00074-3

Bioorganic & Medicinal Chemistry 10 (2002) 2171–2175
Enantioselective Hydrolysis of Naproxen Ethyl Ester
Catalyzed by Monoclonal Antibodies
Zhen-Dan Shi,a Bing-Hui Yang,a,* Jing-Jing Zhao,a Yu-Lin Wu,a,*
Yong-Yong Jib and Ming Yehb
aState Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, China
bShanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China
Received 10 December 2001;accepted 9 February 2002
Abstract—This report described that a hapten of racemic phosphonate 3 designed as the mimic of the transition state of hydrolysis
of naproxen ethyl ester was successfully synthesized from easily available 2-acetyl-6-methoxy-naphthalene 5. Then BALB/C mice
were immunized and one of the monoclonal catalytic antibodies, N116-27, which enantioselectively accelerated the hydrolysis of the
R-(À)-naproxen ethyl ester was given. The Michaelis–Menton parameter for the catalyzed reaction was KM=6.67 mM and kcat
/
kuncat=5.8Â104. This enantioselective result was explained by the fact that the R-isomer of rac-hapten was more immunogenic than
the S-isomer. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
other applications.8À10 Therefore, more research on
catalytic antibodies will promote deeper understanding
of the reaction mechanism and molecular recognition
and broaden their applications in organic, biotechnol-
ogy and medicine.
From the hypothesis of Pauling1 that the binding forces
utilized by both enzymes and antibodies are fundamen-
tally identical to the two monumental reports2,3
appearing on an antibody catalysis, the catalytic anti-
body opened a new field between chemistry and immu-
nology. The most common strategy for producing
catalytic antibodies needs designing and synthesizing for
suitable transition state analogues as haptens. Then,
after conjugation to carrier proteins to form antigens,
the desired monoclonal antibodies were afforded by
immunizing the animals with antigens and screening
through hybridoma technology. Up to now, catalytic
antibodies have shown the ability to catalyze a variety
of organic reactions, such as hydrolytic, concerted, car-
bon–carbon bond forming and redox. Catalytic anti-
bodies can even reverse kinetic control reactions,
including ring formation, ring cycloaddition, cationic
cyclization and elimination.4À7 Moreover, they have
Naproxen is an important non-steriod anti-flammatory
drug, of which the S-isomer is 28 times more effective
than R-isomer.11 Chemical synthesis of the S-isomer
involving diastereomeric crystallization from a racemic
acid mixture has been exploited.12 Considerable efforts
have been made in improving the methods of its asym-
metric synthesis.13À16
Recently, we reported a polyclonal antibody that cata-
lyzed the hydrolysis of naproxen ethyl ester 1a–c to
naproxen 2a–c, but the value of rate enhancement (kcat
/
kuncat) was rather too low.17,18 Somewhat later, Janda et
al. reported the hydrolysis of naproxen p-methylsulfo-
nylphenyl ester by antibody prepared by the reactive
immunization.19 In continuing our program, herein, we
report an enantioselective hydrolysis of naproxen ethyl
ester catalyzed by monoclonal antibody with the satis-
fied rate enhancement. This result also demonstrates
that antibodies generated by racemic phosphonate
hapten 3 could be used for the kinetic resolution of
naproxen ethyl ester.
*Corresponding authors. Tel.: +8621-6416-3300-1405;fax: +8621-
6416-6128;e-mail: yangbh@pub.sioc.ac.cn (B. H. YangT);el.:
+8621-6416-3300-1415;fax: +8621-6416-6128 e-mail: ylwu@pub.
sioc.ac.cn (Y. L. Wu).
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00074-3

2172
Z.-D. Shi et al. / Bioorg. Med. Chem. 10 (2002) 2171–2175
Scheme 1. Substrate 1a–c, product 2a–c, hapten 3, inhibitor 4 used for monoclonal antibodies catalyzed reactions.
Scheme 2. Reagents and conditions: (a) NaClO, 1,4-dioxane, NaOH, H2O, 90 ꢁC, 48 h, 83%;LiAlH , THF, rt, 8 h, 96%;(b) PBr , THF, À40 ꢁC,
4
3
2 h;P(OEt) 3, 120 ꢁC, 10 h;89%;(c) n-BuLi, MeI, THF, À78 ꢁC, 2 h, 87%;(d) NaOH, EtOH, reflux, 16 h, 82%;(e) 1N BBr ꢀ CH2Cl2, CH2Cl2,
3
À78 ꢁC, 2 h, 88%;(f) Br(CH ) CO2Et, K2CO3, acetone, reflux, 40 h, 82%;(g) NaOH, EtOH, reflux, 24 h, 85%;(h) EDCI, DMF, 10 mmol/L PBS
2 3
buffer (pH 7.2), rt, 5 h.
Results and Discussion
Arbuzov reaction in the presence of triethyl phosphite to
afford compound 7 in 89% yield. Phosphonate 7 was
methylated with iodomethane under n-BuLi to produce
compound 8 in 87% yield. Then, the intermediate 8 was
allowed to synthesize inhibitor 4 and hapten 3 via two
different routes. Inhibitor 4 was prepared by selective
hydrolysis of the phosphonate ester 8 under sodium
hydroxide in only one step in 82% yield. For hapten 3, at
first, the intermediate 8 was demethylated on the methyl
Preparation of hapten, inhibitor and immunogen
A tetrahedral structure of phosphonates in compounds
3 and 4, which mimicked the transition state of the
hydrolysis of naproxen ethyl ester 1a–c, was designed as a
hapten and inhibitor (Scheme 1). They were synthesized
from easily available material, 2-acetyl-6-methoxy-naph-
thalene 5 (Scheme 2).20 After oxidation and reduction
from compound 5, the 6-methoxy-2-naphthyl-methanol
6 was brominated by phosphorus tribromide followed by
ꢁ
ꢀ
ether group under 1 N BBr3 CH2Cl2 solution at À78 C
followed by tethering the spacer with the reagents of

Z.-D. Shi et al. / Bioorg. Med. Chem. 10 (2002) 2171–2175
2173
ethyl g-bromobutyrate and potassium carbonate to give
compound 10 in 82% yield. Subsequently, the hapten 3
was prepared by the chemically selective hydrolysis of
the triester 10 with sodium hydroxide in 85% yield.
The monoethyl phosphonate 3 (hapten) was coupled to
the carrier proteins, bovine serum albumin (BSA) or
fowl g-globulin (FgG) with 1-ethyl-3-(3-dimethylami-
nopropyl)- carbodiimide (EDCI) as condensation agent
to afford the immunogens 11 and 12.
Production of monoclonal antibodies capable of catalytic
power
Figure 1. Plot of the concentration of substrates versus the time of
hydrolysis. ^, Â, Control experiment of 1b and 1c with non-specific
antibodies; &, ~, hydrolysis of 1b and 1c by monoclonal antibody
N116–27.
Among the 10 hybridomas antibodies elicited from
immunogens 11 and 12, seven of them were found to be
able to catalyze the hydrolysis of rac-naproxen ethyl
ester. By comparison, three of them were allowed
to be the best catalysis which were amplified by
making ascitic fluid. Based on the establishment of the
hybridoma clones against the transition state analogue
of hydrolysis of naproxen ethyl ester, the hybridoma
cells were injected into the peritoneal cavity of BALB/C
mice to induce ascites. The monoclonal antibodies
were purified from the ascite as described.21
Kinetic evaluation
Our experiment showed that all of the subsequent five
monoclonal antibodies could catalyze the hydrolysis of
R-naproxen ethyl ester. Among them, the most efficient
monoclonal catalytic antibody, N116–27, greatly accel-
erated the hydrolysis of rac-naproxen ethyl ester and the
R-isomer ester, but not the S-isomer ester (Fig. 1).
Figure 2. Lineweaver–Burke plot for the reaction of hydrolysis of
R-(À)-1c by N116–27.
could highly enantioselectively catalyze the hydrolysis of
R-naproxen ethyl ester. This result was contrary to that
of the commercial and semipurified lipase of Candida
cyclindracea, which enantioselectively catalyzed the
hydrolysis of S-naproxen ethyl ester. These phenomena
could be explained that the R-isomer of the rac-hapten
was more immunogenic than the S-isomer, just as the
d-amino acids of the hapten were more immunogenic
than the l-amino acid. Of course, our results could be
further tested by comparing the catalytic activity of
antibodies elicited from the R-isomer phosphonate of
the hapten with that from the S-isomer.
From Figure 1, the ester of R-isomer was 20% con-
sumed during 1 h while the ester of S-isomer and back-
ground accounted for less than 1% of the total
hydrolysis. The rates of the reactions were measured by
following the decrease in absorbance of the R-isomer
ester 1c at 240 nm by HPLC. The obtained data were
corrected for the background reaction and employed to
construct a Lineweaver–Burke plot, from which all the
kinetic parameters were given (Fig. 2).
The results were observed to follow the Michaelis–Menton
kinetic equation of pseudo-first-order reaction. kM/ꢀmax is
determined as the slope of the line and 1/ꢀmax is also
determined as the intercept of the vertical axis in Figure 2.
The values of the kinetic parameters are: kM=6.67 mM,
Conclusion
ꢀ
max=32.3 mM, kcat=ꢀmax/[Ig]=10.1 minÀ1. Comparison
We have demonstrated that the racemic hapten 3 and
inhibitor 4 were designed and successfully synthesized.
Then, the racemic hapten conjugates were immunized
and monoclonal antibodies were generated under the
standard protocol. The acquired monoclonal antibody
N116–27 enantioselectivly catalyzed the hydrolysis of
R-naproxen ethyl ester with high rate enhancement.
Moreover, our experiment affords an example that it
should be possible to employ monoclonal antibodies
generated from the racemic hapten to effect enantiose-
lectively catalytic results in a variety of reactions, such
as acyl-transfer, formation and cleavage of carbon–
carbon bonds.
the kcat for the hydrolysis of the R-isomer ester with
kuncat (1.27Â10À4 min) for the control experiments gave
the rate enhancement of 5.8Â104-fold. This enhance-
ment was considered to be high for the acceleration rate of
hydrolysis of the inactive ester. The catalytic hydrolysis of
the naproxen ethyl ester exhibited competitive inhibition
by phosphonate 4, which revealed that catalysis took
place at the antibody binding sites.
By exploiting the known enantiomeric discrimination
inherent with the immune system, the monoclonal anti-
body induced from the racemic hapten phosphonate

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Z.-D. Shi et al. / Bioorg. Med. Chem. 10 (2002) 2171–2175
Experimental
Diethyl 1-(6-hydroxy-2-naphthyl)-ethylidene phosphonate
9. To a stirred solution of 8 (1.420 g, 4.41 mmol) in
30 mL of CH2Cl2 was added dropwise BBr3 CH2Cl2
General
ꢀ
solution (30 mL, 30 mmol) at À78 ꢁC under argon. The
mixture was stirred for 2 h and fragments of ice and
water were added. The aqueous layer was extracted with
EtOAc (150 mLÂ3) and the combined organic layer was
washed with brine, dried over Na2SO4. The solvent was
removed in vacuo and the residue was purified by flash
chromatography (ethyl acetate/petroleum ether 2:1) to
give 9 (1.212 g, 88%) as a yellow oil. MS (EI) m/z 308
(M)+; 1H NMR (300 MHz, CDCl3) d 7.58–6.81 (6H, m,
Ar), 4.16–3.93 (4H, m, both OCH2CH3), 3.36 (1H, m,
CHAr), 1.68 (3H, m, CH3), 1.34 (3H, t, J=6.8 Hz,
OCH2CH3), 1.26 (3H, t, J=7.2 Hz, OCH2CH3). Anal.
calcd for C16H21O4P: C, 62.33;H, 6.87. Found: C,
62.36;H, 6.97.
1
IR spectra were recorded on a FT-IR instrument. H
NMR spectra were recorded on Bruker AM-300
(300 MHz) and assigned in ppm (d) downfield relative to
TMS as internal standard. MS spectra were conducted
on a HP-5989A and VG QUATTRO mass spectro-
meters. Microanalyses were preformed in Micro-
analytical Laboratory at the Shanghai Institute of
Organic Chemistry. Flash column chromatography
were preformed on silica gel (10–40 m) using a mixture
of petroleum ether–ethyl acetate or dichloromethane–
methanol as the eluent. Solvents and reagents were
purified and dried by standard methods prior to use.
Diethyl (6-methoxy-2-naphthyl)-methylene phosphonate
7. A 200 mL flask was charged with a solution of 622
(5.56 g, 29.6 mmol) in anhydrous THF (150 mL) under
argon. The flask was cooled to À40 ꢁC in dry ice/ace-
tone bath, then PBr3 (3.4 mL) in anhydrous THF
(30 mL) was added dropwise for 30 min and stirring
continued for an additional 2 h. The mixture was
warmed to room temperature, and satd NaHCO3 was
added to neutralize the mixture. The aqueous layer was
extracted with EtOAc (150 mLÂ3) and the combined
organic solvent was dried over Na2SO4, concentrated in
vacuo to give the crude product (7.27 g), which was dis-
solved in P(OEt)3 (10 mL) and the mixture was stirred at
120 ꢁC for 10 h. EtOAc (150 mL) was added to the
cooled mixture and the solution was washed with brine,
dried over Na2SO4. The solvent was removed in vacuo
and the residue was purified by flash chromatography
(ethyl acetate/petroleum ether, 2:3) to give 7 (8.13 g,
89%) as a yellow oil. IR (film, cmÀ1): 2985, 1635, 1508,
1485;MS (EI) m/z 308 (M)+; 1H NMR (300 MHz,
CDCl3) d 7.69–7.10 (6H, m, Ar), 4.02–3.97 (4H, m, both
OCH2CH3), 3.90 (3H, s, OCH3), 3.27 (2H, d,
J=21.4 Hz, CH2Ar), 1.22 (6H, t, J=7.2 Hz, both
OCH2CH3). Anal. calcd for C16H21O4P: C, 62.33;H,
6.87. Found: C, 61.89;H, 7.02.
Diethyl 1-[6-(3-ethoxycarbopropoxy)-2-naphthyl]-ethylidene
phosphonate 10. To a solution of 9 (1.428 g, 4.64 mmol)
in 200 mL of acetone was added ethyl 4-bromobutyrate
(1.05 mL) and K2CO3 (4.50 g). The stirred mixture was
refluxed for 40 h followed by evaporated in vacuo. Ice-
water was added and the mixture was extracted with
EtOAc (100 mLÂ3). The organic layer was washed with
brine, dried over Na2SO4. The solvent was removed in
vacuo and the residue was purified by flash chromato-
graphy (ethyl acetate/petroleum ether 1:1). Compound
10 (1.608 g, 82%) was given as a yellow oil. MS (EI) m/z
1
422 (M)+; H NMR (300 MHz, (CD3)2CO) d 7.79–7.14
(6H, m, Ar), 4.17–4.03 (6H, m, three OCH2CH3), 4.00–
3.88 (2H, m, OCH2CH2), 3.37 (1H, m, CHAr), 2.53
(2H, t, J=7.3 Hz, CH2CO), 2.07 (2H, m, CH2CH2CO),
1.59 (3H, dd, J=9.5 Hz, J=18.0 Hz, CH3), 1.26–1.17
(6H, m, both OCH2CH3), 1.26 (3H, t, J=7.2 Hz,
OCH2CH3). HRMS (EI): (M)+, found 422.1858.
C22H31PO6 requires 422.1862.
Ethyl 1-[6-(3-carboxypropoxy)-2-naphthyl]-ethylidene
phosphonate, monoethyl ester 3. To a solution of 10
(181 mg, 0.429 mmol) in ethanol (27 mL) was added
NaOH (725 mg) and the stirred mixture was refluxed for
24 h. Then, the mixture was acidified to pH 5 and con-
centrated in vacuo. The residue was purified by flash
chromatography (CH2Cl2/MeOH 4:1) to afford 3
(133 mg, 85%) as a yellow solid. MS (EI) m/z 366 (M)+;
ESI (CH3OH) 389 (M++Na); 1H NMR (300 MHz,
CD3OD) d 7.89–7.23 (6H, m, Ar), 4.28 (2H, t,
J=6.2 Hz, OCH2), 3.85 (2H, q, J=7.0 Hz, OCH2CH3),
3.32 (1H, m, CHAr), 2.61 (2H, t, J=5.8 Hz, CH2CO),
2.16 (2H, m, CH2CH2CO), 1.62 (3H, dd, J=7.4 Hz,
J=17.3 Hz, CH3), 1.17 (3H, t, J=7.0 Hz, OCH2CH3).
HRMS (EI): (M)+, found 366.1232. C18H23PO6
requires 366.1228.
Diethyl 1-(6-methoxy-2-naphthyl)-ethylidene phosphonate
8. To a solution of 7 (3.550 g, 11.53 mmol) in anhydrous
THF (20 mL) was added dropwise n-BuLi (6.2 mL,
11.53 mmol) at À78 ꢁC under argon. The mixture was
stirred for 45 min and a solution of CH3I (1.8 mL) in
THF (8 mL) was added dropwise. Then the mixture was
stirred overnight. Water was added to quench the reac-
tion and 0.1 N HCl was added to neutralize the mixture.
The aqueous layer was extracted with EtOAc
(100 mLÂ3) and the combined organic layer was
washed with brine, dried over Na2SO4. The solvent was
removed in vacuo and the residue was purified by flash
chromatography (ethyl acetate/petroleum ether 1:2) to
give 8 (3.222 g, 87%) as a yellow oil. IR (film, cmÀ1):
Ethyl (6-methoxy-2-naphthyl)-ethylidene phosphonate,
monoethyl ester 4. To a solution of 8 (210 mg,
0.65 mmol) in ethanol (8 mL) was added NaOH
(170 mg) and the stirred mixture was refluxed for 16 h.
Then the mixture was acidified to pH 5 and con-
centrated in vacuo. The residue was purified by flash
1
2981, 1634, 1392;MS (EI) m/z 322 (M)+; H NMR
(300 MHz, CDCl3) d 7.68–7.07 (6H, m, Ar), 4.03–3.73
(4H, m, both OCH2CH3), 3.84 (3H, s, OCH3), 3.27 (1H,
m, CHAr), 1.62 (3H, dd, J=18.4 Hz, J=7.4 Hz, CH3),
1.24 (3H, t, J=7.1 Hz, OCH2CH3), 1.08 (3H, t,
J=7.0 Hz, OCH2CH3). Anal. calcd for C17H23O4P: C,
63.35;H, 7.14. Found: C, 63.24;H, 7.36.
chromatography (EtOAc/EtOH 1:1) to afford
4
(156 mg, 82%) as a yellow solid. IR (KBr) nmax 2980,

Z.-D. Shi et al. / Bioorg. Med. Chem. 10 (2002) 2171–2175
2175
1663, 1488;MS (EI)
m/z 294 (M)+; 1H NMR
ethyl ester and R-(À)-naproxen ethyl ester over a range
of concentrations were assayed. Initial linear rate were
measure at less than 5% hydrolysis of the total sub-
strate. For antibody N116–27, the catalyzed hydrolysis
rates with five different substrate concentrations from
0.3 to 0.7 mmol/L were measured in the presence of the
antibody concentration (6.0 mmol/L). The analysis of v0
and C0 by the standard Michaelis–Menton equation
gave Michaelis–Menton parameters (KM, Vmax and
kcat).
(300 MHz, (CD3)2CO) d 7.85–7.13 (6H, m), 4.05 (2H,
m, OCH2CH3), 3.90 (3H, s, OCH3), 3.34 (1H, m,
CHAr), 1.61 (3H, m, CH3), 1.17 (3H, m, OCH2CH3).
HRMS (EI): (M)+, found: 294.1021. C15H19PO4
requires 294.1024.
Synthesis of immunogens 11 and 12
To a mixture of bovine serum albumin (BSA) in 4.0 mL
of sodium phosphate buffer (PBS, pH=7.2, 10 mmol/L)
was added a solution of hapten 3 (10 mg) in 2.2 mL of
DMF and 1-ethyl-3-(3-dimethylaminopropyl) carbodii-
mide (EDCI, 15 mg). The mixture was stirred at room
temperature for 5 h and the reaction mixture was dia-
lyzed three times in 3 days with 10 mmol/L PBS buffer
(pH=7.2). After that, the immunogen solution was ali-
quoted, frozen, and stored at À20 ꢁC. Another con-
jugate of FgG was prepared as described above.
Acknowledgements
This work is supported by the State Science and Tech-
nology Commission of China (970211006), the National
Natural Science Foundation of China (39180021) and
the Chinese Academy of Sciences (KJ951-A1–504).
Establishment of mouse lymphocyte hybridoma clones
secreting antibodies
References and Notes
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11. Wechter, W. J.;Loughhead, D. G.;Reischer, R. J.;Van-
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20. Giordan, C.;Villa, M. Syn. Comm. 1990, 20, 383.
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The lymphocytes from spleen cells of BALB/C mouse
immunized with immunogen 12 were fused with cells of
mouse myeloma cell lines Sp2/0 or NS-1 to construct
hybridoma. Five hybridoma clones secreting anti-hydro-
lysis transition state antibodies, named N121–6, N10–16,
N116–27, S35–6 and S1–14, were obtained by screening
through competitive inhibition assay (CIA) with the
inhibitor 4 and indirect enzyme linked immunoabsor-
bent assay (ELISA) with the immunizing conjugate 11
as coating antigen and cloning by method of limiting
dilution. Their immunoglobulin (Ig) subclasses were
IgG1, IgG2a, IgG2b, IgG2b and IgG2b, respectively.
Kinetic measurements
Screening for catalytic antibodies: Purified monoclonal
antibodies were dialyzed against 10 mmol/L PBS buffer
(pH 7.2) three times. Their protein concentrations were
determined by the measurement of the absorbance at
260 and 280 nm. The screening assays of hydrolysis were
performed under 0.4 mmol/L rac-Naproxen ethyl ester
and 10 mmol/L antibody at 37 ꢁC in a pH 8.2 buffer
made from 50 mmol/L Tris–HCl and 136 mmol/L NaCl
with 5% cosolvent acetonitrile. The reaction was mon-
itored by the decrease absorbance of the ester at 240 nm
using a C-18 LiChrosorb RP-18 column with a mobile
phase (55% water, 45% acetonitrile at 1 mL/min). An
aliquot of reaction mixture (1 mL) was removed (50 mL)
and quenched into acetonitrile (50 mL). The background
hydrolysis of the substrate in the absence of catalytic
antibody was also measured at the same conditions. An
internal standard of 6-methoxy-2-naphathyl-methanol
was used to calibrate the ester concentrations.
The buffer system and HPLC assay were the same as
above. To obtain the kinetic parameters, substrate
including rac-naproxen ethyl ester, S-(+)-naproxen
22. Giordano, C.;Villa, M. Syth. Comm. 1990, 20, 383.