CA2190455C - Catalytic antibodies enantioselectively hydrolysing amino acid ester derivatives - Google Patents

Abstract

The present invention is directed to a process for conducting optical resolution of a racemic mixture of an amino acid derivatives and a process for preparing an optically active amino acid using a catalytic antibody enantioselectively hydrolyzing an amino acid ester derivative. The catalytic antibody and hybridoma producing said antibody are also provided. The hybridomas in the present invention are typically produced by stimulation with an antigen comprising as a hapten a compound of the formula:

(see formula 3) wherein CBZ is N-benzyloxycarbonyl.

Description

CATALYTIC ANTIBODIES ENANTIOSELECTIVELY
HYDROLYSING AMINO ACID ESTER DERIVATIVES
The present invention belongs to the field of the art synthesizing optically active amino acids. Specifically, the present invention relates to a process for conducting enantioselective hydrolysis by utilizing catalytic antibodies. More specifically, the invention relates to processes for enantioselectively hydrolyzing amino acid ester derivatives with catalytic antibodies to effect optical resolution of a racemic mixture of amino acid derivatives and to prepare optically active amino acids. Because the present invention allows very easy production of optically active amino acids having various substituted groups, it can contribute to basic studies on the function of living bodies, development of a novel medical drug containing optically active amino acids as an essential component, and development of methodologies for synthesizing various optically and physiologically active substances or functional compounds, which methodologies use optically active amino acids in synthesizing processes.
Proteins, a primary component of the living body, are constructed with optically active amino acids, which results in a chiral environment in the living body. Therefore, many of the physiologically active substances including medical drugs are optically active substances, and it is known that bioactivity is found only in one of the enantiomers, or a difference in bioactivity is observed between the enantiomers. Accordingly, developing a B

methodology for obtaining optically active substances is one of the targets of investigations.
The simplest process usually performed to obtain an optically active amino acids is optical resolution of a racemic mixture of amino acid derivatives. The process for optical resolution includes a process employing an enzyme (e.g. a kinetic optical resolution using aminoacylase, lipase and the like), and a process forming a diastereomer, or the like. However, the suitable process must be selected depending on the particular amino acid to be resolved, because there is no eommon process effective for all the amino acids having a variety of substituted groups.
In such a situation as mentioned above, the inventors of the present invention have succeeded in developing a process for performing optical resolution of a racemic mixture of amino acid derivatvies, using catalytic antibodies, which is applicable for any amino acid. Although reactions using catalytic antibodies usually have drawbacks in that the catalytic antibodies have substrate specificity, and therefore, they have only limited applicablity, the present inventors have overcome such drawbacks by developing catalytic antibodies which enantioselectively hydrolyze ester derivatives of a racemic mixture of any amino acid derivatives to produce corresponding optically active amino acids.
The present invention relates to a process for 2 5 Performing optical resolution of a racemic mixture of amino acid derivatives, comprising using a nonspecific catalytic antibodies enantioselectively hydrolyzing various amino acid ester derivatives.
B

In another aspect, the present invention relates to a process for preparing optically active amino acids from a racemic mixture of amino acid derivatives, characterized by using catalytic antibodies to enantioselectively hydrolyze amino acid ester derivatives.
The processes of the invention can be applied to the synthesis of both L- and D-isomers of amino acids and include two processes each consisting of the following two steps:
(1 ) preparing ester derivatives of a racemic mixture of amino acid derivatives, and then (2) enantioselectively hydrolyzing only L-form of amino acid ester derivatives; and (1) preparing ester derivatives of a racemic mixture of amino derivatives, and then (2) enantioselectively hydrolyzing only D-form of amino acid ester derivatives.
The catalytic antibodies as used herein are typically prepared by immunizing a mammal with, as a hapten, a compound having the formula 3 below:
~.cBz 2 0 ~-~o ~oH
o P.o I ~

wherein CBZ is benzyloxycarbonyl.
Although CBZ in formula 3 is an amino-protecting group, other conventional amino-protecting groups known to those skilled in the art, for example, trichloroacetyl, benzyl, t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, or CBZ having a substituent such as lower alkyl, lower alkoxy, halogen, vitro, etc. in B

the phenyl moiety can also be used instead of CBZ. The compound of the above formula 3 has a 4-nitrobenzyl ester moiety. However, the benzyl ester moiety can have one or two substituents selected from lower alkyl, lower alkoxy, vitro, halogen, or the like in the phenyl moiety. Catalytic antibodies obtained using the haptens having a substituted benzyl moiety can also hydrolyze any corresponding amino acid ester derivatives. Thus, these are also potential haptens.
In one embodiment, the invention aims at antibodies which catalyze enantioselective hydrolysis of L- and D-isomers of N-benzyloxycarbonyl amino acid ester derivatives. N-benzyloxycarbonyl amino acid is an N-protecting amino acid often used for the synthesis of peptides. Such antibodies catalyze hydrolytic reactions as shown in the following Scheme 1. Although the following explanation concerns the catalytic antibodies which are obtained by the use of the compound of the above Formula 3, those skilled in the art will understand that the following explanation is also applicable to catalytic antibodies obtained from the potential haptens mentioned above.
2 0 Scheme 1 ~, CBZ An antibody of , CgZ CH20H
the invention + I
R~-O I ~ R~ COOH
O

, CBZ An antibody of , CBZ
HN the invention ~ CH20H
R~- O I ~ R~ COOH +
i O
NO

wherein CBZ is N-benzyloxycarbonyl and R may be substantially any substituent, and, it is typically alkyl or phenyl group which is optionally substituted with hydroxy, amino, alkylthio, acyloxy or phenyl, and is, more particularly, 5 CH3-, phCH2-, (CH3)2CHCH2-, CH3(CH2)s-, CHsSCH2CH2-, (CH3)2CH-, ph-, or 4-HOph-;
wherein ph- is phenyl group;
alkyl is typically a straight or branched C,-C,o alkyl; and acyloxy is a group of the formula: RCOO- wherein R is alkyl.
The above-noted antibodies are prepared using a known technique for catalytic antibodies reported by Lerner, R. A. et al., Science, 252, 659, 1991. Thus, the antibodies of the invention are produced by immunizing mammals using, as a hapten, the phosphonate derivative of the following formula 3 which is a transition state analog during hydrolysis.
HN~CBZ
HO OH
O P~p ~ w O ~N02 Prer~aration of Ha ten The above phosphonate derivative can be synthesized, for example, according to the synthetic route shown in the following Scheme 2.

Scheme 2 CH2Ph PhCH2NH2 H N
Et0 PDC Et0 C H O P(OEt)3 Et0 OEt 1~~0 H- ~' ---~ ~~P:
O O 4 O 5 0 OEt 1 H2, Pd-C ~CgZ ~CBZ
2; CBZCI H N O H H N
Et0 . NaOH H O
3 TMSBr ~P-O I ~ ~P~O H I \
4~ 4 - nitrobenryl O ~ O
alcohol g N02 3 N02 1 0 N - hydroxy O CBZ .CBZ
succ~ ~N,O~~ O H KLH X-N~~~ O H
11~~0 0~~~~''~_P'O t ~ O
O / N02 8 (X - KL~ / N02 Commercially available ethyl 6-hydroxy hexanoate is oxidized with PDC (pyridinium dichromate), the resultant compound (4) is iminated with benzylamine in situ, and reacted with triethylphosphite to give compound (5). Compound (5) is then debenzylated by catalytic hydrogenation, and the amino group is subsequently benzyloxycarbonylated. After diethylphosphonate is deethylated by bromotrimethylsilane, the resultant phosphonic acid is reacted with 4-nitrobenzyl alcohol in the presence of DCC
(dicyclohexylcarbodiimide) and 1 H-tetrazole to obtain compound (6).
An ethyl ester of compound (6) is alkali-hydrolyzed to obtain hapten (3). Hapten (3) is converted to activated ester (7) with 2 5 hydroxysuccinimide and the latter is reacted with KLH (Keyhole limpet hemocyanin) to obtain antigen (8).
Preparation of Catalytic Antibodies BALB/c mice are immunized with the above antigen (8) to give monoclonal antibodies.
A BALB/c mouse (female, 4-week old) is immunized four times with antigen (8), and spleen cells are removed. After cell fusion is performed in a conventional manner, resultant hybridomas are screened using Enzyme-Linked Immuno Sorbent Assay (ELISA) to obtain a hybridoma producing antibodies which bind to the hapten.
Repeated clonings by limiting dilution are performed to obtain a monoclonal IgG-producing hybridoma. Crude antibodies precipitated by adding (NH4)2S04 to the supernatant of the culture are purified using cation exchange chromatography and protein G affinity chromatography.
Identification of Cata~rtic Antibodies The above-obtained antibodies are screened for catalytic activity thereof. In order to simply and quickly conduct the screening of the catalytic activity, the esters (L-10) and (D-10) shown in the following Scheme 3 are used as substrates, which permit tracing of the progress of hydrolysis by measuring the changes in fluorescence intensity. Fluorescence due to anthranilic acid 2 0 moiety in the esters (L-10) and (D-10) is quenched by 4-nitrobenzyl group existing in the molecules, and when the quenching is dissolved by hydrolysis, fluorescence intensity thereof increases. Namely, the esters (L-11 ) and (,D-11) in the following Scheme 3 are fluorogenic substances.
s s Scheme 3 ,caz ,cBz NH2 O HN NH2 O ~ CH20H
I ~ N O I ~ --.~ I ~ N COOH + I
i H O i i H i ,cBZ ,cBz NH2 O HN NH2 O ~ CH20H
I \ N O I \ I ~ N COOH + I
i H O i i H i D-10 N02 D-11 ' N02 Ester (L-10) is equivalently mixed with ester (D-10) to prepare a fluorogenic substrate (DL-10) and the fluorogenic substrate is used in the screening of the catalytic activity.
At first, changes in fluorescent intensity of the obtained purified antibodies are traced at ~, ex 340 nm and ~, em 415 nm for 10 minutes using the fluorogenic substrate (DL-10). By performing the screening, antibodies hydrolyzing the fluorogenic substrate are identified. Next, a similar procedure is performed using either one of the fluorogenic substrates (L-10) and (D-10) to evaluate enantioselectivity of each antibody.
The antibodies having selective catalytic activities to L- or D-form, as obtained above, are examined for their substrate-specificity and enantioselectivity to various amino acid ester derivatives by HPLC analysis.
2 5 Antibody 7612 (see working Examples hereinafter described) having selective catalytic activity to L-form catalyzed the hydrolysis of L-form (L-1 ) of substrates derived from alanine, phenylalanine, leucine, norleucine, methionine, valine, phenylglycine, 4-hydrophenylglycine, and lysine, but the antibody 7612 did not catalyze the hydrolysis of the corresponding D-form substrates (D-1 ). Namely, the antibody 7612 enantioselectively catalyzed the hydrolysis of various substrates.
Antibody 3G2 (see working Examples hereinafter described), having selective catalytic activity to D-form, catalyzed the hydrolysis of D-form (D-1 ) substrates derived from alanine, phenylalanine, leucine, norleucine, methionine, valine, phenylglycine, and 4-hydrophenylglycine, but the antibody 3G2 did not catalyze the hydrolysis of the corresponding L-form substrates (L-1 ). Thus, the antibody 3G2 enantioselectively catalyzed the hydrolysis of various substrates.
The above results demonstrate that the antibodies of the invention obtained by use of the phosphonate (3) as a hapten enantioselectively catalyze the hydrolysis of various amino acid ester derivatives.
The reason why the antibodies of the invention show such non-specificity to substrates in spite of being antibodies is considered to be as follows. It is know that the total maximum contacting surtace area between hapten and antibody is 700-800 A2, and that the contacting surtace area between low molecular hapten (M.W. up to 350) and antibody is 250-400 A2. This means that the recognition site of the antibody has such size, and it is presumed that, when an antibody derived from the above hapten 3 recognizes CBZ group, 4-nitrobenzyl alcohol, phosphonate, and stereochemistry of a-position of the hapten 3, recognition site of the antibody is sufficiently filled by them, and substituent (R) on a-position of the hapten gets out of the recognition site. Thus, when the antibodies of the invention catalyze the hydrolysis of amino acid ester derivatives, a-substituent (R) thereof will be outside the recognized site, and therefore, it is believed that the antibodies catalyze every substrate irrespective of their substituents (R) so long as 5 said substrates have the same stereochemistry as the enantiomer of the hapten recognized by the antibodies.
Antibodies 7612 and 3G2 catalyzed indeed the enantioselective hydrolysis of a wide range of substrates according to the above-noted design (see working Examinations hereinafter 10 described). Accordingly, it was demonstrated that immunization by the use of the hapten having a structural unit which has a size corresponding to that of the recognition sites of the antibodies, and a structural unit which may protrude outside said recognition site gave the catalytic antibodies the capablility of performing the stereospecific reaction and catalyzing the wide range of substrates thanks to their high recognition abilities.
As mentioned above, the antibodies of the invention can be utilized in a process for preparing optically active amino acids from their racemic mixture. Specifically, such a process for preparing an optically active amino acid comprising the steps of:
a) subjecting a racemic mixture of an amino acid derivatives to an ester-forming reaction;
b) selectively hydrolyzing a desired stereoisomer, D- or L-isomer, of the ester derivative by the use of a catalytic antibody which enantioselectively hydrolyzes an amino acid ester derivative; and c) treating the resultant reaction mixture in a conventional manner, for example, making the .mixture acidic or basic and then extracting with an organic solvent to leave an aqueous layer, from which the desired hydrolyzed stereoisomer is isolated.
The present invention will be illustrated in more detail with reference to the following Examples and Examinations.
However, none are intended to limit the scope of the invention.
Example 1 Synthesis of hapten~31 PhCH2NH2 L.N. CH2Ph 1 O Et0 OH ~ Et0 CHO P(~ Et0 ,OEt O CH2CI2 ~~ EtOH O P' OEt 4 (40%) 5 (48%) O
1) H2, Pd-C ~,CBZ ~.CBZ
HCOOH-MeOH
2) CBZCI, Et3N/THFEtO ,OH NaO~ HO ,OH
3) TMSBr/CH3CN O P'O i ~ H20-MeOH O P'O I
4) 4 - nitrobenryl alcohol O ~ O
DCC, 1 H - tetrazole g (~/o) NO2 3 (c~g%) Np2 1 5 CH2C12 - pyridine 1. Synthesis of compound (4) To a solution of commercially available ethyl 6-hydroxyhexanoate (2.83 g, 17.7 mmol) in methylene chloride (70m1) 20 was added PDC (pyridinium dichromate) (9,95 g, 26.4 mmol) at room temperature. After stirring for 5 hours, the reaction mixture was purified by flash column chromatography over silica gel, eluting with ethyl acetate/hexane (1:2), to obtain compound (4) (1.1276 g, 40%).
2 5 t H-NMR (500 MHz, CDC13): 8 9.78 (t, J=1.4 Hz, 1 H), 4.14 (q, J=7.2 Hz, 2H), 2.50-2.46 (m, 2H), 2.37-2.30 (m, 2H), 1.71-1.63 (m, 4H), 1.26 (t, J=7.2, 3H).

2. Synthesis of compound (5) To a solution of compound (4) (1.1276 g, 7.13 mmol) in ethanol (2.5 mL) were added benzylamine (1.56 rnl, 14.3 mmol) and triethylphosphite (2.44 mL, 14.2 mmol) at room temperature. The mixture was stirred at room temperature for 17.5 hours and then at 40°C for 6 hours. After the solvent was evaporated, the residue was purified by flash column chromatography over silica gel, eluting with ethyl acetate/isopropanol (20:1 ), to obtain compound (5) (1.1276 g, 40%).
~H-NMR (500 MHz, CDC13): 8 7.35-7.23 (m,SH), 4.21-4.09 (m,6H), 3.97 (d, J=13.1 Hz, 2H), 3.88 (dd, J=13.1 Hz, 1.3 Hz, 2H), 2.85 (m, 1 H), 2.30-2.25 (m, 2H), 1.83-1.53 (m, 6H), 1.34 (t, J=7.0 Hz, 6H), 1.25 (t, J=7.1 Hz, 3H).
FBMAS (fast atom bombardment mass) m/z: 386 (M++N).
HR-FABMAS (high resolution-FABMAS) for Ci9H3305NP(M++H):
Calcd.: 386.2132, Found: 386.2092.
3. Synthesis of compound (6) To a solution of compound (5) (473.0 mg, 1.23 mmol) in methanol (3.0 mL) and formic acid (0.40 mL) was added 10% Pb-C
(4.6 mg), and the mixture was stirred at room temperature for 2.5 days under hydrogen. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to obtain a colorless residue. The residue was dissolved in THF (5.0 mL), and carbobenzoxy chloride (700 p.L, 4.90 mmol) and triethyl amine (1.71 2 5 mL, 12.3 mmol) were added thereto at room temperature. After stirring for 3 hours, 1 N hydrochloric acid was added, and the mixture was extracted with ethyl acetate. The combined organic phase was washed with saturated brine, dried, and evaporated. The residue was purified by flush column chromatography over silica gel, eluting with ethyl acetate to obtain colorless gum (120.5 mg).
The gum (120.5 mg) was dissolved in methylene chloride (0.5 mL) and bromotrimethylsilane (222 ~,L, 1.68 mmol) was added thereto at room temperature. After stirring at 35 °C for 2 hours, the solvent was evaporated off, and to the residue were added acetonitrile (1.0 mL) and water (0.2 mL). After 12 hours, the solvent was evaporated and the residue was dried. To a solution of the residue in methylene chloride (1.0 mL) and pyridine (0.5 mL) were added 4-nitrobenzyl alcohol (61.2 mg, 0.40 mmol), 1 H-tetrazole (9.7 mg, 0.14 mmol) and DCC (261.5 mg, 1.27 mmol), and the mixture was stirred at 35°C for 5 hours. After the solvent was evaporated off, acetonitrile was added to the residue, and the mixture was filtered. The filtrate was purified by HPLC (YMCA-323 C-18, 10 mm in diameter x 250 mm in length, acetonitrile/0.1 % aqueous trifluoroacetic acid solution=50/50, 3.0 mL/min, 254 nm) to obtain compound (6) (34.5 mg, 6% from compound (5)).
1 H-NMR (500 MHz, CDC13): S 8.22 (d, J=8.6 Hz, 2H), 7.62 (d, J=8.6 Hz, 2H), 7.38-7.28 (m, 5H), 5.18 (ABX, JAB=13.5 Hz, JAx=JBx= 7.4 HZ, Ov=16.2 Hz, 2H), 5.11 (AB, J=12.6 Hz, Ov=20.2 Hz, 2H), 4.14 (q, J=7.1 2 0 Hz, 2H), 4.06 (m,1 H), 2.33 (t, J =7.3 Hz, 2H), 1.95-1.37 (m, 6H), 1.27 (t, J=7.1 Hz, 3H).
FABMAS m/z: 509 {M++H).
HR-FABMAS for C23HsoO9N2P(M++H):
Calcd.: 509.1689, Found: 509.1688.
2 5 4. Synthesis of compound (3) To a solution of compound (6) (15.0 mg, 0.0295 mmol) in methanol (0.2 mL) and water (0.2 mL) was added 1 N NaOH (0.1 mL, 0.1 mmol) at room temperature. After 6.5 hours, the reaction 'Trade mark mixture was made acidic by addition of trifluoroacetic acid, and the mixture was purified by HPLC (YMCA-323: C-18, 10 mm in diameter x 250 mm in length, acetonitrile/0.1 % aqueous trifluoroacetic acid solution=50/50, 254 nm) to obtain compound (3) (14.0 mg, 99%).
1 H-NMR (500 MHz, CD30D): 8 8.22 (d, J=8.6 Hz, 2H), 7.61 (d, J=8.6 Hz, 2H), 7.37-7.28 (m, 5H), 5.18 (ABX, JAB=13.5 Hz, JAx=JBx= 7.4 HZ, Ov=16.7 Hz, 2H), 5.11 (AB, J=12.5 Hz, Ov=26.6 Hz, 2H), 4.07 (m,1 H), 2.32 (t, J =7.3 Hz, 2H), 1.97-1.38 (m, 6H).
FABMAS m/z: 503 (M++Na), 481 (M++H).
HR-FABMAS for C2~ H2509N2PNa(M++Na):
Calcd.: 503.1196, Found: 503.1202.
Examlhe 2 Condensation of hapten and carrier proteins ~_cBZ o ~.caz HO ,pH N - hydroxy succinimide ~ -O P~ O I ~ WSC, DMAP N
O i CH3CN O O

~ (79%) .CBZ
KLH or BSA H
2 0 PBS x- N off (pH 7.4)-DMF O ~'O I w O i NO2 8 (X = KLH) 9 (X = BSA) 5. Synthesis of compound (7) 2 5 To a solution of compound (3) (6.4 mg, 0.013 mmol) in acetonitrile (0.3 mL) were added N-hydroxysuccinimide (2.5 mg, 0.022 mmol), WSC water-soluble carbodiimide (WSC) (8.3 mg, 0.043 mmol) and dimethylaminopyridine (DMAP) (0.1 mg, 0.0008 mmol) and the mixture was stirred for 2 hours. The reaction mixture was purified by HPLC (YMCA-323: C-18, 10 mm in diameter x 250 mm in length, acetonitrile/0.1 % aqueous trifluoroacetic acid solution=50/50, 3.0 mL/min, 254 nm) to obtain compound (7) (6.1 5 mg, 79%).
iH-NMR (500 MHz, CD30D): 8 8.23 (d, J=8.7 Hz, 2H), 7.62 (d, J=8.7 Hz, 2H), 7.38-7.27 (m, 1 H), 5.19 (ABX, JAB=13.4 Hz, JAx=JBx= 7.5 HZ, Ov=15.9 Hz, 2H), 5.11 (AB, J=12.5 Hz, Ov=16.4 Hz, 2H), 4.08 (m,1 H), 2.85 (s, 4H), 2.66 (t, J=7.2 Hz, 2H), 1.97-1.43 (m, 6H).
10 6. Synthesis of KLH-condensate (8) To a solution of compound (7) (2.5 mg, 0.0043 mmol) in DMF (60 ~,L)-200 mM Na2HP04-NaH2P04, pH7.4 (0.5 mL) was added a solution of KLH in 10 mM NaH2P04, pH 7.4 (11.3 mg/mL, 442 ~.L).
After 21 hours, the reaction mixture was purified by Sephadex* G-15 25M (Pharmacia, PD-10), eluting with PBS, to obtain KLH-condensate (8). The concentration of the protein therein was determined by Bradford's method (1.3 mg/mL).
7. Synthesis of BSA-condensate (9) To a solution of compound (7) (3.0 mg, 0.0052 mmol) in DMF (40 ~.L) was added a solution of BSA (6.0 mg) in 200 mM
Na2HP04-NaH2P04, pH 7.4 (0.5 mL) at room temperature. After 12 hours, the reaction mixture was purified by Sephadex G-25M
(Pharmacia, PD-10), eluting with PBS, to obtain KLH-condensate (9).
The concentration of the protein therein was determined according 2 5 to Bradford's method (1.3 mg/mL). The compound (9) was used for ELISA analysis.
*Trade mark 1g 2190455 Example 3 Immunization A solution of 50 ~.g of the antigen (KLH-condensate), prepared in Example 2, in 50 ~,L of saline was mixed with an equal amount of Freund's complete adjuvant, and the mixture was intraperitoneally injected to BALB/c mice (4-week old, female).
After 10 days, the mice were boostered with a mixture of the antigen-saline solution (50 p.g/50 ~,L) and equal amount of Freund's incomplete adjuvant and, following 10 days, boostered again with the same mixture. After 7 days, blood was taken from the tail vein of the mouse. Antibody titer was assayed using BSA-condensate by ELISA, which uses as a secondary antibody, biotinized anti-mouse IgG antibodies, avidin, and biotinized peroxidase, and the titer measured 2.5 x 105. One month after the second booster, the antigen-saline solution (100 ~.g/100 ~,L) was administered via the tail vein (final immunization).
Example 4 Preparation of h~rbridoma 2 0 Three days after the final immunization, the spleen of the mouse was removed, followed by cell fusion of 1.9 x 108 spleen cells and 2.7 x 10~ myeloma cells (x63/Ag 8.653) using polyethyleneglycol. Using ten 96-well plates each containing HAT
selection medium (RPMI medium containing 0.1 mM hypoxanthine, 0.4 ~,(~ aminopterin, 0.016 mM thymidine, and 10% fetus bovine serum) containing 6 x 105 feeder cells (mouse thymocytes)/well, selection of the hybridomas obtained was conducted (at 37°C, under 10% C02).
Following 8-14 days, supernatants removed from the wells in which a growth of a colony was found were subjected to screening by ELISA. Out of 111 positive wells, 57 wells were subjected to cloning to obtain 39 clones. ELISA analysis using anti-mouse IgG H-chain antibody demonstrated that all of the 39 clones produced IgG.
Example 5 Preparation of monoclonal antibodies Each of 39 hybridomas prepared in Example 4 was cultured in a complete medium (RPMI medium containing 10% fetus bovine serum) for about 7 days. The supernatants were precipitated with an equal volume of aqueous saturated ammonium sulfate solution and then subjected to cation exchange chromatography on a S-Sepharose'~column and affinity chromatography on a protein G
column to obtain 10-20 mg of the purified antibodies.
Preparation 1 Synthesis of fluorogenic substrate (L-10~

BOC NH
1) ~ COO- N
,CBZ ~ BOC ,CBZ
I-N ~ 12 O N..~ HN
2)P - nit~obenzyl alcohol 2 O H2N COOH wgC, DMAP I ~ H O
O ~ N02 L-13 (28%) CBZ

w .N O I w I i H O i 2 5 L-10 (84°i°) No2 1. Synthesis of compound (12) To a solution of commercially available anthranilic acid *Trade mark (1.3737 g, 10.0 mmol) in 0.5N sodium hydroxide (20.0 mL), dioxane (10.0 mL) and acetonitrile (2.0 mL) was added di-t-butyldicarbonate (3.124 g, 14.3 mmol) at 0°C. Following 8-hours of stirring at room temperature, the volatile solvent was evaporated under reduced pressure. To the reaction mixture were added ice and 10% citric acid solution and the mixture was extracted with ethyl acetate. The combined organic phase was washed with saturated brine and dried.
The solvent was evaporated, and the residue was crystallized from ethyl acetate-hexane to obtain t-butoxycarbonyl anthranilic acid 1 0 (1.71 g, 72%).
1 H-NMR (500 MHz, CDC13): 8 10.1 (s, 1 H), 8.47 (d, J=8.0 Hz, 1 H), 8.09 (dd, J=8.0 Hz, 1.4 Hz, 1 H), 7.56 (dt, J=8.0 Hz, 1.4 Hz, 1 H), 7.04 (t, J=8.0 Hz, 1 H), 1.54 (s, 9H).
t-Butoxycarbonylanthranilic acid is known (Nichino, N.;
1 5 Powers, J. C. J. Biol. Chem. 1980, 255, 3482). To a solution of t-butoxycarbonylanthranilic acid (378.9 mg, 1.60 mmol) in methylene chloride (2.5 mL) were added N-hydroxysuccinimide (225.9 mg, 1.96 mmol), WSC (565.9 mg, 2.95 mmol), DMAP (4.7 mg, 0.04 mmol) at room temperature, and the mixture was stirred for 1 hour. To the 20 reaction mixture was added 1% citric acid solution and the mixture was extracted with methylene chloride. The combined organic phase was washed with saturated brine and dried. The solvent was evaporated and the residue was crystallized from ethyl acetate-hexane to give compound (12) (199.7 mg, 30%).
2 5 1 H-NMR (500 MHz, CDC13 ): b 9.49 (s, 1 H), 8.51 (d, J=8.0 Hz, 1 H), 8.17 (d, J=8.0 Hz,1 H), 7.63 (d, J=8.0 Hz,1 H), , 7.07 (d, J=8.0 Hz, 1 H), 2.93 (s, 4H), 1.52 (s, 9H).
A

2. Synthesis of compound (L-13) A mixture of compound (12) (67.3 mg, 0.201 mmol), Na-benzyloxycarbonyl-L-lysine (56.4 mg, 0.201 mmol), and diisopropylethylamine (0.04 mL, 0.230 mmol) was stirred in DMF
(0.2 mL), methanol (0.2 mL), ethyl acetate (0.1 mL) and water (0.1 mL) at room temperature for 16 hours. The volatile solvent was evaporated under reduced pressure and the residue was purified by silica gel flush column chromatography, eluting with ethyl acetate/isopropanol/water (9:1:0-8:2:0.2-5:3:1 ) to give the residue (122.2 mg). To a solution of the residue (122.2 mg) in methylene chloride (1.0 mL) and acetonitrile (0.1 mL) were added 4-nitrobenzylalcohol (34.9 mg, 0.228 mmol), WSC (58.6 mg, 0.306 mmol) and DMAP (1.4 mg, 0.011 mmol) at room temperature and stirred for 4 hours. The reaction mixture was purified by silica gel flush column chromatography, eluting with ethyl acetate/hexane (1:1.1 ), to give compound (L-13) (35.7 mg, 28% from compound (12)).
~ H-NMR (600 MHz, CDC13): 8 10.1 (s, 1 H), 8.33 (d, J=8.0 Hz, 1 H), 8.20 (d, J=8.0 Hz, 2H), 7.52 (d, J=8.0 Hz,2H), , 7.44-7.38 (m, 2H), 7.35-7.28 (m, 5H), 6.95 (t, J=8.0 Hz, 1 H), 6.37 (brs, 1 H), 5.36 (brs, 1 H), 2 0 5.24 (AB, J=13.3 Hz, Ov =16.6 Hz, 2H), 5.06 (AB, J=12.3 Hz, Ov =36.0 Hz, 2H), 4.46 (m, 1 H), 3.43-3.37 (m, 2H), 1.95-1.40 (m, 6H).
3. Synthesis of compound (L-10) To a solution of compound (L-13) (54.1 mg, 0.0858 mmol) in methylene chloride (0.3 mL) was added trifluoroacetic acid 2 5 (0.2 mL) at room temperature and stirred. for 1 hour. The solvent was evaporated under reduced pressure and purified by HPLC (YMCA-323: C-18, 10 mm in diameter x 250 mm in length, acetonitrile/0.1 % aqueous trifluoroacetic acid solution=60/40, 3.0 mUmin, 254 nm) to obtain compound (L-10) (38.1 mg, 84%). The compound (L-10) was crystallized from ethyl acetate-hexane.
1 H-NMR (600 MHz, CDCI3-CD30D): S 8.21 (d, J=8.0 Hz, 2H), 7.49 (d, J=8.0 Hz, 2H), 7.37-7.25 (m, 6H), 7.19 (t, J=8.0 Hz, 1 H), 6.65 (d, J=8.0 Hz, 1 H), 6.64 (t, J=8.0 Hz, 1 H), 5.25 (AB, J=13.3 Hz, 2H), 5.08 5 (AB, J=12.3 Hz, w =24.6 Hz, 2H), 4.40 (m,lH), 3.42-3.34 (m, 2H), 1.94-1.85 (m,1 H), 1.80-1.72 (m,1 H), 1.68-1.54 (m, 2H), 1.51-1.39 (m, 2H).
Preparation 2 10 Syrnthesis of fluorogenic substrate ~D-10) The ester (D-10) was prepared according to the same procedure as that described in Preparation 1, except that the D-form of Na-benzyloxycarbonyl-D-lysine was used.
Physico-chemical properties: ~H-NMR spectrum of D-10 was the 15 same as that of L-10.
Pretaaration 3 Synthesis of substrate compounds ~j -~1) and Method A
2 0 I"N- CBZ 4 - nitrobenzyl alcohol ~. CBZ
WSC, DMAP
R~ COOH CHpCl2 R~ O
O

L_2 L_1 Method B
2 5 . CBZ 4 - nitrobenzyl bromide -CBZ
Et3N
R ~ COOH EtOAc R ~--O I w O

L_2 L_1 General s~rnthesis of com ounds L-1 ) and ,D-1 ) According to the following methods A and B, various amino acid ester derivatives were synthesized.
Method A: A solution of commercially available L- or D-carbobenzoxy amino acid (1 eq.), 4-nitrobenzylalcohol (1.1 eq.), WSC
(1.3-1.6 eq.), and DMAP (0.01 eq.) in methylene chloride was stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure, and ice and 1 N hydrochloric acid were added to the resultant residue, and the mixture was extracted with ethyl acetate. The combined organic phase was washed with saturated brine and dried. The solvent was evaporated, and the residue was purified by silica gel flush column chromatography to obtain compound (L-1 ) or (D-1 ).
Method B: A solution of commercially available L- or D-carbobenzoxy amino acid (1 eq.), 4-nitrobenzylbromide (1.5 eq.), and triethylamine (1.5 eq.) in ethyl acetate was heated under reflux for 2 hours. The reaction mixture was cooled, to which ice and 1 N
hydrochloric acid were added, and the mixture was extracted with ethyl acetate. The combined organic phase was washed with saturated brine and dried. The solvent was evaporated, and the residue was purified by silica gel flush column chromatography to obtain compound (L-1 ) or (D-1 ).
1. Synthesis of N-(benzyloxycarbonyl)alanine 4-nitrobenzyl ester (R=CH3) According to the method A, the titled compound was synthesized, purified by silica gel flush column chromatography, eluting with ethyl acetate/hexane (2:3), and crystallized from ethyl acetate-hexane.

1 H-NMR (500 MHz, CDC13): 8 8.21 (d, J=8.0 Hz, 2H), 7.49 (d, J=8.0 Hz, 2H), 7.37-7.30 (m, 5H), 5.26 (s, 2H), 5.24 (brd, 1 H), 5.12 (s, 2H), 4.47 (m, 1 H), 1.45 (d, J=7.2 Hz, 3H).
FABMAS m/z: 359 (M++H).
HR-FABMAS for C18Hi9OsN2 (M++H):
Calcd.: 359.1244; Found: 359.1244.
2. Synthesis of N-(benzyloxycarbonyl)phenylalanine 4-nitrobenzyl ester (R=PhCH2) According to the method A, the titled compound was synthesized, purified by silica gel flush column chromatography, eluting with ethyl acetate/hexane (2:3), and crystallized from ethyl acetate-hexane.
~ H-NMR (600 MHz, CDC13): 8 8.17 (d, J=8.0 Hz, 2H), 7.38-7.23 (m, 1 OH), 7.08 (m, 2H), 5.23 (brd, J=7.5 Hz, 1 H), 5.19 (s, 2H), 5.09 (s, 1 5 1 H), 4.92 (m, 1 H), 3.12 (d, J=6.8 Hz, 2H).
3. Synthesis of N-(benzyloxycarbonyl)leucine 4-nitrobenzyl ester (R=(CH3)2CHCH2) According to the method A, the titled compound was synthesized, purified by silica gel flush column chromatography, eluting with ethyl acetate/hexane (1:2).
~ H-NMR (600 MHz, CDC13): 8 8.22 (d, J=8.3 Hz, 2H), 7.50 (d, J=8.3 Hz, 2H), 7.40-7.30 (m, 5H), 5.25 (AB, J=13.4 Hz, Ov=11.8 Hz, 2H), 5.11 (AB, J=11.4 Hz, Ov=6.1 Hz, 2H), 5.13-5.11 (1 H), 4.46 (m,1 H), 1.74-1.52 (m, 3H), 0.95 (d, J=7.2 Hz, 3H), 0.94 (d, J=7.2 Hz, 3H).
4. Synthesis of N-(benzyloxycarbonyl)norleucine 4-nitrobenzyl ester (R=CH3CH2CH2CH2) According to the method B, the titled compound was synthesized, purified by silica gel flush column chromatography, eluting with ethyl acetate/hexane (2:5), and crystallized from ethyl acetate-hexane.
~ H-NMR (500 MHz, CDC13): S 8.22 (d, J=8.5 Hz, 2H), 7.50 (d, J=8.5 Hz, 2H), 7.38-7.30 (m, 5H), 5.26 (s, 2H), 5.20 (brd, J=7.9 Hz, 1 H), 5.12 (s, 2H), 4.43 (m, 1 H), 1.85 (m, 1 H), 1.68 (m, 1 H), 1.37-1.23 (m, 4H), 0.87 (t, J=6.9 Hz, 3H).
5. Synthesis of N-(benzyloxycarbonyl)methionine 4-nitrobenzyl ester (R=CH3SCH2CH2) According to the method A, the titled compound was synthesized, purified by silica gel flush column chromatography, eluting with ethyl acetate/hexane (2:3).
~ H-NMR (600 MHz, CDC13): 8 8.22 (d, J=8.0 Hz, 2H), 7.51 (d, J=8.0 Hz, 2H), 7.39-7.32 (m, 5H), 5.42 (brd, J=7.1 Hz, 1 H), 5.27 (AB, J=12.9 Hz, 1 5 Ov=12.4 Hz, 2H), 5.12 (s, 2H), 4.59 (m, 1 H), 2.53 (t, J=7.1 Hz, 2H), 2.19 (m, 1 H), 2.07 (s, 3H), 2.01 (m, 1 H).

6. Synthesis of N-(benzyloxycarbonyl)valine 4-nitrobenzyl ester (R=(CH3)2CH) According to the method A, the titled compound was synthesized, purified by silica gel flush column chromatography, eluting with ethyl acetate/hexane (1:2), and crystallized from ethyl acetate-hexane.
~ H-NMR (500 MHz, CDC13): 8 8.22 (d, J=8.5 Hz, 2H), 7.51 (d, J=8.5 Hz, 2H), 7.39-7.31 (m, 5H), 5.26 (s, 2H), 5.23 (brd, J=8.8 Hz, 1 H), 5.12 (s, 2 5 2H), 4.38 (dd, J=4.8 Hz, 8.8 Hz, 1 H), 2.20 (m, 1 H), 0.98 (d, J=6.8 Hz, 3H), 0.88 (d, J=6.8 Hz, 3H).

7. Synthesis of N-(benzyloxycarbonyl)phenylglycine 4-nitrobenzyl ester (R=Ph) According to the method B, the titled compound was synthesized, purified by silica gel flush column chromatography, eluting with ethyl acetate/hexane (2:5) and crystallized from ethyl acetate-hexane.
~ H-NMR (500 MHz, CDC13): 8 8.12 (d, J=8.5 Hz, 2H), 7.39-7.30 (m, 5H), 7.27 (d, J=8.5 Hz, 2H), 5.77 (brd, J=7.1 Hz, 1 H), 5.44 (d, J=7.1 Hz, 1 H), 5.25 (AB, J=13.4 Hz, Ov=27.4 Hz, 2H), 5.11 (AB, J=12.1 Hz, w=19.8 Hz, 2H).

8. Synthesis of N-(benzyloxycarbonyl) 4-hydroxyphenylglycine 4-nitrobenzyl ester (R=4-HOPh) According to the method B, the titled compound was synthesized, purified by silica gel flush column chromatography, eluting with ethyl acetate/hexane {1:1.25), and crystallized from ethyl acetate-hexane.
~ H-NMR (600 MHz, CDC13): 8 8.14 (d, J=8.5 Hz, 2H), 7.38-7.31 (m, 5H), 7.30 (d, J=8.5 Hz, 2H), 7.22 (d, J=8.5 Hz, 2H), 6.81 (d, J=8.5 Hz, 2H), 5.70 (brd, J=7.1 Hz, 1 H), 5.36 (d, J=7.1 Hz, 1 H), 5.24 (AB, J=13.9 Hz, 0v=24.2 Hz, 2H), 5.11 (AB, J=12.0 Hz, Ov=21.1 Hz, 2H), 5.10 (s, 1 H).

9. Synthesis of Na-(benzyloxycarbonyl)-NE-(tert-butoxycarbonyl)lysine 4-nitrobenzyl ester According to the method B using Na-(benzyloxycarbonyl)-NE-(tert-butoxycarbonyl)lysine, the titled compound was synthesized, purified by silica gel flush column chromatography, eluting with ethyl acetate/hexane (2:3), and crystallized from ethyl acetate-hexane.
~ H-NMR (600 MHz, CDC13): 8 8.22 {d, J=8.3 Hz, 2H), 7.50 (d, J=8.3 Hz, 2H), 7.38-7.30 (m, 5H), 5.40 (brd, J=6.6 Hz, 1 H), 5.25 (AB, J=12.6 Hz, .,..

w=8.1 Hz, 2H), 5.11 (AB, J=12.2 Hz, Ov=12.0 Hz, 2H), 4.55 (m, 1 H), 4.41 (m, 1 H), 3.12-3.07 (m, 2H), 1.88 (m, 1 H), 1.72 (m, 1 H), 1.60-1.31 (m, 4H), 1.42 (s, 9H).

10. Synthesis of Na-(benzyloxycarbonyl)-D-lysine 4-nitrobenzyl 5 ester (R=H2NCH2CH2CH2CH2) To a solution of Na-(benzyloxycarbonyl)-N~-(tert-butoxycarbonyl)-D-lysine 4-nitrobenzyl ester (210.5 mg, 0.408 mmol) in chloroform (1.0 mL) was added trifluoroacetic acid (0.3 mL) at room temperature and the mixture was stirred for 8 hours.
10 The solvent was evaporated under reduced pressure and the residue was crystallized from ethyl acetate-hexane.
~ H-NMR (500 MHz, CDC13-CD30D): 8 8.22 (d, J=8.5 Hz, 2H), 7.51 (d, J=8.5 Hz, 2H), 7.40-7.26 (m, 5H), 5.27 (AB, J=13.3 Hz, Ov=19.9 Hz, 2H), 5.11 (AB, J=12.3 Hz, Ov=19.1 Hz, 2H), 4.35 (m, 1 H), 2.87 (t, 15 J=7.2 Hz, 2H), 1.88 (m, 1 H), 1.77-1.56 (m, 3H), 1.50-1.37 (m, 2H).
Examination 1 Identification of monoclonal antibodies having catal~rtic activity 1. Identification of monoclonal antibodies having hydrolyzing 20 activity by measuring fluorescence To a solution of each of 39 purified antibodies, prepared in Example 5, in 50 mM Tris-buffered solution, pH 8.0 (360 ~.L), was added a mixture (DL-10) of equal amounts of compounds (L-10) and (D-10), which were respectively prepared in Preparations 1 and 2, 2 5 in 150 p,M DMSO solution (40 wL) at 25 °C, and they were mixed with stirring to obtain a reaction mixture containing 1.5 ~.M antibodies and 15 ~,M substrate. Fluorescence intensity was measured 2s immediately after and ten minutes after the mixing at ~, ex 340 nm and ~, em 415 nm to determine the change of intensity in ten minutes. Based on the magnitude of the change in ten minutes, 14 antibodies having the catalytic activity were selected. The results obtained are shown in the following Table 1.
10-minute change in Antibodies fluorescence intensity 7612 12.54 1 OC8 9.40 6C4 8.00 6B 12 7.20 1082 7.10 7H 1 6.40 2E1 5.04 4G9 4.08 8H 10 3.49 8A 1 1 3.40 5H2 2,gg 3G2 2.31 --9F9 2.20 6610 2.08 2. Determination of enantioselectivity of catalytic antibodies by measuring fluorescence To a solution of each of 14 antibodies, which were found to be a catalytic antibody, in 50 mM Tris-buffered solution, pH 8.0 (360 ~,L), was added a solution of one of the compounds (L-10) and (D-10), which were respectively prepared in Preparations 1 and 2, in 150 ~.M DMSO solution (40 ~.L) at 25 °C, and they were mixed with stirring to obtain the reaction mixture containing 1.5 ~M antibody and 15 ~,M substrate. Fluorescence intensity was immediately after and ten minutes after the mixing, at ~, ex 340 nm and ~, em 415 nm to determine the change of the intensity in 10 minutes. Based on the magnitude of the change in ten minutes, 10 antibodies having the catalytic activity selective to the L-form and 4 antibodies having a catalytic activity selective to the D-form were identified. The results obtained are shown in the following Tables 2 and 3.
Table 2 Antibodies 10-min. chanae in fluorescence intensity selective ~o L-form L-10 D-10 7G 12 23.29 0.80 10C8 30.45 0.48 6C4 10.4 0.77 6812 9.67 0.90 1082 7.90 0.96 7H1 8.00 1.03 2E1 7.80 1.28 4G9 5.67 1.12 8H 10 5.05 0.72 6G 10 3.00 0.62 Table 3 Antibodies 10-min. change in fluorescence intensity selective to D-form L-10 D-10 3G2 0.38 4.32 5H2 0.40 5.16 8A1 1 0.30 4.62 9F9 0.48 4.44 Hybridomas producing antibodies 7612 and 3G2 were named hybridomas ZAA 7612 and ZAA 3G2, respectively.
Both hybridomas 7G 12 and 3G2 were deposited at Fermentation Research Institute Agency of Industrial Science and Technology, Japan, on December 21, 1994, under the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure, with accession Nos. FERM BP-4947 and FERM BP-4946, respectively.
DNA-base sequences corresponding to variable regions of 7612 and 3G2 antibodies, in which antigen-binding sites exist, were determined using DyeDeoxy Terminator Cycle Sequencing kits (Applied Biosystems) and the corresponding amino acid sequences were also determined.
For antibody 7612, DNA-base sequence corresponding to 1 5 and the amino acid sequence for the variable region of the heavy chain are shown in SEQ ID NOs. 1 and 2, respectively, and those for variable region of the light chain in SEQ ID NOs. 3 and 4, respectively. For antibody 3G2, DNA-base sequence and the amino acid sequence for the variable region of the heavy chain are shown in SEQ ID NOs. 5 and 6, respectively, and those for variable region of the light chain in SEQ ID NOs. 7 and 8, respectively.

Examination 2 Determination of specificity to substrate and enantioselectivity of monoclonal antibodies having cata~tic activitX
1. Determination of specificity to substrate and enantioselectivity of monoclonal antibody 7612 by HPLC
To a solution of antibody 7612 in 50 mM Tris-buffered solution, pH 8.0 (90 ~.L), was added a solution of a substrate compound, N-(benzyloxycarbonyl)-L-phenylalanine 4-nitrobenzyl ester (L-1 ) (R=PhCH2), synthesized in Preparation 3, in 2mM DMSO
solution (10 ~,L) at 25°C, and they were mixed with stirring to obtain the reaction mixture containing 10.8 ~.M antibody and 200 ~.M
substrate. By means of HPLC (YMCAM-303: C-18, 10 mm in diameter x 250 mm in length, an aqueous solution of acetonitrile/0.1 trifluoroacetic acid=35/65 (0-7 minutes), 35/65-90/10 (7-15 minutes), 90/10 (15-17 minutes), 1.0 mL/minute, 278 nm), the amount of 4-nitrobenzyl alcohol (retention time: 5.9 minutes) produced in the reaction mixture was traced in time course for the purpose of determing initial rate. Next, the same procedure as mentioned above was repeated using N-(benzyloxycarbonyl)-D-phenylalanine 4-nitrobenzyl ester (D-1) (R=PhCH2) instead of N-(benzyloxycarbonyl)-L-phenylalanine 4-nitrobenzyl ester (L-1 ) (R=PhCH2) to determine the initial rate. Background hydrolytic reaction rate was also determined based on the amount of 4-nitrobenzyl alcohol produced by spontaneous degradation in the 2 5 reaction solution free from antibody 7612.
In the above-experiment where antibody 7612 was used, background hydrolytic reaction rate was subtracted from the initial hydrolytic reaction rate for L- and D-forms of the substrate (R=PhCH2) to determine the net initial hydrolytic reaction rate of antibody 7612. The antibody 7612 did not catalyze the hydrolysis of D-form of substrate (D-1 ) (R= PhCH2). Thus, the antibody 7612 enantioselectively catalyzed the hydrolysis of the substrate.
The specificity to substrate and enantioselectivity of the antibody 7612 were extensively studied, according to the same 5 procedure as mentioned above, using other substrates derived from alanine, leucine, norleucine, methionine, valine, phenylglycine, 4-hydroxyphenylglycine and lysine, synthesized in Preparation 3, and it was found that the antibody 7612 enantioselectively catalyzed the hydrolysis of L-form of the substrates. The following Table 4 10 shows the initial hydrolytic rate as well as background reaction rate constants for L-form of the substrates.
Table 44 Hydrolysis of L-amino acid ester derivatives by antibody 7612 Background Concentration for reaction Substrate reaction rate Initial rate L-1 constants xuncat Antibody L-1 V
(R-) (min-~) 7G12 (~,M) (~,M) (~,M/min.) CH3- 1.06X10-4 10 200 3.49X10-1 (CH3)2CHCH2- 1.26X10-5 10 200 6.20X10-CH3(CH2)3- 1.71 X 10-5 5 10 0 3.74X 10-2 CH3SCH2CH2- 3.83X10-5 10 200 7.73X10-~

PhCH2- 2.24X10-5 10 200 3.85X10-(CH3)2CH- 6.47X10-6 5 100 5.54X10-3 Ph- 5.62X10-3 5 100 7.83X10-~

4-HOPh- 1.84X10-4 5 200 3.33X10-ia) H2N(CH2)4- 1.40X10-4 5 100 4.36X10-15 a) Reacted with DL-1 as a substrate.

2. Determination of specificity to substrate and enantioselectivity of monoclonal antibody 3G2 by HPLC
Specificity to substrate and enantioselectivity of antibody 3G2 were determined according to the same procedure as that used for antibody 7612, and it was found that said antibody enantioselectively catalyzed the hydrolysis of D-form of substrates derived from alanine, phenylalanine, leucine, norleucine, methionine, valine, phenylglycine, and 4-hydroxyphenylglycine, synthesized in Preparation 3. The following Table 5 shows the initial hydrolytic rate for D-form of the substrates. The background reaction rate constants are the same as those shown in Table 4.
Table 55 Hydrolysis of D-amino acid ester derivatives (D-1 ) by antibody 3G2.
Substrate Concentration for reaction Initial rate 1 Antibody D-1 V

) M/min.
3G2 (~,M) (~,M) (~' ) CH3- 5 100 9.31 X 10-2 (CH3)2CHCH2- 10 100 1.16X10-CH3(CH2)3- 10 100 9.12X10-2 CH3SCH2CH2- 10 10 0 2.15X 10-1 PhCH2- 5 50 1.54X 10-2 (CH3)2CH- 10 100 1.96X10-3 Ph- 5 100 2.91X10-2 4-HOPh- 1 0 100 4.08X10-~

~~ 9~~55 SEQUENCE LISTING
(1 ) GENERAL INFORMATION
(i)APPLICANT:
(A) NAME: Protein Engineering Research Institute (B) STREET: 2-3, Furuedai 6-chome (C) CITY: Suita-shi 1 0 (D) STATE: Osaka (E) COUNTRY: Japan (ii) TITLE OF INVENTION: CATALYTIC ANTIBODIES
ENANTIOSELECTIVELY HYDROLYSING AMINO ACID ESTER DERIVATIVES
(iii) NUMBER OF SEQUENCE: 8 (v) CURRENT APPLICATION DATA:
APPLICATION NUMBER:
(vi) PRIORY APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
2 5 (2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 640 (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:

CTCGAGTCTG GGACTGAACT GGCAAAACCT GGGGCCTCAG TGAAGATGTC CTGCAAGGCT TCTGGCTACA
GAGCTCAGAC CCTGACTTGA CCGTTTTGGA CCCCGGAGTC ACTTCTACAG GACGTTCCGA AGACCGATGT

CCTTCACTAG CTACTGGATA CACTGGGTAA AACAGAGGCC TGGACAGGGT CTGGAATGGA TTGGATACAT
GGAAGTGATC GATGACCTAT GTGACCCATT TTGTCTCCGG ACCTGTCCCA GACCTTACCT AACCTATGTA

TAATCCTAGT ACTGATTATA CTGAGTACAT TCAGAAGTTC AAGGACAAGG CCACATTGAC TGCAGACAAA
ATTAGGATCA TGACTAATAT GACTCATGTA AGTCTTCAAG TTCCTGTTCC GGTGTAACTG ACGTCTGTTT

. 219055 TCCTCCAGCA CAGCCTACAT GCAACTGAGC AGCCTGACAT CTGAGGACTC TGCAGTCTAT TACTGTGTAA
AGGAGGTCGT GTCGGATGTA CGTTGACTCG TCGGACTGTA GACTCCTGAG ACGTCAGATA ATGACACATT

TGAAGGACTA CTGGGGTCAA GGAACTTCAG TCACCGTCTC CTCAGCCAAA ACGACACCCC CATCTGTCTA
ACTTCCTGAT GACCCCAGTT CCTTGAAGTC AGTGGCAGAG GAGTCGGTTT TGCTGTGGGG GTAGACAGAT

TCCACTGGCC CCTGGATCTG CTGCCCAAAC TAACTCCATG GTGACCCTGG GATGCCTGGT CAAGGGCTAT

GTTCCCGATA

TTCCCTGAGC CAGTGACAGT GACCTGGAAC TCTGGATCCC TGTCCAGCGG TGTGCACACC TTCCCAGCTG
AAGGGACTCG GTCACTGTCA CTGGACCTTG AGACCTAGGG ACAGGTCGCC ACACGTGTGG AAGGGTCGAC

TCCTGCAGTC TGACCTCTAC ACTCTGAGCA GCTCAGTGAC TGTCCCCTCC AGCACCTGGC CCAGCGAGAC
AGGACGTCAG ACTGGAGATG TGAGACTCGT CGAGTCACTG ACAGGGGAGG TCGTGGACCG GGTCGCTCTG

CGTCACCTGC AACGTTGCCC ACCCGGCCAG CAGCACCAAG GTGGACAAGA AAATTGTGCC CAGGGATTGT
GCAGTGGACG TTGCAACGGG TGGGCCGGTC GTCGTGGTTC CACCTGTTCT TTTAACACGG GTCCCTAACA

TGATCA

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 212 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
4 O Leu Glu Ser Gly Thr Glu Leu Ala Lys Pro Gly Ala Ser Val Lys MET Ser Cys (2) INFORMATION FOR SEQ ID N0:2:
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Thr Asp Tyr Thr Glu Tyr Ile Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr MET Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Val MET Lys Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser MET Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Thr Ser (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
2 5 (A) LENGTH: 640 (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear 3 0 (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:

ATGACCTGCA
CTCGAGCACT ACTGGGTCTG AGGTCGTTAG TACAGACGTA GAGGTCCCCT CTTCCAGTGG TACTGGACGT

GTGCCAGCTC AAGTATAAGT TACATGCACT GGTACCAGCA GAAGCCAGGC ACCCCCCCCA AAAGATGGAT

TTTCTACCTA

TTATGGCACA TCCAAACTGA CTTCTGGAGT CCCTGCTCGC TTCAGTGGCA GTGGGTCTGG GACCTCTTTT
AATACCGTGT AGGTTTGACT GAAGACCTCA GGGACGAGCG AAGTCACCGT CACCCAGACC CTGGAGAAAA

TCTCTCACAA TCAGCAGCAT GGAGGCTGAA GATGCTGCCA CTTATTACTG CCATCAGCGG AGTAGTTACC
AGAGAGTGTT AGTCGTCGTA CCTCCGACTT CTACGACGGT GAATAATGAC GGTAGTCGCC TCATCAATGG

CGACGTTCGG TGGAGGCACC AAGCTGGAAA TCAAACGGGC TGATGCTGCA CCAACTGTAT CCATCTTCCC
GCTGCAAGCC ACCTCCGTGG TTCGACCTTT AGTTTGCCCG ACTACGACGT GGTTGACATA GGTAGAAGGG

,...

ACCATCCAGT GAGCAGTTAA CATCTGGAGG TGCCTCAGTC GTGTGCTTCT TGAACAACTT CTACCCCAAA
TGGTAGGTCA CTCGTCAATT GTAGACCTCC ACGGAGTCAG CACACGAAGA ACTTGTTGAA GATGGGGTTT

GACATCAATG TCAAGTGGAA GATTGATGGC AGTGAACGAC AAAATGGCGT CCTGAACAGT TGGACTGATC
CTGTAGTTAC AGTTCACCTT CTAACTACCG TCACTTGCTG TTTTACCGCA GGACTTGTCA ACCTGACTAG

ATGAACGACA
TCCTGTCGTT TCTGTCGTGG ATGTCGTACT CGTCGTGGGA GTGCAACTGG TTCCTGCTCA TACTTGCTGT

TAACAGCTAT ACCTGTGAGG CCACTCACAA GACATCAACT TCACCCATTG TCAAGAGCTT CAACAGGAAT

TGTCCTTACT

GAGTGTTAAT TCTAGA
GTCACAATTA AGATCT
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
2 5 (A) LENGTH: 213 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Glu Leu Val MET Thr Gln Thr Pro Ala Ile MET Ser Ala Ser Pro Gly Glu Lys Val Thr MET Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr MET His Trp Tyr Gln Gln Lys Pro Gly Thr Pro Pro Lys Arg Trp Ile Tyr Gly Thr Ser Lys Leu Thr Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu 4 5 Thr Ile Ser Ser MET Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser MET Ser Ser Thr Leu Thr Leu Thr 1 O Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys . Phe (2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 656 2 0 (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:

CTCGAGTCTG GACCTGAGCT GGTGAAGCCT GGGGGCTCAG TGACGATTTC CTGCAAAGCT TCTGGCTACG

AGACCGATGC

GATTCACTAC CTCTTGGATG AACTGGGTGA GGCAGAGGCC TGGACAGGGT CTTGAGTGGA TTGGACGGAT
CTAAGTGATG GAGAACCTAC TTGACCCACT CCGTCTCCGG ACCTGTCCCA GAACTCACCT AACCTGCCTA

TTATCCTGGA AGTGGGGATA ATAATTACAA TGGGAAGTTC AAGGTCAAGG CCACATTGAC TGCAGAGAGA
AATAGGACCT TCACCCCTAT TATTAATGTT ACCCTTCAAG TTCCAGTTCC GGTGTAACTG ACGTCTCTCT

TCCTCCACCA CAGTCTACCT GCACCTCAGC AGCCTGACCT CTGTAGATTC TGCGGTCTAT TTCTGTGCAA
AGGAGGTGGT GTCAGATGGA CGTGGAGTCG TCGGACTGGA GACATCTAAG ACGCCAGATA AAGACACGTT

TCACCGTCTC
CTAAAGTGAT ACTAATAGCA GCAAGGATAC GATACCTGAT GACCCCAGTT CCTTGAAGTC AGTGGCAGAG

TAACTCCATG
GAGTCGGTTT TGCTGTGGGG GTAGACAGAT AGGTGACCGG GGACCTAGAC GACGGGTTTG ATTGAGGTAC

GTGACCCTGG GATGCCTGGT CAAGGGCTAT TTCCCTGAGC CAGTGACAGT GACCTGGAAC TCTGGATCCC
CACTGGGACC CTACGGACCA GTTCCCGATA AAGGGACTCG GTCACTGTCA CTGGACCTTG AGACCTAGGG

TGTCCAGCGG TGTGCACACC TTCCCAGCTG TCCTGCAGTC TGACCTCTAC ACTCTGAGCA GCTCAGTGAC
ACAGGTCGCC ACACGTGTGG AAGGGTCGAC AGGACGTCAG ACTGGAGATG TGAGACTCGT CGAGTCACTG

TGTCCCCTCC AGCACCTGGC CCAGCGAGAC CGTCACCTGC AACGTTGCCC ACCCGGCCAG CAGCACCAAG

GTCGTGGTTC

GTGGACAAGA AAATTGTGCC CAGGGATTGT ACTAGT
CACCTGTTCT TTTAACACGG GTCCCTAACA TGATCA
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
2 0 (A) LENGTH: 210 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Leu Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Gly Ser Val Thr Ile Ser Cys Lys Ala Ser Gly Tyr Gly Phe Thr Thr Ser Trp MET Asn Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Arg Ile Tyr Pro Gly Ser Gly Asp Asn Asn Tyr Asn Gly Lys Phe Lys Val Lys Ala Thr Leu Thr Ala Glu Arg Ser Ser Thr Thr Val Tyr 4 O Leu His Leu Ser Ser Leu Thr Ser Val Asp Ser Ala Val Tyr Phe Cys Ala Arg Phe His Tyr Asp Tyr Arg Arg Ser Tyr Ala MET Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser MET Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Thr Ser (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 652 (B) TYPE: nucleic acid 1 5 (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:

GAGCTCGTGA TGACCCAGAC TCCATCTTCC ATGTATGCAT CTCTAGGAGA GAGAGTCACT ATCACTTGCA
CTCGAGCACT ACTGGGTCTG AGGTAGAAGG TACATACGTA GAGATCCTCT CTCTCAGTGA TAGTGAACGT

AGGCGAGTCA GGACATTAAT ATCTATTTAA GTTGGTTCCA GCAGAAACCA GGGAAATCTC CTAAGGCCCT
TCCGCTCAGT CCTGTAATTA TAGATAAATT CAACCAAGGT CGTCTTTGGT CCCTTTAGAG GATTCCGGGA

O

GATCTATCGT ACAAACGGATTGGTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGAT

CTAGATAGCA TGTTTGCCTAACCATCTACCCCAGGGTAGTTCCAAGTCACCGTCACCTAGACCCGTTCTA

ATAAGAGAGT GGTAGTCGTCGGACCTTATACTTCTATACCCTTAAATAATAACAGATGTCATACTACTCA

TTCCGTACAC GTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCAT

O

CTTCCCACCA TCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTAC

GAAGGGTGGT AGGTCACTCGTCAATTGTAGACCTCCACGGAGTCAGCACACGAAGAACTTGTTGAAGATG

CCCAAAGACA TCAATGTCAA GTGGAAGATT GATGGCAGTG AACGACAAAA TGGCGTCCTG AACAGTTGGA
GGGTTTCTGT AGTTACAGTT CACCTTCTAA CTACCGTCAC TTGCTGTTTT ACCGCAGGAC TTGTCAACCT

CTGATCAGGA CAGCAAAGAC AGCACCTACA GCATGAGCAG CACCCTCACG TTGACCAAGG ACGAGTATGA
GACTAGTCCT GTCGTTTCTG TCGTGGATGT CGTACTCGTC GTGGGAGTGC AACTGGTTCC TGCTCATACT

ACGACATAAC AGCTATACCT GTGAGGCCAC TCACAAGACA TCAACTTCAC CCATTGTCAA GAGCTTCAAC
TGCTGTATTG TCGATATGGA CACTCCGGTG AGTGTTCTGT AGTTGAAGTG GGTAACAGTT CTCGAAGTTG

AGGAATGAGT GTTAATTCTA GA
TCCTTACTCA CAATTAAGAT CT
1 0 (2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 204 (B) TYPE: amino acid 1 5 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Glu Leu Val MET Thr Gln Thr Pro Ser Ser MET Tyr Ala Ser Leu Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ile Tyr Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Ala Leu Ile Tyr Arg Thr Asn Gly Leu Val Asp Gly 3 0 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr Glu Asp MET Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly 4 5 Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser MET Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys . Phe

Claims (30)

40

1. A process for optically resolving a racemic mixture of amino acid derivatives, comprising using a nonspecific catalytic antibody which enantioselectively hydrolyzes various amino acid ester derivatives.

2. The process of Claim 1 wherein the amino acid ester derivative is a 4-nitrobenzyl ester.

3. The process of Claim 1 or 2 wherein the amino acid ester derivative is amino-protected.

4. The process of Claim 1, 2 or 3 wherein the amino acid ester derivative has the formula (L-1):
wherein CBZ is N-benzyloxycarbonyl and R is an alkyl or phenyl group which is unsubstituted or substituted with hydroxy, amino, alkylthio, acyloxy, or phenyl.

5. The process of Claim 4 wherein the R of the formula (L-1) is CH3-, phCH2, (CH3)2CHCH2, CH3(CH2)3, CH3SCH2CH2, (CH3)2CH-, ph-, or 4-HOph-.

6. The process of Claim 1, 2 or 3 wherein the amino acid ester derivative has the formula (D-1):
wherein CBZ is N-benzyloxycarbonyl and R is an alkyl or phenyl group which is optionally substituted with hydroxy, amino, alkylthio, acyloxy, or phenyl.

7. The process of Claim 6 wherein the R of the formula (D-1): is CH3, phCH2-, (CH3)2CHCH2-, CH3(CH2)3-, CH3SCH2CH2-, (CH3)2CH-, ph-, or 4-HOph-.

8. The process of Claims 1 or 2 wherein the catalytic antibody is produced by stimulation with an antigen comprising a compound of the formula (3):
wherein CBZ is N-benzyloxycarbonyl.

9. The process of Claim 8 wherein the catalytic antibody is produced by hybridoma ZAA 7G12.

10. The process of Claim 8 wherein the catalytic antibody is produced by hybridoma ZAA 3G2.

11. A process for preparing an optically active amino acid comprising the steps of:
a) subjecting a racemic mixture of amino acid derivatives to an ester forming reaction to form amino acid ester derivatives;
b) selectively hydrolyzing a desired stereoisomer, D- or L-isomer, of the amino acid ester derivatives with a nonspecific catalytic antibody which enantioselectively hydrolyzes various amino acid ester derivatives; and c) treating the resulting hydrolyzed mixture to isolate the desired hydrolyzed stereoisomer of the amino acid ester derivatives thereby preparing the optically active amino acid.

12. The process of Claim 11 wherein the resulting hydrolized mixture is treated by being made acidic or basic, followed by extracting with an organic solvent to obtain an aqueous layer from which the desired hydrolyzed stereoisomer is isolated.

13. The process of Claim 11 or 12 wherein the amino acid ester derivative is 4-nitrobenzyl ester.

14. The process of Claim 11 or 12 wherein the amino acid ester derivative is amino-protected.

15. The process of Claim 11, 12, 13 or 14 wherein the amino acid ester derivative is a compound having the formula (L-1):
wherein CBZ is N-benzyloxycarbonyl and R is an alkyl or phenyl group which is unsubstituted or substituted with hydroxy, amino, alkylthio, acyloxy, or phenyl.

16. The process of Claim 15 wherein the amino acid ester derivative has the formula (L-1):
wherein CBZ is N-benzyloxycarbonyl and R is CH3-, phCH2-, (CH3)2CHCH2- , CH3(CH2)3-, CH3SCH2CH2-, (CH3)2CH-, ph-, or 4-HOph-.

17. The process of Claim 11, 12, 13 or 14 wherein the amino acid ester derivative has the formula (D-1):
wherein CBZ is N-benzyloxycarbonyl and R is an alkyl or phenyl group which is optionally substituted with hydroxy, amino, alkylthio, acyloxy, or phenyl.

18. The process of Claim 17 wherein the amino acid ester derivative has the formula (D-1):
wherein CBZ is N-benzyloxycarbonyl and R is CH3-, phCH2-, (CH3)2CHCH2-, CH3(CH2)3-, CH3SCH2CH2-, (CH3)2CH-, ph-, or 4-HOph-.

19. The process of Claims 11 or 13 wherein the catalytic antibody is produced by stimulation with an antigen comprising a compound of the formula:
wherein CBZ is N-benzyloxycarbonyl.

20. The process of Claim 19 wherein the catalytic antibody is produced by hybridoma ZAA 7G12.

21. The process of Claim 19 wherein the catalytic antibody is produced by hybridoma ZAA 3G2.

22. A catalytic antibody capable of enantioselectively hydrolyzing various amino acid ester derivatives, produced by stimulation with an antigen comprising a compound of the formula:
wherein CBZ is N-benzyloxycarbonyl.

23. The catalytic antibody of Claim 22 produced by hybridoma ZAA
7G12.

24. The catalytic antibody of Claim 22 produced by hybridoma ZAA
3G2.

25. A hybridoma producing the catalytic antibody of Claim 22.

26. The hybridoma of Claim 25 designated ZAA 7G12.

27. The hybridoma of Claim 25 designated ZAA 3G2.

28. A catalytic antibody produced by stimulation with an antigen comprising a compound of the formula:
wherein S is an amino protecting group.

29. The catalytic antibody of Claim 28 wherein S is trichloroacetyl, benzyl, t-butoxycarbonyl, or 9-fluorenylmethoxycarbonyl.

30. A catalytic antibody produced by stimulation with an antigen comprising a compound of the formula:
wherein S is a phenyl moiety which is substituted with lower alkyl, lower alkoxy, halogen or nitro.