Relaxing substrate specificity in antibody-catalyzed reactions: Enantioselective hydrolysis of N-Cbz-amino acid esters

Article abstract of DOI:10.1021/ja952220w

For a catalytic antibody to be generally useful for organic synthetic chemistry, it must be able to accept a broad range of substrates, yet retain high selectivity. In this work, we propose a hapten design to endow antibody catalysts with two opposing qualities, such as high enantioselectivity and broad substrate specificity. Racemic hapten 2 induced two separate classes of catalytic antibodies to hydrolyze either the L- or D-isomers of N-Cbz-amino acid esters 1. In the kinetic resolution of racemic ester 9, antibodies 7G12 and 3G2 gave 96% ee of L-10 and 94% ee of D-10, respectively. In addition, antibody 7G12 displayed broad substrate specificity, hydrolyzing the L-esters of Ala (1a), Leu (1b), Norleu (1c), Met (1d), Phe (1e), Val (1f), and phenylglycine (1g) with high enantioselectivity. Antibody 3G2 also hydrolyzed the D-isomers of these esters without sacrificing the enantioselectivity. This observation suggests that the use of haptens that fit snugly into the antigen-combining site, and leave the linker moiety outside, is an effective approach for the generation of catalytic antibodies with high selectivity and broad substrate applicability.

Full text of DOI:10.1021/ja952220w

2332
J. Am. Chem. Soc. 1996, 118, 2332-2339
Relaxing Substrate Specificity in Antibody-Catalyzed
Reactions: Enantioselective Hydrolysis of N-Cbz-Amino
Acid Esters
Fujie Tanaka, Keiko Kinoshita, Ryuji Tanimura, and Ikuo Fujii*
Contribution from the Protein Engineering Research Institute, 6-2-3 Furuedai,
Suita, Osaka 565, Japan
ReceiVed July 6, 1995. ReVised Manuscript ReceiVed December 30, 1995X
Abstract: For a catalytic antibody to be generally useful for organic synthetic chemistry, it must be able to accept
a broad range of substrates, yet retain high selectivity. In this work, we propose a hapten design to endow antibody
catalysts with two opposing qualities, such as high enantioselectivity and broad substrate specificity. Racemic hapten
2 induced two separate classes of catalytic antibodies to hydrolyze either the L- or D-isomers of N-Cbz-amino acid
esters 1. In the kinetic resolution of racemic ester 9, antibodies 7G12 and 3G2 gave 96% ee of L-10 and 94% ee of
D-10, respectively. In addition, antibody 7G12 displayed broad substrate specificity, hydrolyzing the L-esters of Ala
(1a), Leu (1b), Norleu (1c), Met (1d), Phe (1e), Val (1f), and phenylglycine (1g) with high enantioselectivity. Antibody
3G2 also hydrolyzed the D-isomers of these esters without sacrificing the enantioselectivity. This observation suggests
that the use of haptens that fit snugly into the antigen-combining site, and leave the linker moiety outside, is an
effective approach for the generation of catalytic antibodies with high selectivity and broad substrate applicability.
Introduction
Scheme 1
Since antibody-catalyzed reactions display high regio- and
stereoselectivity, according to the reaction pathway programmed
in the hapten,1 catalytic antibodies have the potential to be
powerful tools in the field of organic synthetic chemistry.
However, the applicability to a broad range of substrates is still
restricted within narrow limits, due to the inherent binding
specificity of antibodies. For a catalytic antibody to be generally
useful, it must be able to accept a broad range of substrates,
yet retain high selectivity. Therefore, we have focused on the
issue of whether catalytic antibodies can be developed to
combine the aforementioned two opposing qualities. Recently,
we described catalytic antibodies that discriminate between
chemically identical functional groups in the same molecule to
catalyze regioselective deprotection of a variety of acylated
carbohydrates,2 and others have shown antibody-catalyzed
chemoselective reactions with various substrates.3 Herein, we
demonstrate the generation of two separate classes of catalytic
antibodies that enantioselectively hydrolyze either the L- or
D-isomers of amino acid ester derivatives possessing various
R-substituents (Scheme 1). Despite the many efforts that have
been mounted to prepare chiral R-amino acids,4,5 simple access
to a wide range of them remains limited.4a These antibody-
catalyzed kinetic resolutions with broad substrate specificity will
provide a convenient route to a wide range of natural and
unnatural, enantiomerically pure R-amino acids.
antibody-catalyzed reaction, it seemed necessary to consider the
molecular basis of antibody-antigen interactions. X-ray struc-
tural analyses of antibody-antigen complexes have shown that
relatively small haptens, such as fluorescein,6a progesterone,6b
and progesterone 11R-hemisuccinate,6b are buried deep in the
antigen-combining sites, with surface areas of 270-353 Å2.
Therefore, we thought that the size range of these haptens would
be sufficient to display highly specific antibody-antigen
(4) For some recent examples of preparing chiral R-amino acid deriva-
tives, see references in: (a) Burk, M. J.; Lee, J. R.; Martinez, J. P. J. Am.
Chem. Soc. 1994, 116, 10847-10848. (b) Lander, P. A.; Hegedus, L. S.
J. Am. Chem. Soc. 1994, 116, 8126-8132. (c) Guo, J.; Huang, W.; Scanlan,
T. S. J. Am. Chem. Soc. 1994, 116, 6062-6069. (d) RajanBabu, T. V.;
Ayers, T. A.; Casalnuovo, A. L. J. Am. Chem. Soc. 1994, 116, 4104-
4102. (e) Mulzer, J.; Schro¨der, F.; Lobbia, A.; Buschmann, J.; Luger, P.
Angew. Chem., Int. Ed. Engl. 1994, 33, 1737-1739. (f) Kazmair, U. Angew.
Chem., Int. Ed. Engl. 1994, 33, 998-999. (g) Burk, M. J.; Feaster, J. E.;
Nugent, W. A.; Halow, R. L. J. Am. Chem. Soc. 1993, 115, 10125-10138.
(h) Blaskovich, M. A.; Lajoie, G. A. J. Am. Chem. Soc. 1993, 115, 5021-
5030. (i) Kawabata, T.; Wirth, T.; Yahiro, K.; Suzuki, H.; Fuji, K. J. Am.
Chem. Soc. 1994, 116, 10809-10810. (j) Vejejs, E.; Fields, S. C.; Schrimpf,
M. R. J. Am. Chem. Soc. 1993, 115, 11612-11613.
(5) For some examples of the enzymatic kinetic resolution of amino acid
derivatives, see references in: (a) Chenault, H. K.; Dahmer, J.; Whitesides,
G. M. J. Am. Chem. Soc. 1989, 111, 6354-6364. (b) Miyazawa, T.;
Iwanaga, H.; Ueji, S.; Yamada, T.; Kuwata, S. Chem. Lett. 1989, 2219-
2222. (c) Chen, S.-T.; Wang, K.-T.; Wong, C.-H. J. Chem. Soc., Chem.
Commun. 1986, 1514-1516.
(6) (a) Davies, D. R.; Padlan, E. A. Annu. ReV. Biochem. 1990, 59, 439-
473 and references cited therein. (b) Arevalo, J. H.; Taussing, M. J.; Wilson,
I. A. Nature 1993, 365, 859-863.
Results and Discussion
Design and Synthesis of Hapten. To design a hapten that
would induce broad substrate specificity in an enantioselective
* To whom correspondence should be addressed.
X Abstract published in AdVance ACS Abstracts, March 1, 1996.
(1) (1) Reviews: (a) Schultz, P. G.; Lerner, R. A. Acc. Chem. Res. 1993,
26, 391-395. (b) Lerner, R. A.; Benkovic, S. J.; Schultz, P. G. Science
1991, 252, 659-667.
(2) Iwabuchi, Y.; Miyashita, H.; Tanimura, R.; Kinoshita, K.; Kikuchi,
M.; Fujii, I. J. Am. Chem. Soc. 1994, 116, 771-772.
(3) (a) Li, T.; Hilton, S.; Janda, K. D. J. Am. Chem. Soc. 1995, 117,
2123-2127. (b) Hsieh, L. C.; Yonkovich, S.; Kochersperger, L.; Schultz,
P. G. Science 1993, 260, 337-339.
0002-7863/96/1518-2332$12.00/0 © 1996 American Chemical Society

Substrate Specificity in Antibody-Catalyzed Reactions
J. Am. Chem. Soc., Vol. 118, No. 10, 1996 2333
Scheme 2a
Figure 1.
interactions and would be enough to occupy only the antigen-
combining site, while leaving the linker moiety free from
antibody-antigen interactions. To induce high enantioselec-
tivity in antibody-catalyzed reactions, an immunizing haptenic
epitope including a chiral center should be within the 270-
353 Å2 range so that the haptenic epitope occupies the antigen-
combining site. In addition, since the linker to the hapten is
outside of the antigen-combining site, the position of attachment
of the linker to the hapten may be chosen to allow broad
substrate specificity.
According to these ideas, we designed haptenic phosphonate
transition state analog 2 to generate catalytic antibodies that
enantioselectively hydrolyze amino acid ester derivatives pos-
sessing various R-substituents. In hapten 2, relatively bulky
protecting groups, carbobenzoxy (Cbz) and 4-nitrobenzyl groups,
were used to adjust the size of the surface area of the
immunizing haptenic epitope, and the linker moiety was attached
at the CR position (Figure 1). In practice, a 1H NMR analysis
of hapten 2 has shown a NOE (0.6%) of the phenyl proton in
the Cbz group, with irradiation of the ortho proton of the nitro
group,7 suggesting a packed conformation of these two aromatic
groups of 2 in aqueous media. The surface area (solvent
accessible area) of the packed conformation of 3, which lacks
the linker moiety, was calculated to be 330 Å2.8
a Reagents: (a) PDC/CH2Cl2, 40%; (b) PhCH2NH2, P(OEt)3/EtOH,
48%; (c) H2, Pd-C/HCOOH-MeOH; (d) CbzCl, Et3N/THF; (e)
TMSBr/CH3CN; (f) 4-nitrobenzyl alcohol, DCC, 1H-tetrazole/CH2Cl2-
pyridine, 6% from 6; (g) NaOH/H2O-MeOH, 99%; (h) N-hydroxy-
succinimide, WSC, DMAP/CH3CN, 79%; (i) KLH or BSA/phosphate
buffer (pH 7.4)-DMF.
Table 1. Kinetic Parameters of Anti-2 Antibody-Catalyzed
Hydrolysis of Substrate 9a
c
antibody substrate Km, µM kcat,b min-1 kcat/kuncat Ki, nM (inhibitor)
7G12
3G2
L-9
D-9
13
7.0 × 10-2 3700
19 ((R)-3)
47 ((S)-3)
5.4 3.3 × 10-2 1700
a Reaction conditions: 10% DMSO/50 mM Tris (pH 8.0), 25 °C.
b See ref 13. c The first-order kinetic constant of the background reaction
(kuncat) was 1.9 × 10-5 min-1
.
protocols.10 Hybridoma supernatants were screened for anti-
bodies with binding affinity to BSA-2 by ELISA. Monoclonal
antibodies were purified from the hybridoma supernatants via
cation-exchange chromatography (Mono S) followed by affinity
chromatography (Protein G), as described previously.11
Catalytic Assay and Kinetics. Thirty-nine monoclonal
antibodies that bound to BSA-2 were screened for the ability
to catalyze the hydrolysis of the racemic fluorogenic substrate
9. The reaction was performed using 1.5 µM of antibody and
15 µM of racemic 9 in 10% DMSO/50 mM Tris (pH 8.0) at 25
°C, and the increase in fluorescence (λex 340 nm, λem 415 nm)
was monitored.12 As a result, fourteen antibodies were found
to be catalytic. These antibodies were examined for their
enantioselectivities in separate experiments with 15 µM of either
chiral L-9 or D-9, under the same conditions described above.
Ten of them were enantioselective for the hydrolysis of L-9,
whereas the other four were enantioselective for the hydrolysis
of D-9. Hydrolysis of less than 3% of the opposite isomer was
detected. Antibodies 7G12 and 3G2 were highly active for the
hydrolysis of L-9 and D-9, respectively, and were characterized
in more detail.
To generate two separate classes of catalytic antibodies that
catalyze the hydrolysis of either the L- or D-isomers of amino
acid esters, hapten 2 was synthesized as a racemic mixture.9
An imine derived from aldehyde 5 and benzylamine was treated
with triethyl phosphite to give (aminoalkyl)phosphonate 6, an
intermediate with a linker moiety to a carrier protein at the CR
position. Deprotection and protection of the amino group of 6,
dealkylation of the diethyl phosphonate, condensation with
4-nitrobenzyl alcohol, and hydrolysis of the ester group afforded
2. Finally, hapten 2 was conjugated to carrier proteins, keyhole
limpet hemocyanin (KLH), and bovine serum albumin (BSA),
via an activated ester method (Scheme 2).
Antibodies 7G12 and 3G2 displayed saturation kinetics
described by the Michaelis-Menten equation in the hydrolysis
of L-9 and D-9, respectively. The kinetic parameters are shown
in Table 1. The rate accelerations (kcat/kuncat) with 7G12 and
3G2 above background hydrolysis were 3700- and 1700-fold,
Antibody Production. Balb/c mice were immunized with
KLH-2, and monoclonal antibodies were generated by standard
(7) A 1H NMR NOE experiment of phosphonate 2 in D2O showed 0.6%
NOE of the Cbz phenyl proton and 9.6% NOE of the meta proton of the
nitro group when the ortho proton of the nitro group was irradiated.
(8) The packed conformation of phosphonate 3 was optimized by
Discover CVFF, and the value was calculated using Insight II (Biosym
Technologies) with a 1.7 Å probe sphere and van der Waals radii.
(9) Janda, K. D.; Benkovic, S. J.; Lerner, R. A. Science 1989, 244, 437-
440.
(10) Kohler, G.; Milstein, C. Nature 1975, 256, 495-497.
(11) Miyashita, H.; Karaki, Y.; Kikuchi, M.; Fujii, I. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 5337-5340.
(12) Nishino, N.; Powers, J. C. J. Biol. Chem. 1980, 255, 3482-3486
and references cited therein.

2334 J. Am. Chem. Soc., Vol. 118, No. 10, 1996
Tanaka et al.
performed using antibody 7G12 (20 µM of active site) and
racemic 9 (150 µM) in 5% DMSO/50 mM Tris (pH 8.0) at 25
°C, 96% ee of L-10 (59 µM conversion after 137 min) was
obtained. Similarly, 94% ee of D-10 (34 µM conversion after
177 min) was obtained in the reaction of antibody 3G2 (40 µM
of active site) and racemic 9 (150 µM) under the same
conditions.
Figure 2. Representative plot of tight-binding inhibition of antibody
7G12 by phosphonate (R)-3. Initial velocities were measured at
increasing concentrations of (R)-3 in the presence of 0.22 µM (active
site) antibody 7G12 and 10 µM L-9. [I] is the (R)-3 concentration. V
and Vo are, respectively, the velocities in the presence and absence of
(R)-3.
Correlation of Hapten and Substrate Chirality. In this
study, racemic hapten 2 was used for immunization, so that each
enantiomer of the hapten could elicit catalytic antibodies specific
to the corresponding enantiomer of the substrate. To correlate
the hapten and substrate chirality, the binding affinities of
antibodies 7G12 and 3G2 to chiral phosphonates (R)-3 and (S)-
3, in which their chiral centers are the same configuration (CR)
as L-9 and D-9, respectively, were determined by competitive
ELISA.15,16 The values of the dissociation constants (Kd) are
shown in Table 2. As expected from the observed enantiose-
lectivity to L-9 in hydrolysis, antibody 7G12 displayed an
affinity to (R)-3 that was approximately 4-fold higher than that
to the opposite isomer (S)-3. Similarly, antibody 3G2 prefer-
entially bound to (S)-3 with 60-fold higher affinity than to (R)-
3. This result suggests that the enantioselectivity observed in
the antibody-catalyzed reactions is associated with binding in
the antibody-combining site and that antibodies 7G12 and 3G2
should be elicited against the corresponding enantiomers of the
transition state analog. However, the molecular mechanism of
the enantioselectivity may not so simple because these catalytic
antibodies bind the corresponding enantiomers of the transition
state analog, with cross-reactivity to the opposite enantiomers.
In the case of antibody 7G12, the difference (∆∆G ) 0.8 kcal/
mol) in the binding energy between (R)-3 and (S)-3 is not
consistent with the difference (∆∆G ) 2.3 kcal/mol) calculated
from the enantiomeric excess of product L-10 in the antibody-
catalyzed kinetic resolution. Probably, antibody 7G12 interacts
with (S)-3 by a binding mode different from that of (R)-3.19
Figure 3. Representative plot of tight-binding inhibition of antibody
3G2 by phosphonate (S)-3. Initial velocities were measured at
increasing concentrations of (S)-3 in the presence of 0.27 µM (active
site) antibody 3G2 and 10 µM D-9. [I] is the (S)-3 concentration. V
and Vo are, respectively, the velocities in the presence and absence of
(S)-3.
Table 2. Binding Affinity of Anti-2 Antibodies with Chiral
Phosphonate 3a
Kd of (R)-3, Kd of (S)-3,
ratio of
antibody
µM
µM
10
0.17
affinititesb ∆∆G,c kcal/mol
7G12
3G2
2.5
11
81:19
2:98
0.8
2.5
a Kds were determined by competition ELISA.15 See ref 16. b Ratio
of affinities ) [1/Kd of (R)-3]:[1/Kd of (S)-3]. c ∆∆G ) |RT ln{[1/Kd
of (R)-3]/[1/Kd of (S)-3]}|.
respectively.13 These antibodies catalyzed the hydrolyses with
multiple turnovers, and no product inhibition was detected in
the reactions. The antibody-catalyzed hydrolysis of L-9 with
7G12 was competitively inhibited with the addition of chiral
phosphonate (R)-3, which possesses the same CR configuration
as L-9 (Figure 2). Similarly, the hydrolysis of D-9 with 3G2
was inhibited with (S)-3 (Figure 3). Henderson plots14 of the
tight-binding inhibition afforded the Ki values for these antibody-
catalyzed reactions (Table 1).
Substrate Specificity and Enantioselectivity. In addition
to having the high enantioselectivity described above, the
antibodies 7G12 and 3G2 were able to accept a broad range of
substrates, as determined using N-Cbz-amino acid esters 1
possessing various R-substituents. The initial velocity of the
antibody-catalyzed hydrolysis of each enantiomer was deter-
mined, and its ratio was used to estimate the enantioselectivity
Kinetic Resolution. To demonstrate that antibodies 7G12
and 3G2 have the potential to be useful for kinetic resolution
of racemic substrates, the enantiomeric excess (ee) of the
reaction product, Cbz-amino acid with 10, was determined in
the antibody-catalyzed hydrolysis of racemic substrate 9 by
HPLC assay with a chiral column. When the reaction was
(15) Friguet, B.; Chaffotte, A. F.; Djavadi-Ohaniance, L.; Goldberg, M.
E. J. Immunol. Methods 1985, 77, 305-319.
(16) Competition ELISA of an antibody often gives lower affinity values
(higher values of Kd) than true binding because of the avidity of the bivalent
IgG.17 However, this method is useful in determining the ratio of the Kds.18
(17) For an example: Tarasow, T. M.; Lewis, C.; Hilvert, D. J. Am.
Chem. Soc. 1994, 116, 7959-7963.
(18) Fujii, I.; Tanaka, F.; Miyashita, H.; Tanamura, R.; Kinoshita, K. J.
Am. Chem. Soc. 1995, 117, 6199-6209.
(19) In the X-ray structural analysis of anti-progesterone antibody, some
of the cross-reactivity of the antibody with structurally different steroids
was observed through different binding modes of the steroids.6b
(13) The kcats were obtained by dividing by the measured active site
concentrations. The active site concentrations were determined by ex-
trapolating from Henderson plots14 of the activity titrations, with phospho-
nate (R)-3 for antibody 7G2 or (S)-3 for antibody 3G2.
(14) Henderson, P. J. F. Biochem. J. 1972, 127, 321-333.

Substrate Specificity in Antibody-Catalyzed Reactions
J. Am. Chem. Soc., Vol. 118, No. 10, 1996 2335
Table 3. Enantioselective Hydrolysis of Esters 1 with Antibody
Table 5. Kinetic Parameters of Antibody 7G12-Catalyzed
Hydrolysis of Ester 1a
7G12a
substrate
R-
velocities,b µM/min
L-1 D-1
2.52 × 10-1 3.44 × 10-2
ratio of velocities
substrate
R-
Km, µM
kcat,b min-1
1
L-1:D-1
L-1a
L-1b
L-1c
L-1e
L-1h
CH3-
13
23
36
2.8 × 10-2
3.7 × 10-2
2.8 × 10-2
2.4 × 10-2
1.2 × 10-1 c
(CH3)2CHCH2-
CH3(CH2)3-
PhCH2-
1a CH3-
88:12
98:2
98:2
95:5
98:2
91:9
>99:<1
1b (CH3)2CHCH2- 2.87 × 10-1 7.30 × 10-3
4.9
1c CH3(CH2)3-
1.83 × 10-1 3.85 × 10-3
4-HOPh-
64c
1d CH3SCH2CH2- 3.58 × 10-1 1.80 × 10-2
1.78 × 10-1 3.44 × 10-3
5.54 × 10-3 5.42 × 10-4
7.83 × 10-1 NDc
a Reaction conditions: 10% DMSO/50 mM Tris (pH 8.0), 25 °C.
b See ref 13. c The Km and kcat values were determined by the use of
DL-1h and by disregarding the presence of D-1h, for example, [DL-1h]
) 50 µM was regarded as [L-1h] ) 25 µM.
1e PhCH2-
1f (CH3)2CH-
1g Ph-
1h 4-HOPh-
1i H2N(CH2)4-
3.33 × 10-1 d 6.86 × 10-3
4.36 × 10-1 1.49 × 10-2
97:3
Table 6. Kinetic Parameters of Antibody 3G2-Catalyzed
Hydrolysis of Ester 1a
a Reaction conditions: [L- or D-substrate] 100 µM for 1a,c,f,g,i and
200 µM for 1b,d,e,h, [antibody 7G12] 10 µM (active site), 10% DMSO/
50 mM Tris (pH 8.0), 25. °C. The first-order kinetic constants of the
substrate
R-
Km, µM
kcat,b min-1
background reactions: 1a, 1.06 × 10-4 min-1; 1b, 1.26 × 10-5 min-1
1c, 1.71 × 10-5 min-1; 1d, 3.83 × 10-5 min-1; 1e, 2.24 × 10-5 min-1
1f, 6.47 × 10-6 min-1; 1g, 5.62 × 10-3 min-1; 1h, 1.84 × 10-4 min-1
;
;
;
D-1a
D-1b
D-1c
D-1e
D-1h
CH3-
16
21
41
5.5
27
2.1 × 10-2
1.0 × 10-2
8.6 × 10-3
3.0 × 10-3
6.2 × 10-2
(CH3)2CHCH2-
CH3(CH2)3-
PhCH2-
1i, 1.40 × 10-4 min-1
.
b Initial velocities of the hydrolysis were
determined by HPLC assay. c Not detected. d This velocity was deter-
4-HOPh-
mined by the use of DL-1h instead of L-1h.
a Reaction conditions: 10% DMSO/50 mM Tris (pH 8.0), 25 °C.
b See ref 13.
Table 4. Enantioselective Hydrolysis of Esters 1 with Antibody
3G2a
lyzed with 3G2, the ꢀ-amino group of lysine may influence the
substrate recognition with antibody 3G2, but the details remain
unclear. Neither antibody 7G12 nor 3G2 catalyzed the hy-
drolysis of proline ester derivatives.
velocities,b
µM/min
ratio of
velocities
substrate
R-
1
L-1
D-1
L-1:D-1
1a CH3-
9.68 × 10-3 1.86 × 10-1
6.67 × 10-3 1.16 × 10-1
3.98 × 10-3 9.12 × 10-2
2.44 × 10-2 2.15 × 10-1
2.46 × 10-3 3.08 × 10-2
5:95
5:95
4:96
6:94
7:93
To examine the effects of the R-substituents of the substrates
in more detail, the Km values of the amino acid esters Ala (1a),
Leu (1b), Norleu (1c), Phe (1e), and 4-hydroxyphenylglycine
(1h), which were chosen on the basis of the size (bulkiness) of
the R-substituents, were determined. As shown in Tables 5 and
6, the Km values for these substrates were found to be in the
narrow ranges of 4.9-64 µM for 7G12 and 5.5-41 µM for
3G2. Thus, alteration of the R-substituents of the substrates
has little effect on the binding affinity to the antibodies. This
observation suggests that the use of haptens that fit snugly into
the antigen-combining site, leaving the linker moiety (involving
Câ) outside, is an effective approach for the generation of
catalytic antibodies with broad substrate applicability. Although
Hirschmann et al. also attempted to generate catalytic antibodies
(for peptide-bond formation) that accomodate the diverse
R-substituents of amino acids, by creating a cavity corresponding
to the haptenic cyclohexyl group in the antigen-combining site,
the size and the hydrophobicity of the cavity limited the diversity
of the R-substituents of amino acids.22 In our case, the
R-substituents of the amino acids should be excluded from the
antigen-combining site, and broader substrate specificity was
achieved.
1b (CH3)2CHCH2-
1c CH3(CH2)3-
1d CH3SCH2CH2-
1e PhCH2-
1f (CH3)2CH-
1g Ph-
5.70 × 10-4 1.96 × 10-3 23:77
NDc
5.83 × 10-2 <1:>99
1h 4-HOPh
1i H2N(CH2)4-
1.84 × 10-1 d 4.08 × 10-1
2.04 × 10-1 2.20 × 10-1 48:52
1j H2NCH2CONH(CH2)4- 3.85 × 10-3 1.63 × 10-1
2:98
a Reaction condditions: [L- or D-substrate] 100 µM, except for 1e
(50 µM), [antibody 3G2] 10 µM (active site), 10% DMSO/50 mM Tris
(pH 8.0), 25 °C. The first-order kinetic constants of the background
reactions: 1a-i, see Table 3; 1j, 1.21 × 10-4 min-1
.
b See Table 3.
c Not detected. d This velocity was determined by the use of DL-1h
instead of L-1h.
(Tables 3 and 4).20 The reaction in 10% DMSO/50 mM Tris
(pH 8.0) at 25 °C was followed by monitoring the production
of 4-nitrobenzyl alcohol by HPLC. Antibody 7G12 displayed
broad substrate specificity, hydrolyzing the L-ester derivatives
of Ala (1a), Leu (1b), Norleu (1c), Met (1d), Phe (1e), and Lys
(1i) with high enantioselectivity (Table 3). In all cases, product
inhibition was not detected. Interestingly, amino acid esters
with bulky substituents on Câ, such as Val (1f), phenylglycine
(1g), and 4-hydroxyphenylglycine (1h), were also enantiose-
lectively hydrolyzed by 7G12.21 Antibody 3G2 also accepted
a broad range of amino acid esters and enantioselectively
hydrolyzed the D-isomers. When the hydrolysis of the lysine
derivative (1i) with antibody 3G2 was examined, we were
surprised to find that no enantioselectivity was detected. Since
the Nꢀ-glycyl-D-lysine ester (1j) was enantioselectively hydro-
Conclusion
In this work, we have proposed a hapten design to endow
antibody catalysts with two opposing qualities, such as high
selectivity and broad substrate specificity. We demonstrated
the generation of antibodies that enantioselectively hydrolyze
a wide range of R-amino acid esters. These catalytic antibodies
yield chiral N-Cbz protected amino acids as products, so they
would be useful for the synthesis of peptides including natural
and unnatural amino acids as components.
(20) Catalytic antibodies with either R or S substrate specificity are
expected to show kinetic resolution: (a) Reference 10. (b) Sinha, S. C.;
Keinan, E.; Reymond, J.-L. J. Am. Chem. Soc. 1993, 115, 4893-4894. (c)
Pollack, S. J.; Hsiun, P.; Schultz, P. G. J. Am. Chem. Soc. 1989, 111, 5961-
5962. (d) Napper, A. D.; Benkovic, S. J.; Tramontano, A.; Lerner, R. A.
Science 1987, 237, 1041-1043.
Experimental Section
General Methods (Synthesis). All oxygen- or moisture-sensitive
(21) Because the background hydrolysis (kuncat) of 1f was lower than
that of the other substrates, the velocity of the L-ester hydrolysis by antibody
7G12 was 2 orders of magnitude lower than that of the other L-esters.
Although ester 1f, with a bulky substituent, was more resistant to hydrolysis,
it was enantioselectively (9:1) hydrolyzed by antibody 7G12.
reactions were carried out under argon. Flash chromatography was
(22) Hirschmann, R.; Smith, A. B., III; Taylor, C. M.; Benkovic, P. A.;
Taylor, S. D.; Yager, K. M.; Sprengeler, P. A.; Benkovic, S. J. Science
1994, 265, 234-237.

2336 J. Am. Chem. Soc., Vol. 118, No. 10, 1996
Tanaka et al.
performed using Kisel gel (230-400 mesh) silica gel (Merck).
Preparative thin-layer chromatography (TLC) was performed on Kisel
gel 60 F254 (0.5 mm) (Merck). Preparative high-performance liquid
chromatography (HPLC) was performed on a Hitachi L-6200 equipped
with a L-4200 UV detector. 1H NMR spectra were recorded on Bruker
AM 500 (500 MHz) and AMX 600 (600 MHz) NMR spectrometers.
The spectra are reported in units of ppm downfield from tetramethyl-
silane. Mass spectra were recorded with JEOL JMS-SX102/102A
(Tandem MS) and JMS-HX/HX110A mass spectrometers.
Compound 5. To a solution of ethyl 6-hydroxyhexanoate (4) (2.83
g, 17.7 mmol) in CH2Cl2 (70 mL) was added PDC (9.95 g, 26.4 mmol)
at room temperature. After 5 h of stirring, the reaction mixture was
flash chromatographed (EtOAc/hexane ) 1/2) to give 5 as a colorless
oil (1.1276 g, 40%). 1H NMR (500 MHz, CDCl3): δ 9.78 (t, J ) 1.4
Hz, 1H), 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 Hz, 3H).
Compound 6. To a solution of 5 (1.1276 g, 7.13 mmol) in EtOH
(2.5 mL) were added benzylamine (1.56 mL, 14.3 mmol) and triethyl
phosphite (2.44 mL, 14.2 mmol) at room temperature. After stirring
at room temperature for 17.5 h, triethyl phosphite (1.0 mL, 5.8 mmol)
was added and the reaction mixture was stirred at 40 °C for 6 h. The
solvent was removed in Vacuo. The residue was flash chromatographed
(EtOAc/2-propanol ) 20/1) to give 6 as a colorless oil (1.4837 g, 48%).
1H NMR (500 MHz, CDCl3): δ 7.35-7.23 (m, 5H), 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, 1H), 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). FABMS: m/z 386 (M+ + H).
HR-FABMS: calcd for C19H33O5NP (M+ + H) 386.2132, found
386.2092.
5.16 (B of ABX, dd, JAB ) 13.5 Hz, JAX ) 7.7 Hz, 1H), 5.14 (A of
AB, d, J ) 12.5 Hz, 1H), 5.08 (B of AB, d, J ) 12.5 Hz, 1H), 4.07
(m, 1H), 2.32 (t, J ) 7.3 Hz, 2H), 1.97-1.38 (m, 6H). FABMS: m/z
503 (M+ + Na), 481 (M+ + H). HR-FABMS: calcd for C21H25O9N2-
PNa (M+ + Na) 503.1196, found 503.1202.
Compound 8. A mixture of 2 (6.4 mg, 0.013 mmol), N-hydroxy-
succinimide (2.5 mg, 0.022 mmol), WSC (8.3 mg, 0.043 mmol), and
DMAP (0.1 mg, 0.0008 mmol) in CH3CN (0.3 mL) was stirred at room
temperature for 2 h. The mixture was purified by HPLC (YMC
A-323: C-18 reverse-phase column, φ 10 × 250 mm, CH3CN/0.1%
aqueous TFA ) 50/50, 3.0 mL/min, 254 nm, retention time 8.2 min).
The CH3CN and TFA were removed in Vacuo, and the water was
removed by lyophilization to give 8 as a colorless residue (6.1 mg,
79%). 1H NMR (500 MHz, CD3OD): δ 8.23 (d, J ) 8.7 Hz, 2H),
7.62 (d, J ) 8.7 Hz, 2H), 7.38-7.27 (m, 5H), 5.20 (A of ABX, dd,
JAB ) 13.4 Hz, JAX ) 7.5 Hz, 1H), 5.17 (B of ABX, dd, JAB ) 13.4
Hz, JAX ) 7.5 Hz, 1H), 5.13 (A of AB, d, J ) 12.5 Hz, 1H), 5.09 (B
of AB, d, J ) 12.5 Hz, 1H), 4.08 (m, 1H), 2.85 (s, 4H), 2.66 (t, J )
7.2 Hz, 2H), 1.97-1.43 (m, 6H).
KLH-2. To a solution of 8 (2.5 mg, 0.0043 mmol) in DMF (60
µL)-200 mM Na2HPO4-NaH2PO4 (pH 7.4) (0.5 mL) was added a
KLH solution in 10 mM Na2HPO4-NaH2PO4 (pH 7.4) (11.3 mg/mL,
442 µL) at room temperature. After 21 h, the mixture was purified by
Sephadex G-25M (Pharmacia, PD-10)(PBS buffer) to give KLH-2. The
concentration of KLH-2 was determined by Bradford analysis (1.3 mg/
mL). The KLH-2 was used for immunization.
BSA-2. To a solution of 8 (3.0 mg, 0.0052 mmol) in DMF (40 µL)
was added a BSA solution in 200 mM Na2HPO4-NaH2PO4 (pH 7.4)
(6.0 mg in 0.5 mL) at room temperature. After 12 h, the mixture was
purified by Sephadex G-25M (Pharmacia, PD-10) (PBS buffer) to give
BSA-2. The concentration of BSA-2 was determined by Bradford
analysis (0.9-2.3 mg/mL, three fractions). The BSA-2 was used for
the ELISA experiment.
Compound 7. A mixture of 6 (473.0 mg, 1.23 mmol) and 10%
Pd-C (4.6 mg) in MeOH (3.0 mL)-formic acid (0.40 mL) was stirred
at room temperature for 2.5 days under H2. The mixture was filtered.
The filtrate was concentrated in Vacuo to give a colorless residue. The
resulting residue was dissolved in THF (5.0 mL), and to this were added
carbobenzoxy chloride (700 µL, 4.90 mmol) and triethylamine (1.71
mL, 12.3 mmol) at room temperature. After 3 h, 1 N HCl was added
and the mixture was extracted with EtOAc. The combined organic
layers were washed with brine, dried over MgSO4, filtered, concentrated
in Vacuo, and flash chromatographed (EtOAc, TLC: Rf 0.51) to give
a colorless gum (120.5 mg). The resulting gum was dissolved in CH2-
Cl2 (0.5 mL), and bromotrimethylsilane (222 µL, 1.68 mmol) was added
at room temperature. After the reaction mixture was stirred at 35 °C
for 2 h, the volatile solvent was removed in Vacuo and CH3CN (1.0
mL) and H2O (0.2 mL) were added to the reaction mixture. After the
mixture was stirred for 12 h, the solvent was removed in Vacuo and
the residue was dried. A mixture of the residue, 4-nitrobenzyl alcohol
(61.2 mg, 0.40 mmol), 1H-tetrazole (9.7 mg, 0.14 mmol), and DCC
(261.5 mg, 1.27 mmol) in CH2Cl2 (1.0 mL)-pyridine (0.5 mL) was
stirred at 35 °C for 5 h. The reaction was concentrated in Vacuo, diluted
with CH3CN, and filtered. The filtrate was purified by HPLC (YMC
A-323: C-18 reverse-phase column, φ 10 × 250 mm, CH3CN/0.1%
aqueous TFA ) 50/50, 3.0 mL/min, 254 nm, retention time 11.5 min).
The CH3CN and TFA were removed in Vacuo, and the water was
removed by lyophilization to give 7 as a colorless residue (34.5 mg,
6% from 6). 1H NMR (500 MHz, CD3OD): δ 8.22 (d, J ) 8.6 Hz,
2H), 7.62 (d, J ) 8.6 Hz, 2H), 7.38-7.28 (m, 5H), 5.19 (A of ABX,
(R)-[1-(N-Carbobenzoxyamino)ethyl]phosphonic Acid. To a so-
lution of (R)-(-)-(1-aminoethyl)phosphonic acid (500.8 mg, 4.00 mmol)
in 4 N NaOH (3.3 mL) was added carbobenzoxy chloride (742 µL,
5.20 mmol) at 0 °C, and the reaction mixture was stirred at room
temperature. After 12 h, the mixture was acidified with 2 N HCl and
purified by HPLC (YMC A-323: C-18 reverse-phase column, φ 10 ×
250 mm, CH3CN/0.1% aqueous TFA ) 30/70, 3.0 mL/min, 254 nm,
retention time 7.7 min). The CH3CN and TFA were removed in Vacuo,
and the water was removed by lyophilization to give (R)-[1-(N-
carbobenzoxyamino)ethyl]phosphonic acid as a colorless solid (680.8
mg, 66%). 1H NMR (500 MHz, CD3OD): δ 7.41-7.31 (m, 5H), 5.14
(s, 2H), 4.01 (m, 1H), 1.39 (dd, J ) 7.4 Hz, 16.2 Hz, 3H). FABMS
m/z: 260 (M+ + H). [R]D -14.4° (c 0.9, MeOH).
(S)-[1-(N-Carbobenzoxyamino)ethyl]phosphonic Acid. [R]D
+14.0° (c 1.3, MeOH).
26
26
(R)-[1-(N-Carbobenzoxyamino)ethyl]phosphonic Acid 4-Nitroben-
zyl Ester [(R)-3]. A mixture of (R)-[1-(N-carbobenzyloxyamino)ethyl]-
phosphonic acid (83.0 mg, 0.320 mmol), 4-nitrobenzyl alcohol (57.5
mg, 0.375 mmol), 1H-tetrazole (4.8 mg, 0.069 mmol), and DCC (293.1
mg, 1.42 mmol) in CH2Cl2 (1.0 mL)-pyridine (1.0 mL) was stirred at
35 °C for 10 h. The reaction was concentrated in Vacuo, diluted with
CH3CN and TFA, and filtered. The filtrate was purified by HPLC
(YMC A-323: C-18 reverse-phase column, φ 10 × 250 mm, CH3CN/
0.1% aqueous TFA ) 40/60, 3.0 mL/min, 254 nm, retention time 10.1
min). The CH3CN and TFA were removed in Vacuo, and the water
was removed by lyophilization to give (R)-3 as a colorless solid (51.8
mg, 41%). 1H NMR (600 MHz, CD3OD): δ 8.22 (d, J ) 8.6 Hz,
2H), 7.63 (d, J ) 8.6 Hz, 2H), 7.40-7.29 (m, 5H), 4.15 (m, 1H), 1.44
(dd, J ) 7.2 Hz, 16.6 Hz, 3H). FABMS: m/z 395 (M+ + H). HR-
FABMS: calcd for C17H20O7N2P (M+ + H) 395.1008, found 395.1007.
dd, JAB ) 13.5 Hz, JAX ) 7.4 Hz, 1H), 5.16 (B of ABX, dd, JAB
)
13.5 Hz, JAX ) 7.4 Hz, 1H), 5.13 (A of AB, d, J ) 12.6 Hz, 1H), 5.09
(B of AB, d, J ) 12.6 Hz, 1H), 4.14 (q, J ) 7.1 Hz, 2H), 4.06 (m,
1H), 2.33 (t, J ) 7.3 Hz, 2H), 1.95-1.37 (m, 6H), 1.27 (t, J ) 7.1 Hz,
3H). FABMS: m/z 509 (M+ + H). HR-FABMS: calcd for
C23H30O9N2P (M+ + H) 509.1689, found 509.1688.
Hapten 2. To a solution of 7 (15.0 mg, 0.0295 mmol) in MeOH
(0.2 mL)-H2O (0.2 mL) was added 1 N NaOH (0.1 mL, 0.1 mmol) at
room temperature. After 6.5 h, the reaction was acidified with
trifluoroacetic acid and purified by HPLC (YMC A-323: C-18 reverse-
phase column, φ 10 × 250 mm, CH3CN/0.1% aqueous TFA ) 50/50,
3.0 mL/min, 254 nm, retention time 6.2 min). The CH3CN and TFA
were removed in Vacuo, and the water was removed by lyophilization
to give 2 as a colorless residue (14.0 mg, 99%). 1H NMR (500 MHz,
CD3OD): δ 8.22 (d, J ) 8.6 Hz, 2H), 7.61 (d, J ) 8.6 Hz, 2H), 7.37-
7.28 (m, 5H), 5.19 (A of ABX, dd, JAB ) 13.5 Hz, JAX ) 7.7 Hz, 1H),
26
[R]D +4.6° (c 0.6, MeOH).
26
(S)-3. [R]D -4.6° (c 0.7, MeOH).
2-[(tert-Butoxycarbonyl)amino]benzoic Acid. A mixture of an-
thranic acid (1.37 g, 10.0 mmol) and (Boc)2O (3.12 g, 14.3 mmol) in
0.5 N NaOH (20.0 mL)-dioxane (10.0 mL)-CH3CN (2.0 mL) was
stirred at room temperature for 8 h. After the volatile solvent was
removed in Vacuo, ice and 10% citric acid were added and the mixture
was extracted with EtOAc. The combined organic layers were washed
with brine, dried over MgSO4, filtered, concentrated in Vacuo, and

Substrate Specificity in Antibody-Catalyzed Reactions
J. Am. Chem. Soc., Vol. 118, No. 10, 1996 2337
crystallized from EtOAc-hexane to give 2-[(tert-butoxycarbonyl)-
amino]benzoic acid as colorless crystals (1.71 g, 72%). 1H NMR (500
MHz, CDCl3): δ 10.1 (s, 1H), 8.47 (d, J ) 8.0 Hz, 1H), 8.09 (dd, J )
8.0 Hz, 1.4 Hz, 1H), 7.56 (dt, J ) 8.0 Hz, 1.4 Hz, 1H), 7.04 (t, J ) 8.0
Hz, 1H), 1.54 (s, 9H).
Compound 11. A mixture of 2-[(tert-butoxycarbonyl)amino]benzoic
acid (378.9 mg, 1.60 mmol), N-hydroxysuccinimide (225.9 mg, 1.96
mmol), WSC (565.9 mg, 2.95 mmol), and DMAP (4.7 mg, 0.04 mmol)
in CH2Cl2 was stirred at room temperature for 1 h. To the reaction
mixture was added 1% citric acid, and the mixture was extracted with
CH2Cl2. The combined organic layers were washed with brine, dried
over MgSO4, filtered, concentrated in Vacuo, and crystallized from
EtOAc-hexane to give 11 as colorless crystals (199.7 mg, 30%). 1H
NMR (500 MHz, CDCl3): δ 9.49 (s, 1H), 8.51 (d, J ) 8.0 Hz, 1H),
8.17 (d, J ) 8.0 Hz, 1H), 7.63 (d, J ) 8.0 Hz, 1H), 7.07 (d, J ) 8.0
Hz, 1H), 2.93 (s, 4H), 1.52 (s, 9H).
Compound L-12. A mixture of 11 (67.3 mg, 0.201 mmol), NR-
carbobenzoxy-L-lysine (56.4 mg, 0.201 mmol), and diisopropylethyl-
amine (0.04 mL, 0.230 mmol) in DMF (0.2 mL)-MeOH (0.2 mL)-
EtOAc (0.1 mL)-H2O (0.1 mL) was stirred at room temperature for
16 h. After the volatile solvent was removed in Vacuo, the residue
was flash chromatographed (EtOAc/hexane/H2O ) 9/1/0-8/2/0.2-5/
3/1) to give a residue (122.2 mg). A mixture of this residue (122.2
mg), 4-nitrobenzyl alcohol (34.9 mg, 0.228 mmol), WSC (58.6 mg,
0.306 mmol), and DMAP (1.4 mg, 0.011 mmol) in CH2Cl2 (1.0 mL)-
CH3CN (0.1 mL) was stirred at room temperature for 4 h. The mixture
was flash chromatographed (EtOAc/hexane ) 1/1.1) to give L-12 (35.7
mg, 28%). 1H NMR (600 MHz, CDCl3): δ 10.1 (s, 1H), 8.33 (d, J )
8.0 Hz, 1H), 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, 1H), 6.37 (brs,
1H), 5.36 (brs, 1H), 5.26 (A of AB, d, J ) 13.3 Hz, 1H), 5.23 (B of
AB, d, J ) 13.3 Hz, 1H), 5.09 (A of AB, d, J ) 12.3 Hz, 1H), 5.03 (B
of AB, d, J ) 12.3 Hz, 1H), 4.46 (m, 1H), 3.43-3.37 (m, 2H), 1.95-
1.40 (m, 6H). FABMS: m/z 635 (M+ + H).
residue was dissolved in MeOH (0.5 mL) and 1 N NaOH (0.3 mL)
was added. After the mixture was stirred for 12 h, the reaction was
acidified with trifluoroacetic acid and purified by HPLC (YMC
A-323: C-18 reverse-phase column, φ 10 × 250 mm, CH3CN/0.1%
aqueous TFA ) 55/45, 3.0 mL/min, 254 nm, retention time 5.2 min).
The CH3CN and TFA were removed in Vacuo, and the water was
removed by lyophilization to give L-10 as a pale yellow gum. 1H NMR
(500 MHz, CD3OD): δ 7.57 (d, J ) 8.0 Hz, 1H), 7.40-7.30 (m, 6H),
6.97 (d, J ) 8.0 Hz, 1H), 6.90 (t, J ) 8.0 Hz, 1H), 5.11 (s, 2H), 4.21
(m, 1H), 3.89 (t, J ) 6.9 Hz, 2H), 1.97-1.88 (m, 1H), 1.81-1.62 (m,
3H), 1.59-1.46 (m, 2H). FABMS: m/z 400 (M+ + H). HR-
FABMS: calcd for C21H26O5N3 (M+ + H) 400.1872, found 400.1884.
Synthesis of Nr-Carbobenzoxy Amino Acid 4-Nitrobenzyl Esters
(1). Esters (1a-h) were prepared from either L- or D-N-carbobenzoxy
amino acid and 4-nitrobenzyl alcohol using WSC (method A), or L- or
D-N-carbobenzoxy amino acid and 4-nitrobenzyl bromide using tri-
ethylamine (method B). The general procedures were as follows:
Method A: A mixture of N-carbobenzoxy amino acid (1 equiv),
4-nitrobenzyl alcohol (1.1 equiv), WSC (1.3-1.6 equiv), and DMAP
(0.01 equiv) in CH2Cl2 was stirred at room temperature for 1 h. The
solvent was removed in Vacuo, ice and 1 N HCl were added, and the
mixture was extracted with EtOAc. The combined organic layers were
washed with brine, dried over MgSO4, filtered, concentrated in Vacuo,
and flash chromatographed to give a N-carbobenzyoxy amino acid
4-nitrobenzyl ester.
Method B: A mixture of N-carbobenzoxy amino acid (1 equiv),
4-nitrobenzyl bromide (1.5 equiv), and triethylamine (1.5 equiv) in
EtOAc was refluxed for 2 h. The reaction was quenched with ice and
1 N HCl, and the mixture was extracted with EtOAc. The combined
organic layers were washed with brine, dried over MgSO4, filtered,
concentrated in Vacuo, and flash chromatographed to give a N-
carbobenzoxy amino acid 4-nitrobenzyl ester.
Enantiomeric Purity of Esters 1. The enantiomeric purity of the
substrate esters was determined by HPLC analysis using DAICEL
CHIRALCEL OB (1a,b, hexane/2-propanol), CHIRALCEL OJ-R (1c,d,
CH3CN/0.1% aqueous TFA), and CHIRALCEL OD-R (1e-g, and
D-1h, CH3CN/0.1% aqueous TFA): all of the substrates, except L-1g
(92% ee), were >99% ee. L-1i: [R]D22 -12.4°, c 0.4, MeOH). D-1i:
22
[R]D +12.3° (c 0.2, MeOH). Isomerization of L-1e to D-1e was not
detected under the assay conditions by HPLC analysis using CHIRAL-
CEL OD-R.
N-Carbobenzoxy-L-alanine 4-Nitrobenzyl Ester (L-1a). According
to method A, the reaction was flash chromatographed (EtOAc/hexane
) 2/3) and crystallized from EtOAc-hexane to give colorless crystals.
1H NMR (500 MHz, CDCl3): δ 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, 1H), 5.12
(s, 2H), 4.47 (m, 1H), 1.45 (d, J ) 7.2 Hz, 3H). FABMS: m/z 359
(M+ + H). HR-FABMS: calcd for C18H19O6N2 (M+ + H) 359.1244,
found 359.1244.
Compound L-9. A mixture of L-12 (54.1 mg, 0.0858 mmol) and
trifluoroacetic acid (0.2 mL) in CH2Cl2 (0.3 mL) was stirred at room
temperature for 1 h. The solvent was removed in Vacuo, and the residue
was purified by HPLC (YMC A-323: C-18 reverse-phase column, φ
10 × 250 mm, CH3CN/0.1% aqueous TFA ) 60/40, 3.0 mL/min, 254
nm, retention time 10.1 min). The CH3CN and TFA were removed in
Vacuo, and the water was removed by lyophilization to give L-9 as a
colorless gum (38.1 mg, 83%). The gum was crystallized from
EtOAc-hexane to give L-9 as colorless crystals. 1H NMR (600 MHz,
CDCl3-CD3OD): δ 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, 1H), 6.65 (d, J ) 8.0
Hz, 1H), 6.64 (t, J ) 8.0 Hz, 1H), 5.26 (A of AB, d, J ) 13.3 Hz,
1H), 5.24 (B of AB, d, J ) 13.3 Hz, 1H), 5.10 (A of AB, d, J ) 12.3
Hz, 1H), 5.06 (B of AB, d, J ) 12.3 Hz, 1H), 4.40 (m, 1H), 3.42-
3.34 (m, 2H), 1.94-1.85 (m, 1H), 1.80-1.72 (m, 1H), 1.68-1.54 (m,
2H), 1.51-1.39 (m, 2H). FABMS: m/z 535 (M+ + H). HR-
FABMS: calcd for C28H31O7N4 (M+ + H) 535.2193, found 535. 2182.
The ees of L-9 and D-9 were determined to be >99% by HPLC
(CHIRALCEL OJ-R, φ 4.6 × 150 nnm, CH3CN/0.1% aqueous TFA
) 55/45, 0.5 mL/min, 254 nm, retention time: L-9 13.7 min, D-9 12.8
min).
The ee was determined to be >99% by HPLC (CHIRALCEL OB,
φ 4.6 × 250 mm, hexane/2-propanol ) 10/90, 0.5 mL/min, 254 nm,
retention time: L-1a 33.0 min, D-1a 75.8 min).
N-Carbobenzoxy-L-leucine 4-Nitrobenzyl Ester (L-1b). According
to method A, the reaction was flash chromatographed (EtOAc/hexane
) 1/2) to give a colorless gum. 1H NMR (600 MHz, CDCl3): δ 8.22
(d, J ) 8.3 Hz, 2H), 7.50 (d, J ) 8.3 Hz, 2H), 7.40-7.30 (m, 5H),
5.26 (A of AB, d, J ) 13.4 Hz, 1H), 5.24 (B of AB, d, J ) 13.4 Hz,
1H), 5.12 (A of AB, d, J ) 11.4 Hz, 1H), 5.11 (B of AB, d, J ) 11.4
Hz, 1H), 5.13-5.11 (1H), 4.46 (m, 1H), 1.74-1.52 (m, 3H), 0.95 (d,
J ) 7.2 Hz, 3H), 0.94 (d, J ) 7.2 Hz, 3H). FABMS: m/z 401 (M+
+
H). HR-FABMS: calcd for C21H25O6N2 (M+ + H) 401.1712, found
401.1713. The ee was determined to be >99% by HPLC (CHIRAL-
CEL OB, φ 4.6 × 250 mm, hexane/2-propanol ) 30/70, 0.5 mL/min,
254 nm, retention time: L-1b 20.9 min, D-1b 31.8 min).
N-Carbobenzoxy-L-norleucine 4-Nitrobenzyl Ester (L-1c). Ac-
cording to method B, the reaction was flash chromatographed (EtOAc/
hexane ) 2/5) and crystallized from EtOAc-hexane to give colorless
crystals. 1H NMR (500 MHz, CDCl3): δ 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, 1H), 5.12 (s, 2H), 4.43 (m, 1H), 1.85 (m, 1H), 1.68 (m,
1H), 1.37-1.23 (m, 4H), 0.87 (t, J ) 6.9 Hz, 3H). EIMS: m/z 400
(M+). HR-EIMS: calcd for C21H24O6N2 (M+) 400.1634, found
Compound L-10. A mixture of L-12 (21.8 mg, 0.0346 mmol) and
trifluoroacetic acid (0.2 mL) in CH2Cl2 (0.4 mL) was stirred at room
temperature for 20 min. After the solvent was removed in Vacuo, the

2338 J. Am. Chem. Soc., Vol. 118, No. 10, 1996
Tanaka et al.
400.1639. The ee was determined to be >99% by HPLC (CHIRAL-
CEL OJ-R, ßlc 4.6 × 150 mm, CH3CN/0.1% aqueous TFA ) 60/40,
0.5 mL/min, 2.54 nm, retention time: L-1c 15.7 min, D-1c 16.9 min).
N-Carbobenzoxy-L-methionine 4-Nitrobenzyl Ester (L-1d). Ac-
cording to method A, the reaction was flash chromatographed (EtOAc/
hexane ) 2/3) to give a colorless gum. 1H NMR (600 MHz, CDCl3):
δ 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, 1H), 5.28 (A of AB, d, J ) 12.9 Hz, 1H),
5.26 (B of AB, d, J ) 12.9 Hz, 1H), 5.12 (s, 2H), 4.59 (m, 1H), 2.53
(t, J ) 7.1 Hz, 2H), 2.19 (m, 1H), 2.07 (s, 3H), 2.01 (m, 1H).
FABMS: m/z 419 (M+ + H). The ee was determined to be >99% by
HPLC (CHIRALCEL OJ-R, φ 4.6 × 150 mm, CH3CN/0.1% aqueous
TFA ) 60/40, 0.5 mL/min, 254 nm, retention time: L-1d 14.2 min,
D-1d 15.9 min).
N-Carbobenzoxy-L-phenylalanine 4-Nitrobenzyl Ester (L-1e).
According to method A, the reaction was flash chromatographed
(EtOAc/hexane ) 2/3) and crystallized from EtOAc-hexane to give
colorless crystals. 1H NMR (600 MHz, CDCl3): δ 8.17 (d, J ) 8.0
Hz, 2H), 7.38-7.23 (m, 10H), 7.08 (m, 2H), 5.23 (brd, J ) 7.5 Hz,
1H), 5.19 (s, 2H), 5.09 (s, 1H), 4.92 (m, 1H), 3.12 (d, J ) 6.8 Hz,
2H). FABMS: m/z 435 (M+ + H). HR-FABMS: calcd for C24H23O6N2
(M+ + H) 435.1556, found 435.1556. The ee was determined to be
>99% by HPLC (CHIRALCEL OD-R, φ 4.6 × 250 mm, CH3CN/
0.1% aqueous TFA ) 60/40, 0.5 mL/min, 254 nm, retention time: L-1e
39.7 min, D-1e 43.2 min).
washed with brine, dried over MgSO4, filtered, concentrated in Vacuo,
and flash chromatographed (EtOAc/hexane ) 2/3) to give NR-
carbobenzoxy-Nꢀ-(tert-butoxycarbonyl)-D-lysine 4-nitrobenzyl ester
(272.4 mg, 76%). The ester was crystallized from EtOAc-hexane to
give colorless crystals. 1H NMR (600 MHz, CDCl3): δ 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, 1H), 5.26 (A of AB, d, J ) 12.6 Hz, 1H), 5.25 (B of AB,
d, J ) 12.6 Hz, 1H), 5.12 (A of AB, d, J ) 12.2 Hz, 1H), 5.10 (B of
AB, d, J ) 12.2 Hz, 1H), 4.55 (m, 1H), 4.41 (m, 1H), 3.12-3.07 (m,
2H), 1.88 (m, 1H), 1.72 (m, 1H), 1.60-1.31 (m, 4H), 1.42 (s, 9H).
FABMS: m/z 516 (M+ + H).
Nr-Carbobenzoxy-D-lysine 4-Nitrobenzyl Ester (D-1i). A mixture
of NR-carbobenzoxy-Nꢀ-(tert-butoxycarbonyl)-D-lysine 4-nitrobenzyl
ester (52.5 mg, 0.101 mmol) and trifluoroacetic acid (0.2 mL) in CHCl3
(1.0 mL) was stirred at room temperature for 4 h. The solvent was
removed in Vacuo, and the residue was crystallized from EtOAc-
hexane to give D-1i as colorless crystals (32.6 mg, 77%). 1H NMR
(500 MHz, CDCl3-CD3OD): δ 8.22 (d, J ) 8.5 Hz, 2H), 7.51 (d, J
) 8.5 Hz, 2H), 7.40-7.26 (m, 5H), 5.29 (A of AB, d, J ) 13.3 Hz,
1H), 5.24 (B of AB, d, J ) 13.3 Hz, 1H), 5.13 (A of AB, d, J ) 12.3
Hz, 1H), 5.09 (B of AB, d, J ) 12.3 Hz, 1H), 4.35 (m, 1H), 2.87 (t,
J ) 7.2 Hz, 2H), 1.88 (m, 1H), 1.77-1.56 (m, 3H), 1.50-1.37 (m,
2H). EIMAS: m/z 415 (M+). HR-EIMS: calcd for C21H25O6N3 (M+)
415.1743, found 415.1743.
Nr-Carbobenzoxy-NE-[N-(tert-butoxycarbonyl)glycyl]-L-lysine 4-Ni-
trobenzyl Ester. A mixture of L-1i (31.2 mg, 0.0751 mmol), N-(tert-
butoxycarbonyl)glycine (19.1 mg, 0.109 mmol), and WSC (35.1 mg,
0.183 mmol) in CH2Cl2 (0.5 mL) was stirred at room temperature for
1.5 h. The mixture was flash chromatographed (EtOAc) to give NR-
carbobenzoxy-Nꢀ-[N-(tert-butoxycarbonyl)glycyl]-L-lysine 4-nitrobenzyl
ester (19.6 mg, 46%). 1H NMR (500 MHz, CDCl3): δ 8.21 (d, J )
8.3 Hz, 2H), 7.50 (d, J ) 8.3 Hz, 2H), 7.37-7.29 (m, 5H), 6.18 (brs,
1H), 5.46 (brd, J ) 7.8 Hz, 1H), 5.26 (d, J ) 13.9 Hz, 1H), 5.25 (d,
J ) 13.9 Hz, 1H), 5.12 (d, J ) 12.8 Hz, 1H), 5.11 (d, J ) 12.8 Hz,
1H), 4.39 (m, 1H), 3.78-3.69 (m, 2H), 3.31-3.19 (m, 2H), 1.86 (m,
1H), 1.71 (m, 1H), 1.58-1.33 (m, 4H). FABMS: m/z 573 (M+ + H).
Nr-Carbobenzoxy-NE-glycyl-L-lysine 4-Nitrobenzyl Ester (L-1j).
A mixture of NR-carbobenzoxy-Nꢀ-[N-(tert-butoxycarbonyl)glycyl]-
L-lysine 4-nitrobenzyl ester (11.6 mg, 0.0203 mmol) and trifluoroacetic
acid (0.1 mL) in CHCl3 (0.3 mL) was stirred at room temperature for
3.5 h. The solvent was removed in Vacuo, and the residue was purified
by HPLC (YMC A-323: C-18 reverse-phase column, φ 10 × 250 mm,
CH3CN/0.1% aqueous TFA ) 50/50, 3.0 mL/min, 254 nm, retention
time 5.7 min). CH3CN and TFA were removed in Vacuo, and the water
was removed by lyophilization to give L-1j (7.4 mg, 77%). 1H NMR
(500 MHz, CDCl3-CD3OD): δ 8.21 (d, J ) 8.5 Hz, 2H), 8.14 (brd,
J ) 7.9 Hz, 1H), 7.51 (d, J ) 8.5 Hz, 2H), 7.41-7.25 (m, 5H), 6.23
(brd, J ) 7.9 Hz, 1H), 5.28 (d, J ) 13.4 Hz, 1H), 5.26 (d, J ) 13.4
Hz, 1H), 5.13 (d, J ) 12.3 Hz, 1H), 4.35 (m, 1H), 3.60 (s, 2H), 3.21
(t, J ) 6.6 Hz, 2H), 1.85 (m, 1H), 1.72 (m, 1H), 1.59-1.45 (m, 2H),
1.43-1.35 (m, 2H). EIMS: m/z 472 (M+). HR-EIMS: calcd for
C23H28O7N4 (M+) 472.1957, found 472.1957.
N-Carbobenzoxy-L-valine 4-Nitrobenzyl Ester (L-1f). According
to method A, the reaction was flash chromatographed (EtOAc/hexane
) 1/2) and crystallized from EtOAc-hexane to give colorless crystals.
1H NMR (500 MHz, CDCl3): δ 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, 1H), 5.12 (s, 2H), 4.38 (dd, J ) 4.8 Hz, 8.8 Hz, 1H), 2.20 (m,
1H), 0.98 (d, J ) 6.8 Hz, 3H), 0.88 (d, J ) 6.8 Hz, 3H). EIMS: m/z
386 (M+). HR-EIMS: calcd for C20H22O6N2 (M+) 386.1476, found
386.1476. The ee was determined to be >99% by HPLC (CHIRAL-
CEL OD-R, φ 4.6 × 250 mm, CH3CN/0.1% aqueous TFA ) 70/30,
0.5 mL/min, 254 nm, retention time: L-1f 13.9 min, D-1f 15.8 min).
N-Carbobenzoxy-L-phenylglycine 4-Nitrobenzyl Ester (L-1g).
According to method B, the reaction was flash chromatographed
(EtOAc/hexane ) 2/5) and crystallized from EtOAc-hexane to give
colorless crystals. 1H NMR (500 MHz, CDCl3): δ 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, 1H), 5.44 (d, J ) 7.1 Hz, 1H), 5.28 (A of AB, d, J ) 13.5 Hz,
1H), 5.22 (B of AB, d, J ) 13.5 Hz, 1H), 5.13 (A of AB, d, J ) 12.1
Hz, 1H), 5.09 (B of AB, d, J ) 12.1 Hz, 1H). FABMS: m/z 421 (M+
+ H). The ee was determined to be 92% by HPLC (CHIRALCEL
OD-R, φ 4.6 × 250 mm, CH3CN/0.1% aqueous TFA ) 70/30, 0.5
mL/min, 254 nm, retention time: L-1g 19.8 min, D-1g 22.2 min).
N-Carbobenzoxy-4-hydroxyphenylglycine 4-Nitrobenzyl Ester
(1h). According to method B, the reaction was flash chromatographed
(EtOAc/hexane ) 1/1.25) and crystallized from EtOAc-hexane to give
colorless crystals. 1H NMR (600 MHz, CDCl3): δ 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, 1H),
5.36 (d, J ) 7.1 Hz, 1H), 5.26 (A of AB, d, J ) 13.9 Hz, 1H), 5.22 (B
of AB, d, J ) 13.9 Hz, 1H), 5.12 (A of AB, d, J ) 12.0 Hz, 1H), 5.09
(B of AB, d, J ) 12.0 Hz, 1H), 5.10 (s, 1H). EIMS: m/z 436 (M+).
HR-EIMS: calcd for C23H20O7N2 (M+) 436.1271, found 436.1277. The
ee of D-1h was determined to be >99% by HPLC (CHIRALCEL OD-
R, φ 4.6 × 250 mm, CH3CN/0.1% aqueous TFA ) 45/55, 0.5 mL/
min, 254 nm, retention time: L-1h 77.3 min, D-1h 81.4 min).
Nr-Carbobenzoxy-NE-(tert-butoxycarbonyl)-D-lysine 4-Nitroben-
zyl Ester. A mixture of NR-carbobenzoxy-D-lysine (194.7 mg, 0.695
mmol) and (Boc)2O (176.0 mg, 0.806 mmol) in 1 N NaOH (765 µL)-
MeOH (0.1 mL) was stirred at room temperature for 10 h. The reaction
was quenched with ice and 10% citric acid, and the mixture was
extracted with EtOAc. The combined organic layers were washed with
brine, dried over MgSO4, filtered, and concentrated in Vacuo to give
NR-carbobenzoxy-Nꢀ-(tert-butoxycarbonyl)-D-lysine as a residue. A
mixture of this residue, 4-nitrobenzyl bromide (231.3 mg, 1.07 mmol),
and triethylamine (145 µL, 1.04 mmol) in EtOAc was refluxed for 2
h. The reaction was quenched with ice and 10% citric acid, and the
mixture was extracted with EtOAc. The combined organic layers were
N-Carbobenzoxy-L-proline 4-Nitrobenzyl Ester (E:Z ) 1:1).
According to method A, the reaction was flash chromatographed
(EtOAc/hexane ) 2/3) to give colorless gum. 1H NMR (600 MHz,
1
CDCl3): δ 8.20 (d, J ) 8.2 Hz, 2H × /2), 8.10 (d, J ) 8.2 Hz, 2H ×
1
1
1/2), 7.45 (d, J ) 8.2 Hz, 2H × /2), 7.38-7.23 (m, 5H + 2H × /2),
1
5.30-5.02 (m, 4H), 4.38 (dd, J ) 3.8 Hz, 8.5 Hz, 1H × /2), 4.43 (dd,
1
J ) 3.8 Hz, 8.5 Hz, 1H × /2), 3.67-3.50 (m, 2H), 2.33-2.22 (m,
1H), 2.06-1.88 (m, 3H). EIMAS: m/z 384 (M+). HR-EIMAS: calcd
for C20H20O6N2 (M+) 384.1321, found 384.1321.
Antibody Production. Five Balb/c mice (4-week-old females) each
received an intraperitoneal injection of 50 µg of KLH-2 and Freund’s
complete adjuvant on day 1. On days 11 and 21, each mouse received
an intraperitoneal injection of 50 µg of KLH-2 and Freund’s incomplete
adjuvant. On day 28, serum was taken from the mice and the titer
was determined by ELISA. On day 60, the mouse with the highest
titer was injected intravenously with 100 µg of KLH-2 in saline (final
boost). After 3 days, the spleen was removed from the mouse, and
the cells (1.9 × 108) were fused with X63/Ag8653 myeloma cells (2.7
× 107) according to standard protocols.10 The cells were plated into
ten 96-well plates; each well contained 200 µL of HAT selection media

Substrate Specificity in Antibody-Catalyzed Reactions
J. Am. Chem. Soc., Vol. 118, No. 10, 1996 2339
(RPMI1640, 10% fetal bovine serum, 0.1 mM hypoxanthine, 0.4 µM
aminopterin, 0.016 mM thymidine) containing mouse thymus cells (6
× 105/well). The culture supernatants in wells containing macroscopic
colonies were assayed by ELISA for binding to BSA-2. A goat
antimouse IgG peroxidase conjugate was used as the secondary
antibody. Colonies that initially produced antibodies that bound BSA-2
were subcloned two or three times, after which 39 remained active.
The hybridomas were grown in RPMI1640 medium containing 10%
fetal bovine serum. Monoclonal antibodies (IgG) were purified from
the culture supernatants of the hybridomas by salt precipitation, cation-
exchange chromatography (Mono S), and affinity chromatography
(Protein G), as described previously.11 All antibodies were then
concentrated and dialyzed into 50 mM Tris (pH 8.0). The antibodies
were judged to be homogeneous (>95%) by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis. For screening, the antibody
concentration was determined by absorbance at 280 nm as ꢀ ) 1.4
(0.1%, 1 cm), with a molecular weight of 150 000 for IgG. For the
kinetics and the determination of velocities using antibodies 7G12 and
3G2 and various substrates, the active site concentrations were
determined by extrapolating from Henderson plots14 of the activity
titrations.
Fluorogenic Catalytic Assays and Kinetic Measurements. Reac-
tions were initiated by adding 40 µL of a stock solution of the ester 9
in DMSO to 360 µL of the antibody solution in 50 mM Tris (pH 8.0)
at 25 °C. The increase in the fluorescence was monitored for 10 min,
using a Hitachi F-4000 fluorescence spectrophotometer. The excitation
and emission wavelengths were 340 and 415 nm, respectively.
Hydrolysis of substrate 9 results in a 45-fold linear increase in the
fluorescence under the assay conditions. The observed rate was
corrected for the uncatalyzed rate of hydrolysis in the absence of
antibody. The kinetic parameters, kcat and Km, were determined by a
linear least-squares fitting of the Lineweaver-Burk plots, as described
by the Michaelis-Menten equation. The rate constants, kcats, were
obtained by dividing by the active site concentration. The background
rates (kuncat) were determined in the absence of antibody under otherwise
identical conditions.
zyl alcohol with a 10 µL injection of the reaction mixture. The
analytical HPLC was performed on a Hitachi L-6200 equipped with a
L-4000H UV detector, using a YMC ODS AM-303 analytical column
eluted with CH3CN/0.1% aqueous TFA (35:65) at a flow rate of 1.0
mL/min, with detection at 278 nm. The retention time of 4-nitrobenzyl
alcohol is 6.0 min. The initial velocities were determined from the
linear range of the rate. The observed rate was corrected for the
uncatalyzed rate of hydrolysis in the absence of antibody. The kinetic
parameters, kcat and Km, were determined by a linear least-squares fitting
of the Lineweaver-Burk plots described by the Michaelis-Menten
equation. The rate constants, kcats, were obtained by dividing by the
active site concentration. The background rates (kuncat) were determined
in the absence of antibody under otherwise identical conditions.
Determination of Enantiomeric Excess of Antibodies 7G12- and
3G2-Catalyzed Reactions. Reactions were initiated by adding 5 µL
of a 3 mM solution of racemic substrate 9 in DMSO to 95 µL of
antibody solution in 50 mM Tris (pH 8.0) at 25 °C. Final concentra-
tions: [racemic 9] 150 µM and [7G12] 20 µM (active site) or [3G2]
40 µM (active site). Hydrolysis rates were measured by HPLC
detection of 4-nitrobenzyl alcohol with a 5 µL injection of the reaction
mixture described above. The enantiomeric excess of the product acid
10 was determined by HPLC, with either a 20 µL (7G12) or a 30 µM
(3G2) injection of the reaction mixture, using a SUMICHIRAL OA-
3200 column eluted with MeOH containing 0.01 M ammonium acetate
at a flow rate of 1.0 mL/min, with detection at 254 nm. The retention
times of L-10 and D-10 are 15.0 and 13.0 min, respectively. In the
7G12-catalyzed reaction, after 137 min, the hydrolysis generated 59
µM of a 96% ee of L-10 without correction for the background reaction.
In the 3G2-catalyzed reaction, after 177 min, the hydrolysis generated
34 µM of a 94% ee of D-10 without correction for the background
reaction.
Competitive ELISA. Competitive ELISA was performed by using
BSA-2 and inhibitors, (R)-3 and (S)-3, as described previously.15,16,18
The Kd was determined by a Klotz plot.15
Acknowledgment. We would like to thank Dr. M. Ikehara
Active Site Concentration and Inhibition Assays (Activity Ti-
tration). The active site concentrations of antibodies 7G12 and 3G2
were determined by extrapolating from Henderson plots14 of the activity
titrations according to the fluorogenic assays with phosphonate (R)-3
and substrate L-9 for antibody 7G2 or (S)-3 and substrate D-9 for
antibody 3G2: [I]/(1 - V/Vo) ) [Ab] + Ki(1 + [S]/Km)Vo/V, where [I]
is the phosphonate concentration, V and Vo are, respectively, the
velocities in the presence and absence of the phosphonate, [Ab] is the
active site concentration, Ki is the inhibitor constant, [S] is the substrate
concentration, and Km is the Michaelis-Menten constant.
for encouragement and useful discussions.
Supporting Information Available: Figures of the fluo-
rescence assays, kinetics, and inhibition of the antibody-
catalyzed reactions and HPLC charts of the determination of
the enantiomeric excess of the hydrolysis product 10 (7 pages).
This material is contained in many libraries on microfiche,
immediately follows this article in the microfilm version of the
journal, can be ordered from the ACS, and can be downloaded
from the Internet; see any current masthead page for ordering
information and Internet access instructions.
HPLC Catalytic Assays and Kinetic Measurements. Reactions
were initiated by adding 10 µL of a stock solution of substrate 1 in
DMSO to 90 µL of antibody solution in 50 mM Tris (pH 8.0) at 25
°C. Hydrolysis rates were measured by HPLC detection of 4-nitroben-
JA952220W