10466 J. Am. Chem. Soc., Vol. 121, No. 45, 1999
Datta et al.
6.9 Hz, 2H, CH2), 1.83 (m, 4H, CH2), 1.75 (dd, J2 ) 19.0 Hz, J )
HP
7.3 Hz, 3H, CH3); 13C NMR (125 MHz, CDCl3) δ 173.37, 166.11,
162.39, 157.11, 136.12, 133.84, 133.81, 132.93, 132.87, 132.75, 129.24,
128.97 (d, J2 ) 3.6 Hz), 128.58, 128.56, 128.21, 127.23, 127.18,
CP
127.08, 126.96, 119.22, 105.55, 66.35, 44.58, 44.31, 43.71 (d, J1
)
CP
27 Hz), 33.91, 28.69, 21.75, 17.75 (d, J2 ) 6.5 Hz); 31P NMR (101
CP
MHz, CDCl3) δ 23,38; HRFABMS calcd for C25H29O8PS (M)+
520.1321, obsd 520.1323.
Antibody Production and Purification. As described previously.11
Kinetics. (a) Screening for Catalytic Antibodies. The screening assays
were performed in a Bicine (100 mM, pH 8.0) 1% Tween 80 buffer
system with 4% DMF as cosolvent. The reaction was followed by
monitoring formation of enantiomeric naproxen acids S-(+)-4a and
R-(-)-4b by reversed phase HPLC at 254 nm using a C-18 VYDAC
201TP54 column with an isocratic mobile phase [57% water (0.1%
TFA), 43% acetonitrile at 1 mL min-1]. The reaction was initiated by
addition of the substrate rac-3a, as a solution in DMF (7.5 mM, 20
µL) into a buffer solution with or without antibody (0 µM e [Ab] e
20 µM; 480 µL). At time periods throughout the assay, an aliquot of
the reaction mixture was removed (30 µL) and quenched into a solution
of the external standard (4-methyl-3-nitroanisole) in water (0.1% TFA)/
acetonitrile (1:1; 30 µL). The product concentrations were determined
by comparison with product standards.
(b) Kinetic Parameters. The buffer system and HPLC assay were
the same as described Vide supra. To determine the kinetic parameters,
the individual enantiomers either S-(+)-3b or R-(-)-3c were used as
substrates over a range of concentrations up to at least 3 × Km where
possible (1-900 µM) in DMF with an antibody concentration of 0.01-
1.5 µM in the aqueous buffer system. The reaction was followed for
no more than 5% of the reaction, and as described Vide supra for only
1-2% of the reaction for the transition state analogue antibodies at
high substrate concentrations, during which time the progress curves
were linear. Kinetic parameters were calculated using a combination
of nonlinear regression and Lineweaver-Burke analyses of the raw
data with the EnzymeKinetics v1.1 computer program, Trinity Software
(copyright 1990-1991).
(c) Inhibition Constant, Ki Determinations. The Ki values of
inhibitors 7 and 8 and phenol 9 (6G6) were determined from their ability
to inhibit the hydrolysis of S-(+)-3b by each member of the reactive
immunization and transition state analogue antibody libraries. Because
of the problems associated with measuring tight-binding inhibitors
where [I] ≈ [Ab], the assays were performed so as to measure the
inhibitor concentration required to reduce the enzyme activity to half
its original value (IC50) over a broad range of inhibitor concentrations.25
The antibodies (at concentrations suitable to give good linear rate data)
were preincubated with the inhibitor 8 ([6G6] ) 30 nM; 1-100 nM 8
and 1-200 µM 9, [9B1] ) 100 nM; 10-1,000 nM 8, [12C8] ) 40
nM; 1-100 nM 8, [12D9] ) 40 nM; 1-100 nM 8, [11C10] ) 500
nM; 100-5000 nM 8) for 20 min at room temperature. The assay was
started by addition of the substrate S-(+)-3b (either 1.1 × Km, where
possible, or 800 µM) and followed by the HPLC assay described Vide
supra. The Ki was then determined from the IC50 by incorporation into
eq 1.25
(d) Product Inhibition Determination. The effect of phenol 9 (50
µM) and carboxylate 4a (50 mM) on the catalytic rate of the reactive
immunization and transition state analogue antibodies were studied at
one concentration of S-(+)-3b (200 µM). In a typical assay the
antibodies (0.01-1.5 µM) in the aqueous buffer solution vide supra
(480 µL) were preincubated with either the phenol 9 or acid S-(+)-4a
(2.5 mM in DMF; 10 µL) for 15 min at room temperature. The reaction
was initiated by addition of S-(+)-3b (10 mM in DMF; 10 µL). The
rates were measured as described above.
(e) Equilibrium Binding Constant, Kdapp Determinations. Bind-
ing constants were measured in a competition enzyme-linked immu-
nosorbent assay.36 Polystyrene 96-well plates (Costar) were coated
overnight at 4 °C, with a solution of the BSA-5 or BSA-6 conjugate
(25 µL of 5 µg mL-1) in PBS (50 mM, pH 7.4). The plates were then
blocked with Blotto [4% dry nonfat milk in PBS (50 mM, pH 7.4); 50
Figure 3. Antibody, 12C8, catalyzed kinetic resolution of rac-3a. The
two lines represent formation of (S)-(+)-naproxen 4a. The lower line
represents percent conversion up to the maximum (50%). The top line
is percent ee. Conditions: rac-3a, 600 µM, 12C8 (0.5 µM), pH 8.0
(100 mM bicine), 4% DMF, 1% Tween-80, 22 °C.
catalysts with wide potential in biochemistry and organic
synthesis.35 This paper presents the first direct comparison of
reactive immunization and transition state analogue hapten
manifolds for the elicitation of antibody catalysts. It has revealed
that the antibodies that can be generated by each strategy, even
for the same reaction, exhibit quite different catalytic behavior.
This study has shown that the transition state analogue approach
has furnished better biocatalysts in terms of turnover numbers
and enantiomeric discrimination, but which possess varying
degrees of product inhibition by the phenol 9. Thermodynamic
evaluation reveals that their catalytic power is derived almost
entirely as a function of differential stabilization of the transition
state over the ground state. In comparison, reactive immuniza-
tion has elicited biocatalysts which are ultimately more proficient
because they couple an efficient “catalytic” mechanism and
improved substrate recognition and furthermore do not suffer
from product inhibition. Of course this primary study compares
only ten antibodies, albeit the five most active from each
strategy. Whether the benefits and limitations of each hapten
strategy highlighted in this study will prove to be general can
only be revealed by further experimentation and comparison in
this and other reaction classes.
Experimental Section
General Procedures (Synthesis). As described previously.11
Phosphonic Acid Hapten 6. Phosphonate diester 5 (16 mg, 0.02
mmol) was dissolved in THF (2 mL), to which was added LiOH(aq)
(0.2 mL, 0.1 M). The reaction mixture was stirred at room temperature
for 2 h, then neutralized to pH 3 with 1 N HCl and partitioned with
ether. The organic layer was collected and washed with brine, dried
(MgSO4), and evaporated to dryness to give the title compound 6 as a
1
white solid (14 mg, 98%): mp 207 °C; H NMR (400 MHz, CDCl3)
δ 7.90 (d, 2H, J ) 7.5 Hz, Ar-H), 7.67 (m, 3H, Ar-H), 7.36 (d, J 7.5
Hz, 1H, Ar-H), 7.05 (m, 2H, Ar-H), 6.90 (dd, J2 ) 7.8 Hz, Ar-
HP
H), 4.25 (t, J 5.6 Hz, 2H, CH2), 3.81 (dq, 1H, J2HP ) 14.5 Hz, J ) 7.5
Hz, CHP), 3.01 (s, 3H, SO2CH3), 2.91 (s, 3H, SO2CH3), 2.45 (t, J )
(35) Janda, K. D.; Benkovic, S. J.; McLeod, D. A.; Schloeder, D. M.;
Lerner, R. A. Tetrahedron 1991, 47, 2503-2506.
(36) Harlow, E.; Lane, D. P. Antibodies. A Laboratory Manual; Cold
Spring Harbor Laboratory: Cold Spring Harbor, NY, 1988.