29G12 Antibody Catalyzed 1,3-Dipolar Cycloaddition Reaction
TABLE 2. Temperature Dependence of the
the lower polarity transition state relative to the ground
state, programmed by hapten 3b, coupled with the
fortuitous potential for hydrogen bonding at the amide
2
9G12-Catalyzed, kcat, and Noncatalyzed, kuncat,1,3-DPC
Reaction between 1a and 2m
T/K
kcat/s-
1
kuncat × 10 /M s-1
3
-1
carbonyl carbon of 2m may well be assisting the 29G12
,
268
277
282
288
297
0.13 ( 0.02
0.22 ( 0.02
5.67 ( 0.5
13.3 ( 0.1
19.1 ( 0.2
31.0 ( 0.1
50.6 ( 0.4
mechanism. These effects, both of which are enthalpic
in nature, will destabilize the entropy of the process by
necessitating solvent ordering and restriction of antibody
binding-site residues, respectively, in the transition state.
a
n.d.
0.36 ( 0.03
a
n.d.
q
The clear effect of enthalpic stabilization (∆∆H 4.4
a
Not determined.
-1
kcal mol ) by 29G12 as a route to catalysis, coupled with
q
a net increase in the entropy (∆∆S 10.4 eu) of activation,
is clearly counterintuitive to the notion of how one might
anticipate a biocatalyst evolving to catalyze a bimolecular
reaction. However, 29G12 clearly exhibits a preference
for hydrophobic dipolarophiles. Therefore, the catalytic
process seems to involve a desolvation phenomenon, i.e.,
the antibody is partitioning the hydrophobic dipolaro-
philes from the aqueous buffer system into the favorable
environment of the binding site, thus having the positive
effect on enthalpy. Desolvation has previously been
More interestingly, when the larger dipolarophile 2m
is a cosubstrate with 1a, 29G12 actually lowers the K
m
of 1a from 3.4 mM to 0.8 mM. Such an increase in sub-
strate affinity has evolved as a regulatory mechanism for
activation of enzymes in vivo but has not previously been
observed with catalytic antibodies. Generally, in the case
of enzymes, the increase in substrate affinity is precipi-
tated by binding of the “activator” molecule to an allos-
teric site, leading to a structural reorganization of the
q
shown to account for the reduction in ∆∆H in antibody-
active site as is the case with protein kinase C δ1 acti-
vation by free fatty acids binding to a peripheral site.29
catalyzed unimolecular elimination and decarboxylation
processes.3
1,32
However, in the end, the composite char-
However, in the case of 29G12, it seems that a reorga-
nization of the combining site may be occurring upon
binding of 2m leading to an increase in the affinity of
acter of these quantities and the probable role of solvation
effects in determining them exclude drawing more firm
conclusions.
2
9G12 for 1a and presumably vice versa because the
Conclusions. A substrate tolerance study of the
9G12-catalyzed enantioselective 1,3-dipolar cycloaddi-
kinetics of the reactions supports completely random, se-
quential, bi-uni kinetic mechanism. This improved bind-
ing of 1a with 2m suggests that “substrate-initiated bind-
ing site reorganization” may be an unrecognized approach
to improving the catalytic efficiency of multisubstrate
catalytic antibodies. The ability of protein receptors to
be able to reorganize hydrophobic domains to facilitate
2
tion reaction has revealed the presence of an unoptimized
pocket that accepts a range of bulky hydrophobic dipo-
larophiles. Steady-state kinetic parameters with the most
efficient cosubstrate, 2m, revealed the remarkable phe-
nomena that the affinity of 29G12 for the larger dipo-
larophile 2m is much higher than for its native dipolaro-
phile 2a and that when 2m is a cosubstrate, the affinity
of 29G12 for dipole 1a is also increased. This “substrate
optimization” suggests that there is significant reorga-
nization occurring within the active site of 29G12 during
the catalytic process. A thermodynamic analysis of the
binding of hydrophobic ligands is now being recognized
from SAR analyses of drug-receptor interactions.30
Temperature Dependence of the 29G12-Cata-
lyzed Process. To determine the relative importance of
entropic and enthalpic stabilization to the catalysis of this
bimolecular reaction, the temperature dependence of the
2
9G12-catalyzed process offers support for this assertion
2
9G12-catalyzed and -uncatalyzed 1,3-DPC reactions
between 1a and 2m were investigated. The steady-state
kinetic parameters K (1a), K (2m), and kcat for the
9G12-catalyzed process and the uncatalyzed rate con-
stant kuncat between -5 and 24 °C are compiled in Table
. An Arrhenius analysis of the temperature dependence
of kuncat and kcat reveals that the activation parameters
with there being a significant increase in the entropy of
activation coupled with a decrease in enthalpy of activa-
tion. The nature of the structural reorganization of the
antibody combining site will be ultimately assessed using
X-ray crystallography.
m
m
2
2
Acknowledgment. We thank the NIH (GM43858
to K.D.J.) and The Skaggs Institute for Chemical
Biology for financial support. J.D.T. acknowledges the
FCAR Qu e´ bec for a predoctoral fellowship. P.W. thanks
all members of the Scripps Hybridoma Laboratory for
antibody production and purification and Asher Shafton
and Sangheetha Tripurenani for resynthesis and analy-
sis of compounds 5a-t.
q
-1
for the uncatalyzed reaction are ∆H ) 11.5 kcal mol
q
and ∆S ) -25.1 eu and the antibody-catalyzed reaction
are ∆H ) 7.1 kcal mol and ∆S ) -35.5 eu in aqueous
buffer [50 mM N-morpholinoethanesulfonic acid (MES),
q
-1
q
1
50 mM NaCl, pH 6.5] (Figure 5).
Clearly, the antibody functions by lowering the en-
q
-1
thalpy of activation (∆∆H 4.4 kcal mol ) rather than
the anticipated effects on entropy. This observation is in
line with the observed enthalpy dependence of the 29G12-
catalyzed reaction with dipolarophile 2a11 and supports
the notion that, while the observed rate of the reaction
for 2m is significantly higher than 2a under identical
conditions, the mechanism of catalysis appears to be
analogous. Thus, antibody-binding and stabilization of
Supporting Information Available: All synthetic infor-
mation for dipolarophiles (2a-t) and isoxazolines (5a-t),
2
9G12 antibody production and purification methods, kinetics
assay methods, and X-ray structural information of R-5m. An
X-ray crystallographic file (CIF). This material is available free
of charge via the Internet at http://pubs.acs.org.
JO050410B
(
31) Hsieh, L. C.; Stephans, J. C.; Schultz, P. G. J. Am. Chem. Soc.
(
29) Kobayashi, M.; Kannan, M. R.; Feng, J.; Roberts, M. F.;
Lomasney, J. W. Biochemistry 2004, 43, 7522.
30) Bohm, H.-J.; Stahl, M. Med. Chem. Res. 1999, 9, 445.
1994, 116, 2167.
(32) Grate, J. W.; McGill, R. A.; Hilvert, D. J. Am. Chem. Soc. 1993,
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J. Org. Chem, Vol. 70, No. 20, 2005 7815