Chemistry Reference
In-Depth Information
Scheme 4.5 Cationic iminium ion 14 was used to generate
antibody 4B2, which has an active site carboxylate at L34 and also
promotes the decomposition of benzisoxazoles shown in
Scheme 4.4.
also accelerates the decomposition of benzisoxazoles [43], using a glutamate as the
catalytic base [44]. However, despite hydrophobic surroundings and an elevated
pK
a
of 5.8, this residue is two to four orders of magnitude less proficient at proton
abstraction than Glu
H50
in 34E4 [39]. Bearing little resemblance to the amidinium hap-
ten, the 5-nitrobenzisoxazole substrate presumably binds at the relatively spacious 4B2
binding pocket [44] in various orientations that are suboptimal for proton transfer.
The properties of 34E4 are impressive, demonstrating that functional groups can be
exploited to excellent effect in antibody binding pockets. In fact, as judged by its EM,
the catalytic carboxylate appears to be as effective at proton abstraction as analogous
groups in highly evolved natural enzymes [39, 45]. Nevertheless, the overall efficiency
of the antibody is still several orders of magnitude lower than enzymes such as triose
phosphate isomerase, which also promote proton abstractions. In contrast to its nat-
ural counterparts, 34E4 relies on only one functional group, namely Glu
H50
, for cat-
alysis. Calculations suggest that provision of an additional residue to stabilize the de-
veloping negative charge at the leaving group might augment activity by several orders
of magnitude [46]. Unfortunately, initial attempts to introduce such a residue by site-
directed mutagenesis have been unsuccessful [39].
Construction of active sites containing several functional groups remains a signifi-
cant challenge for antibody catalysis. The likelihood of eliciting multiple catalytic re-
sidues spontaneously is vanishingly small. Likewise, engineering additional groups
into existing pockets, as seen with 34E4, has met with little success. No doubt this
explains a relative dearth of good antibody catalysts for energetically demanding reac-
tions, such as amide or glycoside hydrolysis. Heterologous immunization with two
different but structurally related haptens, each containing a different charged moiety,
was developed to address this problem [47]. Promising results with antibody esterases
were reported [48], but catalysts with truly enzyme-like activities have yet to emerge.
4.6
Covalent Catalysis
Reactive immunization is a fundamentally different approach to selecting antibody
pockets that contain functional groups. This method employs mechanism-based in-
hibitors as haptens; these molecules react covalently with appropriately functionalized
antibodies, allowing direct selection of active clones from large pools of inactive var-
iants. When a suitable substrate is used in place of the inhibitor, reactive residues in
the selected antibodies can often mediate its conversion into product.
This strategy has yielded catalysts for several reactions. For example, phosphonylat-
ing agents have been used to generate antibodies that promote ester hydrolysis by a
