Reactive immunization

The antibodies generated in this programme were initially found to accept a broad range of substrates including acetone, fluoroacetone, 2-butanone, 3-pentanone and dihydroxyacetone. The list has now been expanded to include 

While this example of the Robinson annulation is clearly not enantioselec-tive, the same antibody converts the mero-ketone [120] into the Wieland-Miescher (WM) decalenedione product kcM = 0.086 min-1 and Km = 2.34 mM at 25°C, parameters that give an impressive ER of 3.6 x 106. Good evidence suggests that the mechanism of the reaction involves the formation of a ketimine with the e-amino group of a buried lysine residue in the antibody, as shown in Fig. 39. Most significantly, the reaction delivers the ( )-(+)-WM product in 96% ee (by polarimetry) and in 95% ee by nmr and hplc analysis for a 100 mg scale reaction. A recent report tells that this antibody is to be made commercially available at a cost of 100 for 10 mg. The realization of that objective would mark the start of a new era of application of abzymes to organic stereoselective synthesis. 

Finally, the whole process of reactive immunization opens up the opportunity of using mechanism-based inhibitors as haptens, capable of actively promoting a desired mechanism by contrast to their conventional use as irreversible enzyme inhibitors. 

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Wirsching, P, et al., 1995. Reactive immunization. Science 270 1775-1783. Description of reactive immunization, in which a highly reactive compound is used as antigen. Antibodies raised against such an antigen show catalytic activity for tlie chemical reaction that the antigen undergoes. 

Like many other antibodies, the activity of antibody 14D9 is sufficient for preparative application, yet it remains modest when compared to that of enzymes. The protein is relatively difficult to produce, although a recombinant format as a fusion vdth the NusA protein was found to provide the antibody in soluble form with good activity [20]. It should be mentioned that aldolase catalytic antibodies operating by an enamine mechanism, obtained by the principle of reactive immunization mentioned above [15], represent another example of enantioselective antibodies, which have proven to be preparatively useful in organic synthesis [21]. One such aldolase antibody, antibody 38C2, is commercially available and provides a useful alternative to natural aldolases to prepare a variety of enantiomerically pure aldol products, which are otherwise difficult to prepare, allovdng applications in natural product synthesis [22]. 

Scheme 2. Reactive immunization using a 1,3-diketone as hapten. (R (= -(CH2)3CO-) makes the connection to the carrier protein).

Figure 17 Generation, by the reactive immunization strategy, of antibody SP049H that catalyzes the hydrolysis of phosphonate diester 24 and that of ester 25.

Figure 19 A success of the reactive immunization strategy. Aldolization reaction catalyzed by antibody 38C2 raised against a /3-, 3-diketone hapten.

In order to improve aldolase antibodies, Zong et al employed reactive immunization in combination with transition state theory. Based on hapten 30, a hybrid, hapten 31, was designed, recruiting not only a sulfone... 

Antibody Catalysis. Recent advances in biocatalysis have led to the generation of catalytic antibodies exhibiting aldolase activity by Lemer and Barbas. The antibody-catalyzed aldol addition reactions display remarkable enantioselectivity and substrate scope [18]. The requisite antibodies were produced through the process of reactive immunization wherein antibodies were raised against a [Tdiketone hapten. During the selection process, the presence of a suitably oriented lysine leads to the condensation of the -amine with the hapten. The formation of enaminone at the active site results in a molecular imprint that leads to the production of antibodies that function as aldol catalysts via a lysine-dependent class I aldolase mechanism (Eq. 8B2.12). 

G. Zhong, R. A. Lerner, and C. F. Barbas III, Enhancement of the repertoire of catalytic antibodies with aldolase activity by combination of reactive immunization and transition state theory, Angew. Chem. Int. 

The use of reactive immunization to generate catalytic antibodies (or abzymes) that catalyze aldolase reactions has been described, offering additional utility for this synthetically useful transformation.260 Two such abzymes, 38C2 and 84G3, are available commercially and their respective, diverse activities have been described.261-262... 

C. 84G3 Combining TSA and Reactive Immunization 1. Hapten Design... 

Reactive immunization is a fundamentally different approach to selecting antibody pockets that contain functional groups. This method employs mechanism-based inhibitors as haptens these molecules react covalently with appropriately functionalized antibodies, allowing direct selection of active clones from large pools of inactive variants. When a suitable substrate is used in place of the inhibitor, reactive residues in the selected antibodies can often mediate its conversion into product. 

Aldolase antibodies obtained by reactive immunization are notable for high activity, broad substrate specificity, and high selectivities [53]. Rate accelerations are typically in the range 105 to 107-fold over background. Although the k /K values are 102 to 104 lower than those of aldolase enzymes, these are among the most efficient antibody catalysts described to date. Their efficacy is all the more notable in light of the inherently complex, multistep process they catalyze. 

Scheme 4.8 Hapten 17, designed to combine transition state mimicry and reactive immunization strategies, produced an aldolase antibody (84C3) that promotes aldol reactions with typically higher rates and selectivitiesthan antibodies raised against 15. The retro-aldol reaction of 18 is catalyzed with notable efficiency by this antibody.

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