Aldolase catalytic antibody

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]. 

The development of the concept of reactive immunization yielded more effective antibody aldolases.119-120 In this new approach, rather than raise antibodies against an unreactive hapten designed to mimic the transition state, the antibodies were raised against a reactive moiety. Specifically, a p-diketone that serves as a chemical trap to imprint a lysine residue in the active site of the Ab (Scheme 5.65) was used.340 A reactive lysine is a requirement of the type I aldolase mechanism. By this method two aldolase catalytic antibodies, 38C2 and 33F12 were identified.119... 

Scheme 1.2 Fluorogenic substrates for aldolase catalytic antibodies and related biocatalysts.

C.F. (1995) Efficient aldolase catalytic antibodies that use the enamine mechanism of natural enzymes. Science, 270,1797-1800. 

J.L. (1998) A stereoselective fluorogenic assay for aldolases detection of an anti-selective aldolase catalytic antibody. Tetrahedron Letters, 39, 9415-9418. 

Zhong G, Lemer RA, Barbas CF 111. Broadening the aldolase catalytic antibody repertoire by combining reactive immunization and transition state theory new enantio- and diastereoselectivi-ties. Angew. Chem. Int. Ed. Engl. 1999 38(24) 3738-3741. Schowen RL. The elicitation of carboxylesterase activity in antibodies by reactive immunization with labile organophos-phorus antigens a role for flexibility. J. Immunol. Methods 2002 269(l-2) 59-65. 

Mu YQ, Gibbs RA. Design and synthesis of chiral and racemic phosphonate-based haptens for the induction of aldolase catalytic antibodies. Biooig. Med. Chem. 1997 5(7) 1327—1337. 

Wagner, J. Lemer. R.A. Barbas. C.F.. III. Efficient aldolase catalytic antibodies that use the enamine mechanism of natural enzyme. Science 1995. 270. 1797-1800. Arnold. F.H. Combinatorial and computational challenges for biocatalyst design. Nature 2001, 409. 253-257. Penning, T.M. Jez, J.M. Enzyme redesign. Chem. Rev. 2001. 101. 3027-3046. 

Wagner, J., Lerner, R. A., and Barbas, C. F., Ill, 1995. Efficient adolase catalytic antibodies that use tlie enamine mechanism of natural enzymes. Science 270 1797-1800. See also tlie discussion entitled Aldolase antibody in Science 270 1737. 

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... 

The availability of 38C2 as a broad scope, enantioselective, efficient aldolase enzyme has had a significant impact on organic synthesis. Some of the molecules we have synthesized with 38C2 include the natural products ( + ) —frontalin [( + )— 27] (List et al., 1999), some brevicomins [( —) —28 and (—) —29] (List etal., 1998a), epothilones A (30) and C (31) (Sinha et al., 1998), and the Wieland-Miescher ketone [( ) — ( + )—32] (Hoffmann et al., 1998 Zhong et al., 1997). The brevicomin examples represent the first use of a catalytic antibody to decrease the total number of synthetic steps and increase the enantioselectivity of natural product syntheses. 

Furthermore, the catalytic efficiency (K t/KM) of 84G3 for this substrate, 3.3 X 105 s-1M-1, is comparable with the efficiency of natural muscle aldolase, 4.9 X 104 s 1M 1, in the retro-aldolization of its substrate fructose 1, 6-bisphosphate (Morris and Tolan, 1994). However, these two enzymes use different substrates, and the rates were recorded at different temperatures (22°C for 84G3, 4°C for the natural aldolase). Despite this, we believe that it will be possible to develop a catalytic antibody that, under identical conditions, has a faster cat and lower KM than a natural enzyme for the same substrate. 

In theory, the programmable stereoselectivities of catalytic antibodies makes them well suited for asymmetric synthesis. Several such transformations have been carried out on a preparative scale. Kinetic resolution of the epothilone precursor 19 with the aldolase antibody 38C2 is instructive (Scheme 4.9) [57]. The reaction proceeds in good yield (37 %) and high enantiomeric excess (90 %). However, so much catalyst is needed (0.5 g of IgG antibody was used for the resolution of 0.75 g 19) that large-scale production is likely to be impractical in many cases. As most antibody catalysts are much less efficient than the aldolases, catalyst costs will generally be appreciable. 

Prior to the determination of the aldolase mechanism and the development of catalytic antibodies for the aldol reaction, Hajos and Parrish and independently Wiechert et al. discovered that (5)-proline catalyzes the intramolecular aldol reaction of cyclic triketones (Scheme 6.7). This is not only a catalytic effect the reaction proceeds with high yields and large enantiomeric excess. 

Perhaps the most significant contribution of the catalytic antibody field is the realization that enzyme catalysis is not simply transition state stabilization. Reactive immunization has enabled a mimic of the dynamics involved in enzyme catalysis, and an aldolase antibody that approaches enzymatic rates has been developed using this technique (59, 60). However, only a few types of reactions have been catalyzed using this hapten... 

In an effort to induce catalytic residues during immunization, Lerner et al. reported that efficient catalytic antibodies can be generated by a process called reactive immunization . In this method a chemically reactive hapten is used to create a covalent bond with a catalytic residue in the antibody binding pocket, thereby allowing its selection and amplification in the course of immunization. Aldolase antibodies were obtained using a reactive 1,3-diketone, which formed a covalent bond with a lysine residue in the antibody binding pocket (see Sect. 2.8). A similar experiment yielded esterase antibodies by immunization... 

Antibodies can be utifized under a variety of non-physiological conditions and are excellently suited for preparative applications due to their intrinsic stability. We have found that antibodies catalyzing the retro-Diels-Alder reaction of 63 function equally well between pH 4 and pH 11. Aldolase antibody 72D4 operates in the presence of 10% acetone. Janda et al. have used an immobilized esterase antibody with up to 40% dimethylsulfoxide [110]. Esterase catalytic antibodies have been used in reverse micelles and in lipid-coated form to transform lipophilic substrates [111]. Catalytic antibodies can also be used in a biphasic alkane/water system [112]. The lipophilic substrate remains in the alkane phase where it does not undergo any reaction, which suppresses any uncatalyzed reaction. In case that the reaction product is still lipophilic and returns to the alkane phase, product inhibition is also suppressed under these conditions. 

Most antibody-catalyzed reactions are hydrolytic processes, which are highly exergonic in aqueous environment. / -Elimination of /J-hydroxyketones to form a,/ -unsaturated ketones is a rare case of a dehydration reaction proceeding exothermically in aqueous environment. Catalytic antibodies promoting -elimination of /1-hydroxy and /1-halo-carbonyl compounds have been described [52]. The most interesting from a synthetic viewpoint concerns compound 30, which can be dehydrofluorinated to Z-olefin 32 using catalytic antibody 1D4 raised against hapten 29, while the uncatalyzed reaction exclusively yields the more stable E-olefin 31 (Scheme 11) [53]. Dehydratase activity also arises in aldolase antibodies and will be discussed below in that context. 

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