Aldolase antibody

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. 

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 most interesting developments involve catalysis of simple aldol reactions. The key to reactive immunisation is the use of a hapten that is chemically reactive, rather than a passive template. This means that (i) relevant chemistry is going on during the course of antibody induction, which thus happens in the presence of intermediates involved in the reaction, and so may be modified to favor the formation of antibodies which bind these intermediates (and perhaps transition states leading to them). Furthermore (ii) it becomes possible to select for antibodies that react with, rather than just bind, to the hapten. The system used for the development of aldolase antibodies is outlined in Scheme 2... 

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... 

On the basis of encouraging work in the development of L-proline-DMSO and L-proline-ionic liquid systems for practical asymmetric aldol reactions, an aldolase antibody 38C2 was evaluated in the ionic liquid [BMIM]PF6 as a reusable aldolase-ionic liquid catalytic system for the aldol synthesis of oc-chloro- 3-hydroxy compounds (288). The biocatalytic process was followed by chemical catalysis using Et3N in the ionic liquid [BMIM]TfO at room temperature, which transformed the oc-chloro-(3-hydroxy compounds to the optically active (70% ee) oc, (3-epoxy carbonyl compounds. The aldolase antibody 38C2-ionic liquid system was also shown to be reusable for Michael additions and the reaction of fluoromethylated imines. 

Notes Chiral preparations include the proline-catalyzed reactions2 and recently an aldolase antibody 38C2 method has been reported.3 See also 4... 

III. Directing Evolution at the Level of Chemical Mechanism Aldolase Antibodies and Asymmetric Catalysis... 

It soon became clear that aldolase antibody 38C2 catalyzed the aldol reaction with both efficiency and broad scope. For example, compounds 7 to 13 could all be synthesized in antibody 38C2 catalyzed aldol reactions (Hoffmann et al, 1998). 

We routinely use these substrates for kinetic characterizations of new aldolase catalysts. However, they are not suitable for cell-based screenings because both substrate and product are readily cell permeable. The solution to this problem came when we discovered that our aldolase antibodies catalyze the -elimination (or r ro-Michael reactions) of (3-hetero substituted ketones 37 [Scheme 8 (1)]. 

In our original hapten design for aldolase antibodies, the /3-diketone functionality of hapten 4 was used as a reactive immunogen to trap a chemically reactive lysine residue in the active site of an antibody as a stable enaminone. The chemical mechanism leading up to the stabilized enaminone should match that of Class I aldolases over this portion of the reaction coordinate. 

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. 

Despite the absence of stereochemical information in the reactive immunogen, the aldolase antibodies promote carbon-carbon bond formation with surprisingly high selectivity. For instance, the enamine formed from acetone adds to the si face of various aldehydes with ee s in excess of 95% [53], In other examples, Robinson annula-tions have been carried out with high enantioselectivity [55], tertiary aldols and other compounds have been successfully resolved [56], and enantiopure intermediates have been prepared for the synthesis of various natural products [57, 58]. 

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.

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. 

Since the preparation of enantiomerically pure tertiary aldols remains a challenge, aldolase antibody 38C2 was investigated as a catalyst for the kinetic resolution of racemic tertiary aldols. Ab38C2 was demonstrated to be an efficient catalyst for the retro-aldol reaction of the fluorogenic tertiary aldol /m-methodol (Scheme 5.68) and exhibited an E value of >159 50. At 50% conversion, (R)-terMnethodol is obtained with an enantiomeric excess of >99% ee. Consequently the ability of Ab38C2 to resolve tertiary alcohol was exploited in the enantioselective synthesis of ( )-frontalin (Scheme 5.69).125... 

ALDOLASE ANTIBODY 38C2 PROMOTED ALDOL REACTION... 

Although the use of enzymes in ionic liquids has been explored, the enzymatic carbon-carbon bond forming the reactions in ionic liquids has not been studied in detail. Since aldol reactions catalyzed by the aldolase antibody 38C2 in buffer... 

A proposed mechanism for the Michael addition reaction is shown in Scheme 10.7. Note that enamine, generated from the reaction of hydroxyacetone and aldolase antibody 38C2, reacts with the activated methylene group in 2-(phenyl)ethyl-2-(tri-fluoromethyl)acrylate. 

---