Transition-state analogues, selection

Many of the 60 known reactions catalyzed by monoclonal antibodies involve kinetically favored reactions e.g., ester hydrolysis), but abzymes can also speed up kinetically disfavored reactions. Stewart and Benkovic apphed transition-state theory to analyze the scope and limitations of antibody catalysis quantitatively. They found the observed rate accelerations can be predicted from the ratio of equilibrium binding constants of the reaction substrate and the transition-state analogue used to raise the antibody. This approach permitted them to rationalize product selectivity displayed in antibody catalysis of disfavored reactions, to predict the degree of rate acceleration that catalytic antibodies may ultimately afford, and to highlight some differences between the way that they and enzymes catalyze reactions. 

Despite the trigonal anomeric center and the consequential 4H3 conformation, both inhibitors are too weak to constitute transition-state analogues, making them good examples among glycosidases as protonation trajectory-selective probes for in-plane... 

Fig. 6.1. Summary of methods for selecting phage-displayed enzymes on die basis of catalytic activity (S substrate P product SS suicide substrate Bt biotin SV streptavidin TSA transition state analogue).

A transition-state analogue for an acetal hydrolysis has been used to select and amplify the production of a macrocycle from a dynamic combinatorial library of disulfides in water. The macrocycle gives a modest acceleration of the acetal hydrolysis reaction.6... 

Selection by affinity chromatography on a transition-state analogue has also been used to extract active enzymes from a library of phage-displayed GST Al-1 mutants. Four residues in the electrophilic substrate binding site were randomly mu-... 

Fig. 5.4. Phage-displayed glutathione-S-transferase mutants were selected with an immobilised transition-state analogue [51].

Selection with transition-state analogues has also been used to extract catalytic antibodies from libraries of phage-antibodies. McCafFerty et al. [52] have shown that antibodies with nanomolar affinities for a phosphonate transition-state analogue could easily be extracted from a library derived from immunized mice. 

Antibodies possessing glycosidase activity have been induced by immunization of mice with conjugates of the transition-state analogue 21 (Fig. 5.10A). A combinatorial phage-Fab library was then constructed by PCR amplification of the heavy and light chains. Selection with a BSA-conjugated suicide inhibitor 22 (Fig. 5.10B) was then... 

The selection techniques described above are indirect as they rely on inhibitors. They have important limitations. Transition-state analogues are frequently unable to mimic adequately the essential features of true transition states. For many enzymatic activities, there are simply no transition-state analogues or suicide substrates. 

Figure 10.19 Catal)Tic antibodies structures of the transition state analogue/selection substrate (10.9), the catalyzed reaction substrate (10.10) and product (10.11), and a modified poor substrate for the esterol)dic antibody Mab 17E11.

Figure 10.21 Direct selection of catal)Tic antibodies from phage libraries the selection/ann-plification process to an improved stereoselective hydrolytic antibody from the library LIO using the transition state analogue/selection substrate 10.17.

A similar approach was employed in our laboratory for the development of molecularly imprinted catalysts for the enantio-selective reduction of prochiral ketones with borane (CBS reaction) [122]. A stable, polymerisable transition state analogue of this reaction leading to the formation of one particular enantiomer product was prepared (Fig. 4.9). After polymerisation, the template molecule can... 

Fig. 4.9. Enantio-selective reduction of prochiral ketones using oxazaborolidines as catalysts. The transition state of the reaction and a stable transition state analogue are represented.

For the production of an effective monoclonal antibody it is crucial to raise it from a well-designed transition state analogue. Besides not resembling the product too much in order to avoid product inhibition, this hapten should mimic the transition state as closely as possible to maximize its stabilization by the antibody generated. Utilizing the extraordinary specificity of the immune system by this means, the combining site of the antibody is programmed to exhibit a specific catalytic activity. This technique has been directed toward the development of selective reactions which are chemically difficult to achieve, for example disfavored , and for which no suitable enzyme is available. 

G. E. Whitworth, M. S. Macauley, K. A. Stubbs, R. J. Dennis, E. J. Taylor, G. J. Davies, I. R. Greig, and D. J. Vocadlo, Analysis of PUGNAc and NAG-thiazoline as transition state analogues of human O-GlcNAcase Mechanistic and structural insights into inhibitor selectivity and transitions state poise,... 

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