Ribonuclease cyclodextrin model

Breslow s P-cyclodextrine ribonuclease model system represents one of the best examples concerning the construction of small enzyme-like molecules [33]. Breslow functionalized the P-cyclodextrine with two imidazole moieties (Figure 10.1). Selectively, catechol cyclic phosphate carrying a 4-tert-butyl group (Figure 10.1a) binds into the cavity of the catalyst (Figure 10. lb) in water solution, and is then hydrolyzed by the... 

With the existence of this new cyclodextrin lock, it was again important to select a key to fit it and to serve as substrate. For this we wanted a cyclic phosphate ester that this cyclodextrin bisimidazole could hydrolyze. The enzyme ribonuclease hydrolyzes cyclic phosphates as the second step in the hydrolysis of RNA, and cyclic phosphates are used as assay substrates for the enzyme. The advantage to us of such a substrate was that the geometry of the transition state would be relatively well-defined, so that it should be possible to design congruence between the catalyst and the transition state. Molecular model building indicated that a possible substrate was the cyclic phosphate derived from 4-f-butylcatechol (VIII). Indeed, the cyclodextrin bisimidazole (VII) is a catalyst for the cleavage of cyclic phosphate (VIII) (14). 

It will be of some interest to learn how to build catalysts to handle the particular substrates that natural enzymes cleave, at a rate comparable to the rates of those enzymatic reactions. However, one of the aims of biomimetic chemistry is to extend the kinds of rates and selec-tivities of enzymatic reactions into reactions for which natural enzymes have not been optimized and to substrates that are neither recognized nor handled by normal enzymes. It is clear that we already have achieved this, even though our ribonuclease model system has some distance to go before it can approach the kinds of rates we have observed in the cyclodextrin ferrocinnamate ester reaction, for instance. In lock and key chemistry, the keys that fit artificial enzymes best are not the same as the keys that open the natural enzyme locks. 

By the covalent addition of two imidazole groups to the cyclodextrin torus, artificial ribonucleases have been formed, and these have notable specificity with respect to both substrates and products (Breslow, 1983). Similar accelerations and specificities were found by coupling pyridoxamine covalently to a cyclodextrin to give an artificial transaminase. The accelerations produced by these models are only about 200-fold, but work in progress holds out hope for obtaining far greater velocities (Breslow, 1983). 

We applied this test to our ribonuclease mimic, the 6A,6B isomer of cyclodextrin bisimidazole, cleaving the bound cyclic phosphate 21. We found that there was indeed a square dependence of the kinetic isotope effect on the mole fraction of deuterium in the water solvent, and interestingly the values of the isotope effect for the two protons in flight were almost identical with those that had been seen with the enzyme itself and its normal substrate.As described above, the protonation in the model system involves an imidazolium ion putting a proton on the substrate phosphate anion as the imidazole delivers a water molecule to the phosphorus. 

In this regard, Breslow s group (179) synthesized a jS-cyclodextrin-bisimidazole molecule to model ribonuclease A (RNase A) (see Chapter 3). The approach is based on the preparation of a capped disulfonate derivative made earlier by I. Tabushi and co-workers of Kyoto University (180,181). 

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