0-Cyclodextrin bisimidazole

The pyridoxamine units linked to the cyclodextrins in these previous cases are rather flexibly attached, and do not perhaps have the optimum defined geometry. Thus we prepared a new series of compounds in which a pyridoxamine unit carried two sulfurs in a well-defined geometry so that they could link to the A and B primary carbons of )8-cyclodextrin, using the same mechanism by which we had originally made the 6A, 6B bisimidazole cyclodextrin. With the new pyildoxamines we observed very high selectivities for hydrophobic substrates, and excellent control of their detailed geometries. ... 

In both of these cases we made the A,D isomers since we assumed that the function of the imidazolium ion was to protonate the leaving group oxygen. This is what is usually written for the mechanism of the real enzyme. However, when we did a detailed study of the three isomers, we saw that the A,B isomer of bisimidazole cyclodextrin was the best catalyst of all 102 -pjjjg jg consistent with a mechanism in which the function of the imidazolium ion... 

Several model systems related to metalloenzymes such as carboxypeptidase and carbonic anhydrase have been reviewed. Breslow contributed a great deal to this field. He showed how to design precise geometries of bis- or trisimidazole derivatives as in natural enzymes. He was able to synthesize a modified cyclodextrin having both a catalytic metal ion moiety and a substrate binding cavity (26). Murakami prepared a novel macrocyclic bisimidazole compound which has also a substrate binding cavity and imidazole ligands for metal ion complexation. Yet the catalytic activities of these model systems are by no means enzymic. 

At around the same time, Breslow and co-workers described bifunctional cyclo-dextrin-based catalysts that were capable of hydrolysis of a bound phosphate ester [88]. In later studies, an AD isomer (Scheme 4.9) of a P-cyclodextrin bisimidazole catalyst turned out to be the fastest catalyst for enolization of p-tert-butylacetophe-none (Scheme 4.9) [89]. Here, the extra binding is provided by the P-cyclodextrin... 

Either the C -capped or the O -capped material can be heated with an excess of imidazole to produce /3-cyclodextrin bisimidazole (VII). On the basis of the above discussion, we believe that the material produced from the C-capped compound is a 6A,6C and 6A,6D isomeric mixture, while that produced from the O-capped compound is largely the 6A,6D isomer. As it turns out, we have not detected yet significant differences between these two materials as catalysts in our kinetic and product studies. 

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). 

We have done a little lock and key chemistry with cyclodextrin imidazoles. For instance, Bovy (18) has prepared the cyclic phosphate derived from naphthalenediol (XI) and from a tetralindiol (XII). Both of these are hydrolyzed by our 6A,6D cyclodextrin bisimidazole (VII) but these substrates are hydrolyzed less effectively than is the f-butylcatechol cyclic phosphate (VIII). In XI and XII, the phosphorus atom will lie on the axis of the cavity, rather than displaced to one side as with t-butylcatechol, and in particular the attacking water mole-... 

R. Breslow, P. Bovy, C. Lipsey Hersh, Reversing the selectivity of cyclodextrin bisimidazole ribonuclease mimics by changing the catalyst geometry, J. Am, Chem. Soc., 1980, 102, 2115-2117. 

A different kind of selectivity has been observed with cyclodextrin bisimidazoles. These species were synthesized as mimics of the enzyme ribonuclease, and indeed they do perform the cleavage of certain bound phosphate esters with a mechanism closely related to that of the natural enzyme [26]. Of course in the natural enzyme there is considerable selectivity, including regioselectivity. For instance, the natural enzyme can cleave a cyclic phosphate ester between C-2 and C-3 of a ribose unit in such a way as to leave the phosphate group entirely on C-3. Ordinary chemical hydrolysis of such a molecule would lead to a mixture of C-2 and C-3 phosphates. Our cyclodextrin bisimidazole is able to imitate such selectivity. 

In our first studies [9] we used the cyclodextrin 6A,6D-bisimidazole 8 and also the 6A,6C isomer 7, since by the classic mechanism—written for the enzyme in most textbooks—the base B delivers a water to the phosphate group while the acid BH+ protonates the leaving oxygen atom, in a linear displacement mechanism. This... 

The pH vs rate profile showed a bell-shaped curve indicating that this catalyst uses both B and BH+ in a bifunctional mechanism. As with the enzyme, the bis-imidazole catalyst can perform its bifunctional catalysis by a simultaneous mechanism, not the sequential mechanism of simple buffer catalysis. We saw that this was indeed the case, as revealed by the tool called "proton inventory." In this technique the reaction is performed in D2O, in H2O, and in mixtures of the two. If only one proton that can exchange with D2O is moving in the transition state, the points all lie on a straight line between the H2O and slower D2O points. If two (or more) protons are moving, the line is curved. It had been found for the enzyme ribonuclease A [10] that a curved line was seen corresponding to the movement of two protons, and we also saw a curved plot—with very similar data— for our cyclodextrin-6A,6B-bisimidazole catalyst 6 [11]. Controls established that indeed this was a reliable indication that our system is performing simultaneous bifunctional catalysis, just as the enzyme does. In particular, the... 

The availibility of the three isomers of P-cyclodextrin bisimidazole (6, 7, and 8) gives us the opportunity to examine other bifunctional catalyses. Geometric preferences among the three isomers should also give us detailed information about the mechanisms, just as it did for the phosphate ester hydrolysis discussed above. The first reactions we have examined (Scheme 7) concern enolization of a ketone and processes that ensue. 

We examined exchange of deuterium into the methyl group of ketone 12 catalyzed by the three isomers of cyclodextrin bisimidazole and also by p-cyclodextrin monoimidazole (5) in D2O solution with 14% CD3OD, buffered at pH 6.2 with phosphate buffer, at 35 °C [12]. We saw essentially no deuterium incorporation into the methyl group over 10 hours with the buffer alone, or the buffer with added simple cyclodextrin. However, all four of the cyclodextrin derivatives (5,6,7, and 8) catalyzed significant deuterium incorporation into the methyl group over the same period. The AB (6) and AC (7) isomers were only marginally more effective than was the monoimidazole catalyst, but the AD isomer 8 was clearly better. A pH vs rate plot for the reaction catalyzed by the AD isomer showed a bell-shaped curve with a rate maximum near pH 6.2, as expected for bifunctional catalysis, while the reaction catalyzed by cyclodextrin-6-imidazole 5 showed a simple increase to a plateau at pH above 7.5, as expected for monofunctional base catalysis. 

Figure 1.11 The simultaneous bifunctional mechanism, indicated by isotopic studies, by which the A,B yS-cyclodextrin bisimidazole catalyzes the cleavage of compound 21 in water by first forming a phosphorane.

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. 

The cyclodextrin bisimidazoles we created have general use in determining the detailed geometries of reactions in water with bifunctional catalysts. As one example, we showed that the enolization of ketones can be catalyzed by such cyclodextrin bisiinidazoles, but with a different geometric preference fi-om that for phosphate ester hydrolysis. We also showed that a number of aldol reactions could be catalyzed in water with selectivity. ... 

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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