Enzyme cyclodextrin catalysis

Numerous examples of modiflcations to the fundamental cyclodextrin structure have appeared in the literature.The aim of much of this work has been to improve the catalytic properties of the cyclodextrins, and thus to develop so-called artificial enzymes. Cyclodextrins themselves have long been known to be capable of catalyzing such reactions as ester hydrolysis by interaction of the guest with the secondary hydroxyl groups around the rim of the cyclodextrin cavity. The replacement, by synthetic methods, of the hydroxyl groups with other functional groups has been shown, however, to improve remarkably the number of reactions capable of catalysis by the cyclodextrins. For example, Breslow and CO workersreported the attachment of the pyridoxamine-pyridoxal coenzyme group to beta cyclodextrin, and thus found a two hundred-fold acceleration of the conversion of indolepyruvic acid into tryptophan. 

I Cyclodextrins are excellent enzyme models Catalysis and induced fit. Due to their cavities, which are able to accommodate guest (substrate) molecules, and due to the many hydroxyl groups lining this cavity, cyclodextrins can act catalytically in a variety of chemical reactions and they therefore serve as good model enzymes. Thus, benzoic acid esters are hydrolyzed in I aqueous solution by factors up to 100 times faster if cyclodextrins are added. The reaction in- j volves an acylated cyclodextrin as intermediate which is hydrolyzed in a second step of the j reaction, a mechanism reminiscent of the enzyme chymotrypsin. The catalytic efficiency can. be further enhanced if the cyclodextrins are suitably modified chemically so that a whole range of artificial enzymes have been synthesized [551-555, 556, 563, 564]. 

Ihbushi I (1982) Cyclodextrin catalysis as a model for enzyme action. Acc Chem Res 15 66-72... 

Sangwan and Schneider have studied the effect of cyclodextrins on a number of aqueous Diels-Alder reactions between acrylate, fumarate and maleate derivatives of varying hydrophobicities and (mainly) cyclopenta-diene [26]. No simple correlation between substrate hydrophobicity and cyclodextrin-catalyzed rate enhancement was found. However, those systems that did respond to p-cyclodextrin catalysis exhibited enzyme-like saturation kinetics. This led these workers to conclude that the hydrophobic effect can, in fact, be counterproductive to the Diels-Alder reaction if it leads to unproductive orientation of the reactants. The same can be said about the effect of amphiphiles (detergents capable of micellization) on aqueous Diels-Alder reactions since sodium dodecylsulfate (SDS) decelerated the reaction between cyclopentadiene and methyl acrylate. Those cases in the literature claiming micellar catalysis of the aqueous Diels-Alder reaction may simply be benefiting from the solubilizing effect of the amphiphilic additives rather than any bona fide preorganization of the reactants within a micelle [27,28]. 

An artificial metalloenzyme (26) was designed by Breslow et al. 24). It was the first example of a complete artificial enzyme, having a substrate binding cyclodextrin cavity and a Ni2+ ion-chelated nucleophilic group for catalysis. Metalloenzyme (26) behaves a real catalyst, exhibiting turnover, and enhances the rate of hydrolysis of p-nitrophenyl acetate more than 103 fold. The catalytic group of 26 is a -Ni2+ complex which itself is active toward the substrate 1, but not toward such a substrate having no metal ion affinity at a low catalyst concentration. It is appearent that the metal ion in 26 activates the oximate anion by chelation, but not the substrate directly as believed in carboxypeptidase. 

See for example the pioneering work of Breslow Bres-low, R. Dong, S. D. Biomimetic Reactions Catalyzed by Cyclodextrins and their Derivatives Chem. Rev. 1998, 98,1997-2011 and Breslow, R. Biomimetic Chemistiy and Artificial Enzymes - Catalysis by Design Acc Chem. Res. 1995,28,146-153. 

At the end of the review there are some examples involving catalysis by acids and bases, metal ions, micelles, amylose, catalytic antibodies, and enzymes to give the reader a feeling for how Kurz s approach may be usefully applied to other catalysts. Very few of these examples, or those involving cyclodextrins, were discussed in the original literature in the same terms. It is hoped that the present treatment will stimulate further use and exploration of the Kurz approach to analysing transition state stabilization. 

Preparation and catalysis of disubstituted cyclodextrin as an excellent enzyme model is demonstrated by the RNAase model reported by Breslow et al. (68, 83). The enzyme models 10 and II, derived from 1, show a bellshaped pH versus rate profile for the hydrolysis of the cyclic phosphate of 4-terf-butylcatechol, indicating the cooperative catalysis by two imidazole groups (Fig. 21). The reactions catalyzed by 10 and II give exclusively 12 and 13, respectively. This interesting specificity indicates that the geometry of the P—O bond cleavage is quite different from each other. Another interesting enzyme-like kinetic behavior that these hosts exhibited is successful demonstration of the so-called bell-shaped pH profile. 

An important feature of the cyclodextrins is that they can also accelerate chemical reactions, and therefore serve as models for the catalytic as well as the binding properties of enzymes. The rapid reaction is not catalysis, since the dextrin enters reaction but is not regenerated presumably it arises from approximation, where complex formation forces the substrate and the cyclodextrin into intimate contact. In particular, cyclodextrins can increase the rate of cleavage of phenyl pyrophosphate by factors of as much as 100 (Cramer, 1961). More recent work has improved upon this early example. 

Catalyst 17 is effective only with substrates that can bind to the metal ion, so we attached it - coordinated as its Ni2+ derivative - to the secondary face of a-cyclodextrin in catalyst 21 [102]. This was then able to use the metallo-oxime catalysis of our previous study, but with substrates that are not metal ligands, simply those that bind hy-drophobically into the cyclodextrin cavity. As hoped, we saw a significant rate increase in the hydrolysis of p-nitrophenyl acetate, well beyond that for hydrolysis without the catalyst or for simple acetyl transfer to the cyclodextrin itself. Since there was full catalytic turnover, we called compound 21 an artificial enzyme - apparently the first use of this term in the literature. The mechanism is related to that proposed earlier for the enzyme alkaline phosphatase [103]. 

Artificial enzymes with metal ions can also hydrolyze phosphate esters (alkaline phosphatase is such a natural zinc enzyme). We examined the hydrolysis of p-nitro-phenyfdiphenylphosphate (29) by zinc complex 30, and also saw that in a micelle the related complex 31 was an even more effective catalyst [118]. Again the most likely mechanism is the bifunctional Zn-OH acting as both a Lewis acid and a hydroxide nucleophile, as in many zinc enzymes. By attaching the zinc complex 30 to one or two cyclodextrins, we saw even better catalysis with these full enzyme mimics [119]. A catalyst based on 25 - in which a bound La3+ cooperates with H202, not water - accelerates the cleavage of bis-p-nitrophenyl phosphate by over 108-fold relative to uncatalyzed hydrolysis [120]. This is an enormous acceleration. 

Ribonuclease A is a member of a group of enzymes that cleave RNA using general acid-base catalysis without a metal ion in the enzyme. In ribonuclease A, such catalysis is performed by two imidazoles of histidine units, one as the free base (Im) and the other, protonated, as the acid (ImH+). To mimic this in an artificial enzyme, we prepared (3-cyclodextrin bis-imidazoles 41 [124]. The first one was a mixture of the... 

The catalytic activity of artificial chymotrypsin in the hydrolysis of m-tert-butylphenyl acetate (k = 2.8xl02 s 1, KM = 13xl05M) was found to be close to the activity of chymotrypsin in the hydrolysis of p-nitrophenyl acetate (k,.at = l.lxlO2 s 1, KM = 4x105M). Another example of mimicking enzyme catalysis by P-cyclodextrin is general acid-base-catalyzed hydrolysis and nitrosation of amines by alkyl nitrites (Iglesias, 1998). 

Classically, the bell-shaped dependence of rate of the enzymic reaction on pH has been attributed to general acid and base catalysis by the two histidine residues in the active site, His-12 and His-119 (66). Support for this explanation based on the kinetic properties of a model system was first provided by an observation by Breslow and co-workers that 8-cyclodextrin functionalized with two imidazole groups will catalyze the 1,2-cyclic phosphate of 4-rert-butylcatechol (67). The dependence of hydrolysis rate on pH mimics that of RNase A, and this behavior demonstrates that the presence of two imidazole functional groups on a nonionizable framework is the simplest kinetic mimic of the enzyme. 

One of the principal reasons for the utilization of cyclodextrins as models of enzymes is the formation of inclusion complexes between the catalyst and the substrate preceding the catalysis, which is comparable to the formation of a Michaelis-Menten complex in enzymatic reactions. 

Esters can be cleaved by template catalysts that use a metal ion as both a binding group and part of the catalytic system [79-81]. However, metal ion catalysis has also been extended to cases in which the principal substrate binding involves cyclodextrin inclusion indeed, the first catalyst described as an artificial enzyme was. such an example [82]. A cyclodextrin dimer 47 with a bound metal ion between the two cyclodextrins is a particularly effective hydrolytic catalyst for esters that can bind into both cyclodextrin units (Scheme 6-20) [83, 84]. 

Phosphate esters can be cleaved by template catalysts, especially those with cyclodextrin binding groups and linked catalytic groups. Catalysis of the hydrolysis of a bound cyclic phosphate by ribonuclease mimics has been extensively studied [92-98], as has catalysis by enzyme mimics carrying bound metal ions [99-102]. 

In the previous reactions the cyclodextrin acted as a reactant, not a catalyst. However, there are some excellent examples in which true catalysis occurs with simple binding into a cyclodextrin cavity. Here we will describe the cases where the cyclodextrin has not been modified, while in later sections we will discuss cases in which additional catalytic groups have been added to the cyclodextrin, and mimics of metaUoenzymes and of enzymes with co-enzymes have been achieved. 

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