Enzyme cyclodextrins

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. 

We finish this Section with enzymic conversions that are difficult to classify elsewhere Takasweet, a commercial variety of immobilized glucose-iso-merase, converts 6-O-methyl-D-fructose and 6-deoxy-D-fructose into the gluco isomers in not very satisfactory yield.34 A mixture of catalase (75 U/mmol) and glucose oxidase (80 U/mmol) oxidizes xylitol to L-xylose in 50% yield, on the 100- mol scale.106 The enzyme cyclodextrin a-(l—>4)-glucosyltransferase (1000 U immobilized on silica gel-glutaraldehyde) allows107 preparation of cyclomaltohexaose (0.3 g), cyclomaltoheptaose... 

They are prepared by enz3miatic degradation of starch. The enzyme cyclodextrin glucanosyltransferase (CGT) from Bacillus macerans. Bacillus megaterium or other bacterial strains cut the starch helix and join both ends of such destruct forming a cyclic compound. Because enzymes are not very specific, the obtained mixture contains cyclodextrins from 6 to 12 glucose units. The main fi-actions are a-, p- and y-cyclodextrins which correspond to 6, 7 and 8 glucose units. 

Cyclodextrins are manufactured by the enzymatic degradation of starch using specialized bacteria. For example, P-cyclodextrin is produced by the action of the enzyme cyclodextrin glucosyltransferase upon starch or a starch hydrolysate. An organic solvent is used to direct the reaction that produces P-cyclodextrin, and to prevent the growth of microorganisms... 

Cyclodextrins are a special type of starch derivative. They come about as a result of the effect of the enzyme cyclodextrin glucosyltransferase on starch. Cyclic products result with 6,7 or 8 glucose units they are designated as a-, 6-, and y-cyclodextrin (Fig. 5.8). Although cyclodextrins are not naturally present in food, they will be considered here. In countries where they are permitted for food, they are used, e.g. for the encapsulation of flavourings and colourings and for masking off-flavours [24-29]. 

AMP or ADP-analogues - NAD - or NADP -dependent enzymes cyclodextrines - different aromatic compounds... 

Of particular importance for modifications of starch are the enzyme degradation products such as glucose symps, cyclodextrins, maltodextrins, and high fmctose com symps (HFCS). Production of such hydrolysis products requites use of selected starch-degrading enzymes such as a-amylase,... 

Various chemical species influence the rates of hydrolysis of penicillins, e.g. metal ions (Cu >Zn >Ni Co ) (80JCS(P2)1725), carbohydrates (78MI51101), certain amine-containing catechol derivatives (69JPS1102) and /3-cyclodextrin (71JA767). Some of these even show some of the characteristics of enzyme-catalyzed hydrolyses. 

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. 

The above two models together with Tabushi s cyclodextrin bis(histamine)23) are really elabolate ones, each having a substrate binding cavity, but their catalytic activities are yet far behind of those of natural enzymes. They suggest the difficulties associated with the design of a metal ion center inside of a cavity which activates both substrate and catalytic groups. 

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. 

Micellar medium has received great attention because it solubilizes, concentrates and orientates the reactants within the micelle core and in this way accelerates the reaction and favors the regio- and stereoselectivity of the process [68], In addition the micellar medium is cheap, can be reused, is more versatile than cyclodextrins and more robust than enzymes. With regard to Diels Alder reactions, we may distinguish between (i) those in which one or both reagents are surfactants which make up the micellar medium, and (ii) those that are carried out in a micellar medium prepared by a suitable surfactant. 

It has been shown that it is possible to compel regiospecific para substitution by enclosing the substrate molecules in a cavity from which only the para position projects. Anisole was chlorinated in solutions containing a cyclodextrin, a molecule in which the anisole is almost entirely enclosed (see Fig. 3.4). With a high enough concentration of cyclodextrin, it was possible to achieve a para/ortho ratio of 21.6 (in the absence of the cyclodextrin the ratio was only 1.48). This behavior is a model for the regioselectivity found in the action of enzymes. 

From these observations, we have noticed the similarity of the simple lattice inclusions to the more sophisticated assemblies of molecules (e.g. cyclodextrins 76 and proteins 78 where the formation of H-bonded loops was first detected and described. Conclusively the motive for the formation of simple inclusion crystals and of more complex associates between high and low molecular weight compounds, such as enzyme-substrate complexes, can be traced back to the same source. 

As we saw in the previous sections, inclusion compounds have many structural properties which relate them to other systems based on the hierarchy of non-bound interactions, like enzymes or enzyme-substrate complexes. As a matter of fact, most of the so-called artificial enzymes are based on well-known host molecules (e.g. P-cyclodextrin) and are designed to act partly on such bases 108>109). Most of these models, however, take advantage of the inclusion (intra-host encapsulation) phenomena. Construction of proper covalently bound model molecules is a formidable task for the synthetic chemistuo>. Therefore, any kind of advance towards such a goal is welcomed. 

Recent examples of artificial enzyme models based on the P-cyclodextrin skeleton ... 

Sucrase-type (non-Leloir-type) enzymes that operate both regio- and stereo-selectively, using sucrose as a cheap substrate, or, in some cases (such as cyclodextrin (CD) transferases) starch these enzymes are, however, limited to the transfer of only glucose or fructose... 

Pun SH, Tack F, Bellocq NC, Cheng J, Grubbs BH, Jensen GS, Davis ME, Brewster M, Janicot M, Janssens B, Floren W, Bakker A (2004) Targeted delivery of RNA-cleaving DNA enzyme (DNAzyme) to tumor tissue by transferrin-modified, cyclodextrin-based particles. Cancer Biol Ther 3 641-650... 

Cyclodextrins as catalysts and enzyme models It has long been known that cyclodextrins may act as elementary models for the catalytic behaviour of enzymes (Breslow, 1971). These hosts, with the assistance of their hydroxyl functions, may exhibit guest specificity, competitive inhibition, and Michaelis-Menten-type kinetics. All these are characteristics of enzyme-catalyzed reactions. 

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