Synthetic artificial enzymes

Suh, J. (2000) Designing active sites of synthetic artificial enzymes. Adv. Supramol. Chem., 6, 245-286. 

DESIGNING ACTIVE SITES OF SYNTHETIC ARTIFICIAL ENZYMES... 

Artificial enzymes may be divided into two categories semisynthetic artificial enzymes and synthetic artificial enzymes. Semisynthetic artificial enzymes are partly prepared by biological systems. Catalytic antibodies are typical examples of semisynthetic artificial enzymes. Semisynthetic artificial enzymes are also prepared by modification of a known protein or enzyme at a defined site with a cofactor or new functional group. Synthetic artificial enzymes are prepared totally by synthetic methods. Synthetic artificial enzymes may be either relatively small molecules with well-characterized structures or macromolecules. The term syn-zymes has been coined to designate synthetic polymers with enzyme-like activities. In addition, synthetic artificial enzymes are also obtained with molecular clusters such as micelles and bilayer membranes formed by amphiphiles. 

For construction of active sites of synthetic artificial enzymes, it is necessary to combine various concepts and methodologies developed in supramolecular chemistry with the principles of enzymology. Synthetic artificial enzymes as effective as natural enzymes may not be obtained in the near future. Even synthetic artificial enzymes with a modest degree of efficiency, however, may be useful for practical applications, and some primitive forms of synthetic artificial enzymes have been commercialized. 

Major obstacles faced in the early stage of design of synthetic artificial enzymes were the limited solubility of polymer derivatives in water and the lack of specific binding sites on polymer skeletons. Linear polymers containing quaternary ammonium ions and PEI derivatives are reasonably soluble in water. Without specific binding sites for substrates, however, these polymer derivatives may be simply regarded as polymicelles. 

Several homogeneous synthetic artificial enzymes " and catalytic antibod-igsi05,i06 proteinase activity have been reported. The monoclonal catalytic antibody prepared with a phosphinate hapten exhibited optimum activity at pH 9.5. The measured with an amide substrate at pH 9 and 37 °C was 1.65 x 10" s" Thus, the half-life is 49 days when the substrate is fully complexed to the active site of the catalytic antibody. 

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. 

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. 

The wide variety of enzymes available gives for promise enzymatic derivatization to become a potent analytical tool in the future. Better understanding and theoretical formulations will lead to commercial availability of immobilized enzymes and consequently to more ready use of them. Since in such systems a low content of organic cosolvent in the mobile phase can only be tolerated (whereas a compromise has to be made as far as the optimum mobile phase pH is concerned), artificial enzymes, which are synthetic polymer chains having functional groups that mimic the biocatalytic activity of natural enzymes, are currently being synthesized and investigated as a means to overcome such limitations (276). 

Criteria for calling a compound a synthetic enzyme are (i) completion of at least one catalytic cycle (ii) its presence after the catalytic cycle in unchanged form and (iii) a saturation kinetics behavior such as is manifested by Michaelis-Menten kinetics. There is a tetrameric helical peptide that catalyzes the decarboxylation of oxaloacetate with Michaelis-Menten kinetics and accelerates the reaction 103-104-fold faster than n-butylamine as control, a record for a chemically derived artificial enzyme. 

In the mid to late 1980s, many research groups focused on methods and processes to prepare L-phenylalanine (Chapter 3). This was a direct result of the demand for the synthetic, artificial sweetener aspartame. One of the many routes studied was the use of phenylalanine dH (Scheme 19.4, R = C6H5CH2) with phenylpyruvate (PPA) as substrate.57-58 This enzyme from Bacillus sphaericus shows a broad substrate specificity and, thus, has been used to prepare a number of derivatives of L-phenylalanine.59 A phenylalanine dH isolated from a Rhodococcus strain M4 has been used to make L-homophenylalanine (.S )-2-amino-4-pheny I butanoic acid], a key, chiral component in many angiotensin-converting enzyme (ACE) inhibitors.40 More recently, that same phenylalanine dH has been used to synthesize a number of other unnatural amino acids (UAAs) that do not contain an aromatic sidechain.43... 

Biomimetic Chemistry, including that involved in the synthesis and study of artificial enzymes, has grown to enormous proportions. Even the part of the field using cyclo-dextrins as binding groups in synthetic catalysts that mimic enzymes has been the subject of a large review article [1]. Thus in this chapter I will focus mainly, but not exclusively, on work from our own laboratory. Other chapters will help make up for this somewhat narrow focus. I have published several reviews of our work elsewhere [2-51]. 

This new style of synthetic catalysis will of course not replace all normal synthetic methods. For many purposes, the standard methods and rules - e.g. aldehydes are more easily reduced than are ketones - will continue to dominate organic synthesis. However, when we require a synthetic transformation that is not accessible to normal procedures, as in the functionalization of unactivated carbons remote from functional groups, artificial enzymes can play a role. They must compete with natural enzymes, and with designed enzyme mutants, but for practical large-scale industrial synthesis there can be advantages with catalysts that are more rugged than proteins. 

Klotz IM, Suh J. Evolution of synthetic polymers with enzyme-hke catalytic activities. In Artificial Enzymes. Breslow R, ed. 2005. Wiley-VCH. Weinheim, Germany, pp. 63-88. 

Biomimetic Supramolecular receptors, synthetic ligands and inhibitors, MIMs Porphyrins, artificial enzymes... 

Electrocatalytic groups such as porphyrins and phthalocyanines that act as supramolecular hosts for different metals and mimic the active sites of various proteins are commonly used in amperometric sensors [66,67]. A biomimetic sensor based on an artificial enzyme or synzyme has been demonstrated [68]. The artificial enzyme used in this study was a synthetic polymer (quaternised polyethyleneimine containing 10% primary amines) which decarboxylated oxaloacetate. The product carbon dioxide was detected potentiometrically via a gas membrane electrode. 

Several papers have dealt with electrophilic attack at the amide nitrogen of the unsubstituted benzisoselenazol-3(27T)-one. Alkylation using KOH and alkyl or allyl halide or halocarboxylic ester gave 2-substituted benzisoselenazol-3(27T)-ones <1991CZ135, 1989BSB395> and the reaction has synthetic value. 6-[Benzisoselenazol-3(27T)-one]-/3-cyclodextrin, designed as an artificial enzyme, was prepared by reaction of benzisoselenazol-3(2//)-one potassium salt with 6-iodo-/3-cyclodextrin (/3-CD-6-I) (Scheme 12) <2002CAR1309>. 

Construction of such active sites with small synthetic molecules would be very difficult. Several catalytic elements are to be placed on the molecular framework. Furthermore, those catalytic elements should take productive positions and the conformational freedom of the molecular framework should be controlled to maintain the productive conformation. Thus, a large amount of laborious computational and skillful synthetic work is needed to synthesize such active sites. Instead, synthetic as well as natural macromolecules have been frequently chosen as the backbone of artificial enzymes. Nature has adopted polypeptide as the backbone of the catalysts for fine tuning of the positions and the reactivity of the convergent catalytic elements. 

So far, cyclodextrin (CD 4) derivatives have been the most successful artificial enzymes based on small synthetic host molecules. Since CD derivatives form inclusion complexes with various hydrophobic molecules, they have been utilized as binding pockets." " Examples are illustrated by 5 and 6. The CD derivative indicated in 5 is a mimic of ribonuclease in which the two histidyl imidazoles of the active site are believed to act as a general acid and a general base catalyst. The CD dimer indicated in acts as an artificial metalloesterase manifesting selectivity toward esters with two hydrophobic groups. 

Design of catalysts mimicking the catalytic principles of enzymes is among the great challenges of modern chemistry (9, 10). Catalytic antibodies are examples of semisynthetic artificial enzymes (11-14). Fully synthetic molecules also have been designed as enzyme mimics by using either peptidic (15, 16) or nonpeptidic (17-24) molecules. 

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