MINI-REVIEW

Artificial enzymes, “Chemzymes”: current stateand perspectives

Jeannette Bjerre & Cyril Rousseau & Lavinia Marinescu &

Mikael Bols

Received: 16 April 2008 /Revised: 29 July 2008 /Accepted: 1 August 2008 /Published online: 9 September 2008# Springer-Verlag 2008

Abstract Enzymes have fascinated scientists since theirdiscovery and, over some decades, one aim in organicchemistry has been the creation of molecules that mimic theactive sites of enzymes and promote catalysis. Neverthe-less, even today, there are relatively few examples ofenzyme models that successfully perform Michaelis–Menten catalysis under enzymatic conditions (i.e., aqueousmedium, neutral pH, ambient temperature) and for thosethat do, very high rate accelerations are seldomly seen. Thisreview will provide a brief summary of the recent develop-ments in artificial enzymes, so called “Chemzymes”, basedon cyclodextrins and other molecules. Only the chemzymesthat have shown enzyme-like activity that has beenquantified by different methods will be mentioned. Thisreview will summarize the work done in the field ofartificial glycosidases, oxidases, epoxidases, and esterases,as well as chemzymes that catalyze conjugate additions,cycloadditions, and self-replicating processes. The focuswill be mainly on cyclodextrin-based chemzymes sincethey have shown to be good candidate structures to base anenzyme model skeleton on. In addition hereto, othermolecules that encompass binding properties will also bepresented.

Keywords Supramolecular . Cyclodextrin . Biomimetic .

Enzyme model . Catalysis

Introduction

Enzymes are truly outstanding biological catalysts with theability to accelerate the rate of chemical reactions up to1019 times for specific substrates and reactions (Wolfendenand Snider 2001). Only with the help of enzymes can theprocesses of life inside and outside cells take place, asthe underlying chemical reactions could not be executedin the absence of catalysis. Enzymes are required forbasically all major life-sustaining physiological functions,e.g., for digestion of food and nutrients (Whitcomb andLowe 2007); for converting the fuel contents of our diet toappropriate energy forms for brain and muscle; forchemical conversion of toxins and metabolic waste prod-ucts to excretable forms (Thummel et al. 1997); for growth,tissue rebuilding, and for healing processes. The crucialrole of enzymes in the life processes is reflected in the factthat enzymes are found in many everyday commercialproducts including washing detergent (Vasconcelos et al.2006) and contact lens cleaning fluid (Begley et al. 1990).Production of alcoholic beverages (Neelakantan et al. 1999)and dairy products (Linko et al. 1998) are likewiseexamples of commercial utilization of enzymes. Enzymeshave also found their way into chemical synthesis processessince enzymes offer an environment-friendly alternative totoxic chemical reagents; the field of green chemistry is, atpresent, enjoying a high level of attention from thescientific community (Riva 2006). Nevertheless, the useof enzymes in chemical processes is severely limited by thenarrow substrate specificity to mainly hydrolytic reactionsof biomolecules such as carbohydrates and lipids. Obvi-ously, if enzymes could be mimicked by chemists andengineered to more useful reactions, this situation wouldchange.

Appl Microbiol Biotechnol (2008) 81:1–11DOI 10.1007/s00253-008-1653-5

J. Bjerre :C. Rousseau : L. Marinescu :M. Bols (*)Department of Chemistry, University of Copenhagen,DK-2100 Copenhagen, Denmarke-mail: bols@kemi.ku.dkURL: www.ki.ku.dk

Enzyme models

Enzymes bind substrates in their active site and use bindingand proximity effects to achieve their impressive rateenhancements. In some cases, enzymes can make achemical reaction take place in just a few seconds thatwould otherwise take millions of years to complete. Naturalenzymes are large protein structures of extreme molecularcomplexity but their mechanism of catalysis is frequentlyintriguingly simple with only a few amino acids involved incatalysis. Hence, it is inherently possible to build acomparatively simple model of an active site and obtainpowerful and selective catalysis, but as with many, a simpleidea in science it is not so simple to realize. Thisminireview will emphasize some of the latest work onbuilding active-site models. As the word artificial enzymeis occasionally used in connection with engineered proteinsand catalytic antibodies, for the purpose of this review, theword “chemzyme” will be used to describe small-molecular-weight active-site models. Only work on such chemzymesduring the last 10 years is covered, while work on the manyorganic and inorganic catalysts that do not have a bindingsite or where binding has not been shown is not included. Forthe work before 1998, the reader is referred to excellentreviews (Breslow and Dong 1998; Breslow 2005).

The binding cavity

Chemzymes need a binding cavity that is relatively readilyaccessible. While many different binding structures have beenemployed, the most successful binding cavities in chemzymeshave been cyclodextrins (CDs). These are water-soluble ring-shaped oligomers composed of 4C1 chair conformationα(1→4)-D-glucopyranoside units. The most common CDscontain six (α-CD), seven (β-CD), or eight (γ-CD) glucoseunits, which are named alphabetically (α, β, etc.; Fig. 1).

The CDs form a truncated cone-shaped structure whichis hydrophilic on the exterior and lipophilic on the interiorcavity lining. The internal hydrophobicity is mainly causedby 3- and 5-sugar hydrogens pointing into the cavity. Theexternal hydrophilicity is a result of the sugar alcoholgroups, found on the two rims of the cone structure,pointing away from the cavity and towards the aqueousenvironment. The smallest (primary, upper) rim encom-passes the 6-OH’s and the larger (secondary, lower) rimholds the 2- and 3-OH’s. The different number of glucoseunits gives rise to variance in size among the most commonCDs; the small α-CD can bind in its cavity slim molecularstructures like fatty acid chains and non-branched aliphaticchains. The slightly larger β-CD can accommodate phenyl,

H3

H5

HO OH

O

HO

OOH

OH

HO

2 3

6

n

n = 6n = 7n = 8

23

4 5

A D

The sugar units of-cyclodextrin molecule

B C

EF

γβα

α

Fig. 1 Common native form of cyclodextrins

Fig. 2 Mechanism of diacid CD catalysis

(OH)21 (OBn)21 (OBn)19

OHOH

(OBn)19

OO

(OBn)19

FGFG

(OH)19

FGFG

BnClNaH(94%)

DIBAL-HTolueneMol. sieves(81%)

Dess-Martinperiodinane(100%)

modificationTFA (cat.)

H2-Pd/CFunctionalgroup (FG)

FG = COOH, CNOH, CF3OH, etc.

Scheme 1 General route for the synthesis of difunctionalized cyclodextrins

2 Appl Microbiol Biotechnol (2008) 81:1–11

naphthalene, or cholesterol structures, the latter of whichgives rise to a pronounced toxicity of i.v. administered β-CD, probably due to cholesterol extraction from biologicalmembranes. The CDs are otherwise non-toxic when givenorally, and are presently found in many food products, suchas instant coffee, tea, and fine oils, as flavor preservers.Finally, the large γ-CD is big enough to bind the C60

Buckminsterfullerene, affording a beautiful purple inclusioncomplex. The CDs are readily made from starch byenzymatic degradation, making them both decent in a greenchemistry perspective, readily available in large amounts,and quite inexpensive (Szejtli 1998).

The hydrophobic/hydrophilic features are what make CDsso versatile; as supramolecular hosts, they can encapsulatenon-polar structures in their cavity whilst being soluble inwater at the same time. This is reminiscent of the naturalenzymes, which also rely on non-bonding interactions tobind their substrate in the active site, in an aqueous medium.

Selective chemical manipulation of cyclodextrins

Doing selective organic chemistry on a CD moleculecontaining 18 to 21 alcohol functionalities is a worthwhile,but certainly not straightforward, task. Normally, it is carriedout by mono-functionalization; but in the last decade, a newclever selective de-O-benzylation method has emerged(Pearce and Sinaÿ 2000). It is possible to liberate by choice

one or two alcohol functionalities on perpendicular sides (Aand D sugar moieties) of the upper, primary rim of aperbenzylated CD cone structure. The free OH groups can befunctionalized to give catalytic activity, and the rest of theCD molecule can act as an active site, to specifically bind thesubstrates in the cavity and then have the catalytic groups onthe rim exercise the enzymatic reaction. The generalsynthetic route is shown in Scheme 1. Some examples ofthe final compounds synthesized after this method togetherwith their activity will be subsequently summarized.

Glycosidases

The first class of enzymes to mimic was the glycosidases. Theglycosidic bond in aqueous solution, under non-catalyzedconditions, is very stable, with a half time for glycosidiccleavage of as much as 5 million years (Wolfenden andSnider 2001). The cleavage of glycosidic bonds is, therefore,a very important process that enables us to use the energy inpolysaccharide starch products like grain, rice, and potatoes.Natural glycosidases include the well-known lysozyme thatperforms the cleavage of NAG–NAM residues in bacterialcell walls. Both the anomeric carbon inverting and non-inverting natural glycosidases make use of two carboxylicacid residues in their active site, situated ca. 5 to 10 Å apart,to perform general acid/base catalysis, combined withnucleophilic catalysis, in the hydrolysis of the glycosidicbond. Inspired by this, modified α- and β-CDs have beendesigned with two carboxylate groups on the primary rim,situated 5 and 6.5 Å apart, respectively (Fig. 2). Theseenzymes are able to act as artificial glycosidases, affordingcleavage of the glycosidic bond of nitrophenyl glycosides(Rousseau et al. 2004a).

The hydrolysis progress was continuously monitored byUV measurement of the concentration of the cleavedproduct nitrophenol (400 nm). Reaction rates for catalyzedand non-catalyzed hydrolysis, as well as KM values areobtained by non-linear regression fitting to the Michaelis–Menten equation (Eq. 1).

V ¼ Vmax S½ �KM þ S½ � ð1Þ

(OH)19

OHOH

Phosphate bufferpH 8.0, 59 °C

F3C CF3O

O

NO2

HO O

NO2

HOOH

HO

+

Scheme 2 Ditrifluoromethylalcohol β-CD-catalyzed hydrolysis of p-nitrophenyl glycopyranosides

Fig. 3 Mechanism of cyanohydrin CD catalysis

Appl Microbiol Biotechnol (2008) 81:1–11 3

An important difference between the CD glycosidasesand natural glycosidases is that whereas the naturalenzymes recognize mainly the saccharide portion of thesubstrate, the glycosidase CDs recognize the aromatic partof the aryl glycosides. The synthesis of the two diacids wasperformed via the general synthesis route (Scheme 1)wherein the 6A,D-dialdehyde was subjected to NaClO2

oxidation to afford the diacid, which was then de-O-benzylated. Catalysis was proven to require binding of thesubstrate inside the CD cavity, as addition of cyclopentanolor aniline inhibited the reaction. The reaction followedMichaelis–Menten kinetics, as does that of many naturalenzymes. Furthermore, substrate selectivity was observed,and it was found that a variation in the aryl moiety from 4-nitrophenyl to 2-nitrophenyl gave rise to a dramaticdecrease in reaction rate, as is also seen for many of morerecently synthesized CD analogues. Thus, the diacid CDsdisplayed several of the hallmarks associated with naturalenzymes. Catalysis rate (kcat/kuncat) was, however, not asimpressive, and for hydrolysis of 4-nitrophenyl gluco-,manno-, or galactosides, it was found to be between 12–35(pH 7.4, 59 °C, 50 mM phosphate buffer). With ten timeshigher phosphate concentrations, catalysis rates of up to1,000 were achieved with the β-CD diacid, and a linearrelationship could be observed between phosphate concen-tration and catalysis rate. This leads to the assumption that

catalysis was brought about by electrostatic stabilization ofa positively charged transition state, facilitating substitutionwith phosphate (Rousseau et al. 2005a; Fig. 2). Thismechanism requires the presence of both carboxylic groups,and no catalysis arising from the 6-monoacid CD wasobserved.

However, it was discovered that dicyanohydrin CDswere significantly better artificial glycosidases than diacidCDs, with rates of catalysis of up to 8,000 (kcat/kuncat, pH8.0, 59 °C, phosphate buffer) for cleavage of nitrophenylglycosides. The α- and β-CD 6A,D-dicyanohydrins weresynthesized by addition of cyanide to the corresponding 6A,D-dialdehydes, followed by de-O-benzylation (Ortega-Caballero et al. 2005a, b). Enzymatic characteristics wereseen, as for the diacid CDs (substrate cavity binding,Michaelis–Menten kinetics, substrate selectivity). By degra-dation analysis, the stereochemistry of the cyanohydringroup was determined to be R,R and, based on previouswork (Hardlei and Bols 2002), gt-conformation, meaningthat the cyanohydrin alcohol groups point towards the CDcavity, whereas the nitrile group points towards the exterior.This fits well with the assumed mechanism, wherein thealcohol proton is acidified by the electron-withdrawingeffects of the nitrile group, enabling the proton toparticipate in general acid catalysis cleavage of theglycosidic bond (Fig. 3).

The mechanism only requires the presence of onecatalytic group and, as expected, it was found that themonocyanohydrin β-CD displayed a catalysis rate that wasroughly half of that of the dicyanohydrin β-CD (Ortega-Caballero et al. 2005b).

This brought on the idea of including other knownfeatures of natural glycosidases, such as nucleophiliccatalysis. Therefore, a difunctionalized 6A-cyanohydrin-6D-carboxylate β-CD was synthesized, to bring about bothgeneral acid catalysis and nucleophilic catalysis in the samemolecule (Ortega-Caballero and Bols 2006). Chemically,the two 6A,D-alcohols were differentiated by mono-silylation,and then the usual chemical manipulations were performedto achieve the two desired functional groups. Even thoughan initial catalysis rate of up to 1,100 was found for thiscompound, rapid decomposition and subsequent loss of

Fig. 4 Catalytic cycle of ketone-bridged CD epoxidation. Thereaction is performed in water with 30 mol.% CD present, alkeneand oxone being slowly added over 1 h

(OBn)19

OHOH

(OBn)19

OO

(OBn)19

OO

O

(OH)19

OO

O

ClCl

NaH, DMF 2) NaIO4

1) OsO4H2-Pd/C

TFA (cat.)

Scheme 3 Synthesis of diether-bridged ketone CD. The corresponding diester-bridged ketone CD is produced in the same fashion, by startingwith the 6A,D-diacid instead of the 6A,D-dialcohol CD

4 Appl Microbiol Biotechnol (2008) 81:1–11

catalysis occurred, and it was believed it is the intrinsicchemical nature and interaction of the carboxylate with thecyanohydrin that causes the breakdown of the molecule.The lower rate of catalysis, compared to that of the mono-and dicyanohydrin, also indicates that no cooperativecatalysis between the different catalytic groups takesplace.

To elaborate on the cyanohydrin theme, an analogue wassynthesized wherein the nitrile group was replaced with atrifluoromethyl group, since these two groups possessroughly the same magnitude of electron-withdrawing effect(Bjerre et al. 2007). The synthesis was performed byaddition of CF3 units to the 6A,D-dialdehyde, using, as acatalyst, the Arduengo carbene.

The catalytic rate (kcat/kuncat) was only up to 90,however, and it can, in part, be due to some of the CF3-bound OH’s pointing away from the cavity, resulting in acatalytically non-productive enzyme conformer. Bindingconstants (KM) were in the range of 3–8 mM, as is also ingeneral the case for the cyanohydrin CDs, and this is quitesimilar to the binding that many natural enzymes have withtheir substrates (Scheme 2).

Epoxidases

Epoxidation is a well-known reaction in organic chemistrythat can be performed with different epoxidizing reagentssuch as m-CPBA or tBuOOH, but as with many chemicalreagents, controlling the specificity towards substrate andfine-tuning selectivity can be an issue of concern. Intelli-

gently designed epoxidation catalysts that, like enzymes,can recognize substrates, could potentially be engineered tosuit the needs of any chemical reaction and medium.Therefore, it was interesting to craft an artificial epoxidase,based on the CD skeleton, that includes a ketone-containingbridge that spans over the primary rim of the CD. It wasdiscovered that 1,3-dialkoxyacetone CD was able tocatalyze the epoxidation of a number of alkenes, withoxone (KHSO5) as a stoichiometric cooxidant. The mech-anism is assumed to involve initial oxone-mediatedepoxidation of the ketone to a dioxirane, thereby activatingthe catalytic group for epoxidation of alkenes. Uponepoxidation, the ketone returns to its original state (Fig. 4;Rousseau et al. 2004b).

The ketone bridge was both made with a diether or adiester connection between the ketone and the CDprimary rim edges, and it was the diester-bridgedversion that showed the greatest catalytic effect. Inshort, the synthesis was carried out starting either fromthe perbenzylated CD 6A,D-dialcohol (for getting thediether-bridged ketone), or from the 6A,D-diacid (forobtaining the diester-bridged ketone). From either of thesewas formed a bridged CD by reaction with methallylchloride, affording a methylene-diether or -diester bridge,spanning across the primary rim of the CD. The doublebond moiety was then converted to a ketone by OsO4

dihydroxylation followed by NaIO4 cleavage (Scheme 3;Rousseau et al. 2005b).

The ketodiester-bridged β-CD afforded 45% enantio-meric selectivity for the epoxidation of styrene, whichproceeds to full completion in 1 h (Scheme 4).

(OH)19

OO

KHSO5, H2O, 25 °C, 1 h

100% conversion, 45% ee

OO

O

O

Scheme 4 Ketodiester-bridgedCD epoxidation of styrene

(OH)19

OO

Phosphate buffer, H2O2pH 7.0, 25 °C

OO

O

OH

NH2

O

N

O

NH2

Scheme 5 Ketodiester-bridgedCD oxidation of o-aminophenol

Appl Microbiol Biotechnol (2008) 81:1–11 5

Oxidases

Oxidation is one of the cornerstone reactions of bothorganic chemistry and biological processes. In the liver, theoxidase enzymes CYP450 are responsible for metabolizinga wide variety of compounds, including pharmaceuticaldrugs, their conversion to more polar oxidation productsenabling their subsequent excretion in the urine.

The ketodiester-bridged CDs were used for o-aminophenoloxidation affording a tricyclic compound, with hydrogenperoxide as a stoichiometric cooxidant (Scheme 5). Thoughhydrogen peroxide is an inexpensive, environment-friendly,and non-toxic oxidation agent on its own, most oxidationreactions using H2O2 have a high energy of activation, whichjustifies the use of enzymatic catalysis. The reaction displayssubstrate selectivity and follows Michaelis–Menten kinetics;the catalytic rate was found to be up to 1,070. Themechanism of oxidation is assumed to take place via ahydroperoxide adduct formed by reaction of the ketone withH2O2 (Marinescu et al. 2005).

Inspired by the catalytic potential of the ketone moiety, a6A,D-ditrifluoromethyl-ketone β-CD was synthesized(Fig. 5), obtainable from Swern oxidation of the aforemen-tioned ditrifluoromethylalcohol β-CD.

For both amine and alcohol oxidations, assisted byH2O2, catalysis rates of around 100 were obtained (Bjerreet al. 2007). The presence of excessive conformationalrotation in the ditrifluoromethylketone CD could have anegative impact on catalysis, as indicated by the results,which are also confirmed by similar observations for other6A,D-diketo CD oxidases that have been synthesized.

The most prominent catalysis was found for benzyl alcoholoxidation using the ketodiester-bridged CD (Scheme 6).

This reaction displays an astonishing catalysis rate of60,000, KM values being around 1 mM. The reaction

follows Michaelis–Menten kinetics, can be inhibited byaddition of cyclopentanol and is catalyzed neither by β-cyclodextrin nor by 1,3-dichloroacetone. This is this firstexample of a CD-based artificial enzyme being as effectiveper weight unit as some natural enzymes (Marinescu andBols 2006). Synthesis of a series of new cup-shaped α-CDcarbonyl compounds was reported and their ability toaccelerate oxidation reactions was quantified to a catalyticrate (kcat/kuncat) of up to 410 for amine oxidations (Lopez etal. 2007).

Fujita and coworkers have prepared dimeric CDs that, inthe presence of cerium, catalyze the well-known chemilumis-cence reaction of luminol with base and hydrogen peroxide(Yuan et al. 2002, 2007). The synthesis involved the couplingof β-6 amino- or β-3 amino-CDs with EDTA and thecomplexation with cerium from an aqueous solution ofCe(NH4)2(NO3)4 (Fig. 6).

Luminol was almost chemiluminescently mute in theabsence of catalyst and neither the CD-dimer, Ce(IV) ion,nor EDTA–Ce(IV) complex demonstrated obvious influenceon the reaction conditions while the emission of luminolwas remarkably enhanced when CD-catalyst was used(Yuan et al. 2002). Cerium is bound in the EDTA-likelinker and catalysis significantly depends on achieving theright geometry of the cyclodextrin cavity that bringsluminol close to the HOO− metal complex (Fig. 6).

The dimer bridged at the primary rim of CD was oneorder of magnitude more efficient than those bridged at thesecondary rim. Modifications of either the CD rims or theEDTA linker considerably altered the catalytic abilities ofthe dimer–CD–EDTA complex (Yuan et al. 2007).

Fig. 5 Ditrifluoromethylketoneβ-CD

(OH)19

OO

Phosphate buffer, H2O2pH 8.0, 45 °C

OO

O

OH OH

OH O

Scheme 6 Ketodiester-bridgedCD oxidation of benzylicalcohols

4

222

+

Fig. 6 Possible pre-organization of the catalyst with the ceriumoxidant

6 Appl Microbiol Biotechnol (2008) 81:1–11

A third type of oxidase has been reported by Liu et al.,who attached an organoselenium moiety to the primary rimof a CD (Liu et al. 2002) in order to mimic the activities ofsuperoxide dismutases (SODs) and glutathione peroxidise(GPx). Both enzymes are part of the antioxidative defensesystem which can protect cells from the free radicals andthe reactive oxygen species; the main causes of aging,cardiovascular, tumorous, and endemic diseases.

The synthesis of novel β-CD derivatives containing a1,2-benzisoselenazol-3(2H)-one moiety (Fig. 7) is reportedstarting from 6-O-monotosyl-β-CD and their activity asSOD mimics was determined by measuring the auto-oxidation speed of pyrogallol in the absence and presenceof chemzymes (Liu et al. 2002).

The best mimic of SOD activity proved to be compound2 with an activity of 330 U/mg; one tenth of that of naturalSOD from bovine erythrocytes (3,400 U/mg). This type ofcompound also catalyzed glutathione oxidation in the rangeof 0.34–0.86 U/µmol but, nevertheless, the GPx activitiesare lower than that of ebselen (PZ51), one of the best smallmolecular mimics of GPx, but whose low water solubilitylimits its use.

A more efficient glutathione peroxidase mimic has beenreported with 2-deoxy-2-telluro-β-CD (Dong et al. 2006;Fig. 8). A comparative study of this chemzyme withdifferent substrates in the presence of a variety ofstructurally distinct hydroperoxides (H2O2, tBuOOH and,cumene peroxide) as the oxidative reagents, revealed thatthe CD moiety endows the molecule with selectivity forROOH and thiol substrates, just like native GPx exhibitsdifferent ROOH and thiol specificities.

Hydrophobic interactions proved to be the most importantdriving force in 2-telluro-CD complexation. This chemzymeis able to catalyze the reduction of ROOH about 3.4×105

times more efficiently than diphenyl diselenide and itssecond order rate constants for thiol are similar to those ofnative GPx (Dong et al. 2006).

Esterases

A very promising aspartic proteinase has been prepared(Jiang et al. 2005), based on aza-crown ethers. A series ofaza-crown derivatives with or without carboxyl groups inthe side arms were prepared and they showed differentdeacylation activity toward glycine p-nitrophenyl ester

hydrobromide. These chemzymes can catalyze the hydro-lysis of glycin esters with a kcat/kuncat of up to 4,000. Therelationship between structure and deacylation activities ofhost compounds suggests the existence of an anhydrideintermediate (Scheme 7).

The effect of ring size of crown ethers on the catalysishas been studied and the conclusion was that compoundswith 18-membered rings have a higher catalytic efficiencythan the corresponding ones with 15-membered rings, dueto better interaction with the substrate (Jiang et al. 2005).

Ortho ester hydrolysis is promoted by a novel metal ligandassembly in a recent report (Pluth et al. 2007). The tetrahedralcomplex of six molecules of N,N′-bis(2,3-dihydroxyben-zoyl)-1,5-diaminonaphthalene and four gallium ions issoluble in water and has a lipophilic cavity where it canbind ortho esters (Scheme 8). Since the cavity stabilizes thepositively charged transition state of ortho ester hydrolysis,the reaction rate is accelerated with a kcat/kuncat of 890. Thecatalytic reaction obeys Michaelis–Menten kinetics andexhibits competitive inhibition in the presence of the stronglybinding NPr4

+. The catalyst displays substrate size selectiv-ity, shown in that triisopropyl orthoformate is moreefficiently hydrolyzed than triethyl orthoformate whencompeting for the same binding site.

The strategy of using synthetic hosts to modify thechemical properties of the bound substrates in order toenhance their reactivity and to promote the acid-catalyzedhydrolysis in basic solutions proved to be an efficientmethod for these acid-sensitive molecules (Pluth et al. 2007).

Conjugate addition

Móran’s group has worked on a diaminoxanthone-basedreceptor that, when suitably modified with functionalgroups, catalyzes conjugate addition reaction on the boundmolecule in organic solvent (Scheme 9). The ligand for thisreceptor is an α,β-unsaturated-δ-lactam; the rate of additionto the conjugated double bond of the ethyl mercaptan isincreased 50–100 times by the binding to the receptor(Simón et al. 2007a).

A series of xanthone-based receptors containing differentside chains has been prepared and the most reactive haveproved to be the receptor containing proline derivative in theside chain since it is suitable for the proton-switch mechanismproposed for these types of reactions and investigated by

Fig. 7 Organoselenium modi-fied β-CD oxidants

Fig. 8 Structure of 2-deoxy-2-telluro-CD and 4-nitrobenzenethiol assubstrate

Appl Microbiol Biotechnol (2008) 81:1–11 7

computational methods. The asymmetric induction of thereaction was also studied and the highest enantiomeric excesswas obtained with the D-proline-containing receptor.

Different chemzymes based on the same type of receptorwere designed for Michael addition of pyrrolidine to α,β-unsaturated lactams and rate increases approaching 104

have been obtained (Simón et al. 2007b). These results

were similar to the catalytic antibody values (kcat/kuncat=103), but, nevertheless, different from those obtained fornatural enzymes (kcat/kuncat=10

12; Simón et al. 2007b).Related hereto, is the chemistry reported by Hooley and

Rebek on the catalysis performed by a deep cavitand(Hooley and Rebek 2005). This artificial receptor (Fig. 9)binds bicyclic bases like DABCO or quinuclidine and

O O

N

O

O

O

O

HOOC

+

NH3O

OO2N

O O

N

O

O

O

O

HOOC

H2N

RH

O

OAr

+

O O

N

O

O

O

O

H2N

RH

HO OAr

+

O

O

O O

N

O

O

O

O

H2N

RH

+

O

OO

CH3O

CH3OH

O O

N

O

O

O

O

HOOC

H2N

RH

+

O

COCH3

CH3OOCCH2NH3

O O

N

O

O

O

O

HOOC

Scheme 7 Nucleophilic mechanism of an artificial aspartic proteinase system

H2O

OH-

HC(OR)3

HC(OR)3

Resting State

RO

ROOHR

H2O

2 ROH

O

OH+

ROH

OH

ORH

2 OH-

H2O

OH

ORH

Scheme 8 Orthoformate hydro-lysis promoted by a supramo-lecular host

8 Appl Microbiol Biotechnol (2008) 81:1–11

enhances their reactivity towards conjugate addition to anα,β-unsaturated system such as methyl vinyl ketone. Thisreaction is reversible but can be monitored in the rapid α-deuteration of the vinyl ketone, which occurs with rateincreases up to 1,400.

Even in the presence of only 2% cavitand, a 40%increase of the initial reaction rate is observed and theactivity is thought to be due to the stabilization of theaddition intermediate by the well-organized hydrogen-bonded network of the upper rim of the molecule (Hooleyand Rebek 2005).

It has been shown that conjugate addition of benzene-thiol to a modified maleimide substrate can be acceleratedby a designed oligoamide chemzyme. The acceleration

(kcat/kuncat) was 37-fold. A variety of cycloadditions to thesubstrate were also investigated but no significant catalysiswas seen (Cowie et al. 2006). However, the study of asimple model system as a receptor for the transition state ofcycloaddition reactions is very interesting in regard torational design of future catalysts.

Cycloaddition

An interesting approach of using β-CD itself as achemzyme for 1,3-dipolar cycloaddition of nitrile oxidesto alkynes has been reported recently (Barr et al. 2006).

Earlier work had shown that an alkynoyl amide attachedto the primary rim of a CD gave a dramatically differentregioisomeric product ratio in nitrile oxide cycloadditionthan when the CD was replaced with hydrogen. Typically,cycloaddition of nitrile oxides to propiolamide afford morethan 80% of the 3,5-disubstituted cycloadduct while 6-propynamido-β-CD gives mostly the 3,4-regioisomer(Scheme 10).

It was subsequently shown that complexation of thenitrile oxide not only influenced the selectivity, but alsoaccelerated the reaction rate up to 475 times. The release ofcycloaddition products from the CD is generally difficultdue to the stability of amide bond, but the use of CD esterswas considered in an alternative approach for these types ofreactions (Barr et al. 2006).

Self-replicating molecules

An interesting sub-group of chemzymes are self-replicatingcompounds, i.e., molecules that catalyze their own formation.

NH

O

EtSH

DABCONH

O

EtS

O

HN

HN

EtOOC

O

O

Bu

Bu

OO

N

HH

H S

Et

NH

O

Scheme 9 Addition of ethanethiol to lactam and the catalyticcomplex between the receptor and the lactam

Fig. 9 Deep cavitands used for α-deuteration of vinyl ketones

Appl Microbiol Biotechnol (2008) 81:1–11 9

The last decade has seen progress in the area of custom-tailored catalysts, e.g., Ghadiri and collaborators reported apeptide that could act as a template for its own formation fromtwo smaller peptides. The two peptides are joined together bya Kent-type ligase reaction and the helix template increasedthe rate of reaction by over 4,100 times (Kennan et al. 2001;Scheme 11).

The reaction proceeds via an initial trans-thiolesterifica-tion via a nucleophilic attack of the N-terminal cysteinesulfhydryl group from Sj to the C-terminal thiolester ofelectrophile Si. The resulting intermediate P* undergoes arapid rearrangement by an intramolecular S–N acyl transferto produce the desired amide bond in the final peptide P.

The electrophilic Si and the nucleophilic Sj peptidefragments are bound on an α-helical template throughelectrostatic complementarity, forming a complex whichfacilitates the ligation due to higher reactant effectivemolarities. The template has an enzyme-like function; itrecognizes the substrates, binds them and forms a thiolestercomplex where the rearrangement takes place to give the

product. Dissociation of the product–template complex willregenerate the free template.

Rate enhancements up to 4,100-fold were observedwhen using the template compared to the uncatalyzedreaction in absence of the template and the catalysis ishighly sensitive to the precise positioning of reactivefunctional groups as in the case of natural ligase (efficiency105; Kennan et al. 2001).

Another totally different self-replication system has beeninvented more recently (Kassianidis et al. 2005). In thiscase, a Diels Alder reaction is catalyzed by its own product,the latter compound being a template for the cycloaddition,however, with a more modest rate increase. The study wasbased on the reaction between alkylfurans and maleimidesand the formation of endo- and exo-cycloadducts wasfollowed by NMR experiments. The two diastereoisomericcycloadducts were capable of accelerating their ownformation through the assembly of catalytic ternary com-plexes and they did not have any effect on the formation oftheir diastereoisomer (Kassianidis et al. 2005).

Future perspectives

From the work presented in this paper can be envisaged aremarkable progress in the field of chemzymes and a greatfuture prospect for upcoming artificial enzymes.

Selective enzymatic remedies for acute poisoning (Bjerreet al. 2008) or treatment of metabolic diseases are stillneeded and there are many potential areas for future uses ofchemzymes, as is the area of biofuel where selectiveglycosidase degradation of otherwise non-utilizable carbo-hydrate fuel constituents could be explored. Study of re-engineered enzymes or custom-tailored catalysts remain anarea of active research. Enzyme-like systems that arecapable of performing pericyclic transformations with highlevels of efficiency and selectivity are still a challenge fororganic chemists and the work summarized here bringsabout optimism for possible upcoming applications ofchemzymes. The effort should be continued with designingpowerful catalysts that can achieve defined geometricallydirected functionalization, good binding properties, andmore environment-friendly futures.

NH O

NHO

N O

R

NH O

O

NR

H2O+

15 : 1

C NR O

R = 4-tBuPh

Scheme 10 1,3-Dipolar cyclo-addition of nitrile oxides toalkyne-CDs

HN

CH3

O

S

HN

O

H2N

HS

Si

Sj- BnSH

HN

CH3

O

S HN

O

H2N

P*

HN

NH

OSH

O

HN

CH3

P

Scheme 11 A de novo-designed peptide ligase and mechanism.Peptide backbones are shown as cylinders

10 Appl Microbiol Biotechnol (2008) 81:1–11

Acknowledgments This work has been supported by the LundbeckFoundation and The Danish National Science Research Council.

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