Supramolecular Chemistry of Cyclodextrins in Enzyme Technology

Chem. Rev. 2007, 107, 3088−3116

3088

Supramolecular Chemistry of Cyclodextrins in Enzyme Technology Reynaldo Villalonga,† Roberto Cao,* and Alex Fragoso‡ Laboratory of Bioinorganic Chemistry, Faculty of Chemistry, University of Havana, Vedado, Havana 10400, Cuba, Center for Enzyme Technology, University of Matanzas, Matanzas 44740, Cuba, and Nanobiotechnology & Bioanalysis Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Avinguda Paı¨sos Catalans 26, Tarragona 43007, Spain Received June 29, 2006

Contents 1. Introduction 2. Structure and Properties of Cyclodextrins 3. Selective Modification of Cyclodextrins with Enzymes 4. Cyclodextrins in the Biocatalytic Transformation of Organic Compounds through Enzyme-Catalyzed Reactions 4.1. Increased Substrate Solubility and Availability with Cyclodextrins 4.2. Reduced Substrate and Product Inhibition by Association with Cyclodextrins 4.3. Increased Enantioselective Transformation of Substrates by Association with Cyclodextrins 4.4. Other Supramolecular-Based Effects 5. Cyclodextrin−Enzyme Neoglycoconjugates with Improved Functional Properties 5.1. Chemical Modification of Enzymes with Monoactivated Cyclodextrin Derivatives 5.2. Enzymatic Glycosidation of Enzymes with Monoaminated Cyclodextrins 5.3. Covalent Cross-Linking of Enzymes with Polymeric Cyclodextrin Derivatives 6. Cyclodextrins As Chaperones for Enzymes 7. Supramolecular Methods for Immobilizing Enzymes on Macro- and Nanosized Supports 7.1. Macro- and Micrometric Surfaces Modified with Cyclodextrins 7.2. Nanodevices Containing Cyclodextrins 7.2.1. Soft Nanodevices 7.2.2. Hard Nanodevices 8. Enzyme Biosensors with Supramolecular Architecture 8.1. Electrochemical Biosensors 8.2. Optical Biosensors 9. Outlook 10. Abbreviations 11. References

3088 3088 3089 3092 3093 3094 3095 3095 3096 3098 3099 3100 3101 3104 3105 3105 3105 3107 3109 3109 3111 3111 3112 3113

1. Introduction Cyclodextrins (CDs) have captivated many scientists since their discovery back in 1891. The intriguing structure of these * To whom correspondence should be addressed. Phone: +537-8792145. Fax: +537-8733502. E-mail: [email protected]. † University of Matanzas. ‡ Universitat Rovira i Virgili.

materials has attracted chemists, physicists, biologists, engineers, and many others in attempts to take advantage of the unique properties. Cyclodextrins are among the most important molecular receptors studied in supramolecular chemistry. They have raised many questions and provided answers to academic and industrial problems, and as a result of these efforts, there is now what we may call a ‘cyclodextrin science’ that involves the search for improved properties and widespread applications of these molecules. The properties and applications of CDs have been the subject of many reviews1-6 and books.7-11 The last compilation appeared in 1998 in a special issue of Chemical ReViews edited by D’Souza and Lipkowitz.12 Readers interested in this field can find information on the most relevant aspects of CDs, including application of NMR spectroscopy, computational chemistry, and X-ray crystallographysamong other techniquessfor the elucidation of the structures of CDs and their complexes. For this reason, it is not our intention to cover all of these topics but to focus on the most recent applications of CDs in the biotechnological field.

2. Structure and Properties of Cyclodextrins Cyclodextrins are a family of cyclic oligomers composed of R-(1 f 4)-linked D-glucopyranose units in the 4C1 chair conformation (Figure 1). As a consequence of this peculiar structure, the molecule features a conical cavity that is essentially hydrophobic in nature. The most common CDs have six, seven, and eight glucopyranose units and are referred to as R-, β-, and γ-CD, respectively. Larger CDs have also been identified and isolated but have little value in terms of applications.13 The cavity is limited by hydroxyl groups of different chemical character. Those located at the narrower side come from position 6 of the glucopyranose ring (primary side), while those located at the wider entrance are secondary and therefore less prone to chemical transformation (secondary side). The reactivity of the hydroxyl groups strongly depends on the reaction conditions. The nonreducing character of CDs makes them behave as polyols. On the other hand, the large number of hydroxyl groups available implies that careful selection of the reaction conditions is required in order to avoid the substitution of more groups than those needed for a particular purpose. The inner diameter of the cavity in unmodified cyclodextrins varies from 5 to 10 Å, and it is about 8 Å in depth. These dimensions allow the inclusion of several types of guest molecules of appropriate size to form inclusion complexes.8 As a consequence of the inclusion, some properties of the guest molecules changesa phenomenon that constitutes the basis of most CD applications. These ap-

10.1021/cr050253g CCC: $65.00 © 2007 American Chemical Society Published on Web 06/23/2007

Supramolecular Chemistry of Cyclodextrins

Reynaldo Villalonga was born in 1970 in Matanzas, Cuba. He studied Chemistry at the University of Havana, where he graduated with his Gold Diploma in 1993. During 1994 he worked on protein structures at the National Center for Genetic Engineering and Biotechnology and then joined the Laboratory of Bioinorganic Chemistry under the supervision of Professor Roberto Cao. In 1996 he moved to the University of Matanzas, where he founded the Enzyme Technology Group. He completed his Ph.D. degree in neoglycoenzyme synthesis in 2001 and is currently Full Professor and Director of the Center for Enzyme Technology at the University of Matanzas. His research is focused on neoglycoenzymes, application of cyclodextrins and polymers in enzyme technology, drug delivery systems, enzyme biosensors, and nanotechnology. He has been awarded four prizes from the Cuban Academy of Sciences, the Development Cooperation Prize of Belgium, and the Third World Academy of Sciences for Young Chemists. In 2006 he received the National Award in Chemical Sciences. He is Director of the Latin American Network for Enzyme Technology (RELATENZ).

Roberto Cao was born in Havana, Cuba, in 1946. He received his B.Sc. (1971) and M.Sc. (1975) degrees from the University of Havana and his Ph.D. degree (1977) from the Technological Institute of Leningrad (now St. Petersburg), USSR, under the supervision of the late Professor Y. N. Kukushkin. He is Full Professor of the Department of Inorganic Chemistry of the University of Havana and Head of the Laboratory of Bioinorganic Chemistry. Besides this area, he is also very interested in supramolecular chemistry and bionanosciences. At present he is Secretary of the International Relations of the Cuban Chemical Society. He has received several awards from the University of Havana and four from the Cuban Academy of Sciences.

plications include artificial enzymes, sensors, drug formulations, cosmetics, food technology, and textiles.5,8-10 CD complexes can be thermodynamically more or less stable depending on the shape and size of the guest, and the association constants can be measured by a range of physicochemical methods. Absorption and emission spectroscopy along with NMR and calorimetry are the most popular techniques used to study these systems and have provided an understanding of the structure and energetics

Chemical Reviews, 2007, Vol. 107, No. 7 3089

Alex Fragoso was born in Havana, Cuba, in 1970 but was raised in the city of Pinar del Rıó, land of the best tobacco in the world. He obtained his B.Sc. (1993) and Ph.D. (1999) degrees from the University of Havana under the supervision of Professor Roberto Cao working on cyclodextrin-based superoxide dismutase mimics. He was a Research Associate (2000−2002) and Assistant Professor (2003−2004) at the same university working on applications of cyclodextrins in sensing and biotechnology before joining Professor Javier de Mendoza’s group at the Universidad Autońoma de Madrid, Spain (2004−2006), as a postdoctoral fellow. Since 2006 he has held a Ramoń y Cajal Research Professorship at the Department of Chemical Engineering of the Universitat Rovira i Virgili in Tarragona, Spain, and belongs to the Nanobiotechnology and Bioanalysis Group. He is interested in the development of new generation biosensing platforms combining supramolecular architectures, nanotechnologies, and microfabrication. Among other distinctions, he has received the Cuban National Award of Chemistry for Young Researchers (1999) and the Third World Academy of Sciences Prize for Young Chemists (2002).

Figure 1. Structure of cyclodextrins.

of the inclusion process.14 Recently, use of scanning probe techniques such as atomic force microscopy has allowed measurement of the force involved in these interactions at a single-molecule level,15-17 opening new and exciting prospects in supramolecular chemistry.

3. Selective Modification of Cyclodextrins with Enzymes CDs are produced industrially by the action of a family of enzymes called cyclodextrin glucosyltransferases (CGTases) on starch. CGTases are produced by several microorganisms such as Bacillus macerans and Bacillus circulans and catalyze the cleavage and subsequent cyclization of linear R-(1 f 4)-linked polysaccharides. In the last two decades, new types of CGTases that possess higher activity have been produced by genetic engineering.18,19 This advance,

3090 Chemical Reviews, 2007, Vol. 107, No. 7

combined with the optimization of the production technology, has resulted in a dramatic drop in the prices of CDs. CDs can be chemically or enzymatically modified. Chemical synthesis20,21 has produced a large number of derivatives,22,23 and this is the most widely employed method to date despite the inherently complex nature of the CD molecule, which sometimes hampers the selectivity of a given reaction. It is well known that enzymes provide an efficient tool for derivatization of carbohydrates, especially in terms of improved regioselectivity and mild reaction conditions. In the synthesis of CD derivatives, enzymes can be efficiently used in three different ways: (i) to catalyze incorporation of mono- and oligosaccharides into the CD core, (ii) to catalyze esterification of CDs with carboxylic acids, and (iii) to perform the cycloglycosylation of acyclic precursors. These three areas constitute the most important aspects covered in this section in the present review. The search for new and more effective drug carriers derived from CDs prompted the study of the action of several glycosylating enzymes on CDs in the presence of mono- or oligosaccharides as donor groups. In these derivatives, one or more primary hydroxyl groups of the CD core are substituted by sugar units to form a type of CD that is generically called a “branched CD”. This family of CD derivatives shows higher water solubility and lower hemolytic activity than native CDs,24,25 and such systems have found applications in areas such as pharmaceuticals and cosmetics.26 Enzymatically modified CDs can be produced in several ways (Table 1). Pullulanases catalyze the transfer of several kinds of mono- and oligosaccharides to the primary side of CDs in good yields (I in Table 1).27-32 This method is preferred to the action of CGTase on starch, which also produces branched CDs but in much lower yields.31,33 On a laboratory scale, use of R-maltosyl fluoride as a maltose source increases greatly the yield of maltosyl-CDs, although this process is not employed industrially.34,35 In general, monosubstituted derivatives are obtained along with traces of oversubstituted products that can be separated by HPLC. Diglycosylated-CDs, as well as trace amounts of the trisubstituted derivatives, have also been found in the conversion mixtures used for production of branched CDs.36-38 These derivatives show even higher water solubility than monosubstituted CDs, a property that can be used in the development of drug formulations. The combined action of isoamylase and pullulanase on RCD has been employed to produce branched CDs bearing higher maltose oligomers. The branching maltooligosaccharides (from maltotriose to maltohexose) are first generated by isoamylase treatment followed by coupling to RCD to give the oligomaltosyl-CDs in 22-35% yield.39,40 GlucosylCDs have also been prepared by enzymatic degradation of Mal-CDs with glucoamylase.41,42 In turn, the action of some amylases on branched CDs has been employed to synthesize some synthetically or biologically important oligosaccharides.43,44 In this way, 6ω-substituted maltooligosaccharides and 62-R-maltosyl-maltotriose have been prepared in one step with high yields. The hydroxymethyl group of the terminal glucose unit in the branching part of Mal-CDs has been regioselectively oxidized by an alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes to give a family of branched CDs containing a carboxylic acid group (II in Table 1).45 These glucuronic acid CDs show even lower hemolytic activity than the corresponding nonionic CD derivatives and negligible

Villalonga et al.

cytotoxicity on human intestinal epithelial cells. The binding constants for inclusion of the basic drug chlorpromazine at physiological pH decrease in the order glucuronyl-βCD > maltosyl-βCD > βCD. This makes glucuronyl-CDs suitable carrier candidates for basic drugs.46 Receptors selective to mannose, galactose, and N-acetylglucosamine play key roles in cell surface recognition and are the basis of several targeted drug delivery systems. Koizumi and co-workers47 extensively studied formation of heterogeneous branched CDs bearing sugar units other than glucose. The reverse transfer reaction of R-galactosidase, R-mannosidase, and β-N-acetylhexosaminidase has been employed to attach R-galactosyl,48,49 R-mannosyl,50,51 and β-N-acetylglucosaminyl52,53 groups, respectively, to the primary side of the CD core (III in Table 1). Heterogeneous units can also be attached to the side chain of branched CDs (IV in Table 1).54-58 For example, Koizumi reported the selective attachment of mannose and N-acetylglucosamine units to the side chain of maltosyl-βCD using jack bean R-mannosidase47 and lysozyme.59 On the other hand, heterobranched CDs have been prepared by pullulanasecatalyzed transfer of 4-O-β-D-galactosyl-maltose,60 6-O-RD-mannosyl-maltotriose,61 and 6-O-β-D-N-acetylglucosaminylmaltotriose56 to βCD. This strategy takes advantage of the selectivity of pullulanase toward glucose-terminated substrates. Formation of oversubstituted derivatives during the branching reactions can be avoided by introducing an alkyl spacer on the CD core (V in Table 1). Rabiller regioselectively introduced a galactosyl group on mono-6-(2-hydroxyethylamino)-6-deoxy-βCD.62 In the key glycosylation step Rgalactosidase was used to catalyze the regioselective transfer of the sugar unit on the hydroxyl group of the spacer. Products directly substituted on the CD molecule were not detected, indicating that the enzyme shows a preference for the more accessible hydroxyl groups of the substituent. Combination of chemical and enzymatic steps has been successful in the preparation of very complex CD clusters. One interesting example is attachment of the sialyl Lewis X (SLeX) tetrasaccharide [Neu5AcR2,3-D-Galβ1,4-(L-FucR1,3)D-GluNAcβ1-OR] onto a per-substituted βCD platform, as reported by Furuike et al. (Figure 2).63 Since steric hindrance can hamper the extension of the branching substituent, the subsequent glycosylations were carried out enzymatically in the presence of the appropriate glycosyltransferase. The target product was obtained as a monodisperse material in a remarkable overall yield of 67%, revealing the usefulness of the combined strategy employed to build complex oligosaccharides on a CD core. This SLex-branched βCD inhibits the binding of E-selectin to an SLex-BSA biochip and showed an enhanced cluster effect when compared with the native SLex tetrasaccharide.63 Acylation of CDs has been achieved by means of proteases and lipases in organic media (VI in Table 1).64-69 These enzymes catalyze transesterification reactions to CDs with some degree of regioselectivity, although in most cases mixtures of products are produced. Rhizomucor miehei lipase was used to acylate βCD and its methyl and hydroxypropyl derivatives with a series of carboxylic acids, from acetic to stearic acid.66 No correlation between the degree of substitution and the alkyl chain length was observed. Recently, the regioselective esterification of βCD with some fatty acid vinyl esters by means of immobilized preparations of thermolysin and subtilisin was reported.67 Treatment of βCD



Table 1. Overview of the Main Strategies for Enzymatic Modification of Cyclodextrins

with vinyl decanoate as an acyl donor and thermolysin in DMSO resulted in the preferential formation of heptakis(2O-decanoyl)-β-cyclodextrin. In contrast, monoacylated βCDs at the C-2 position have been observed as the major products on using divinyl esters of dicarboxylic acids in the presence of Bacillus subtilis alkaline protease in DMF.68 These derivatives, obtained in up to 80% yield, are useful candidates for the preparation of polymeric materials containing CDs.

Use of an excess of the acyl donor prevented formation of dimeric products through attachment of a CD unit to both ends of the dicarboxylic acid. The same strategy has recently been employed for attachment of nonsteroidal anti-inflammatory drugs such as indometacin, ketoprofen, and etodolac to βCD.69 Although the biological activity of the conjugates has not been reported, this strategy could be extended to construction of other drug-appended CDs.


Villalonga et al.

Figure 2. Stepwise synthesis of sialyl Lewis X branched βCD.

Figure 3. CGTase-catalyzed cyclooligomerization of a R-maltosyl fluoride to produce substituted 6-O-methyl-CDs.

In the examples presented so far, enzymes have been used to attach residues of a different nature to native or previously modified CDs. Another strategy to obtain CD derivatives with the aid of enzymatic reactions has been developed and is based on the cycloglycosylation of activated di- or trisaccharides in the presence of CGTase.70-72 For instance, 62-O-methyl-R-maltosyl fluoride undergoes CGTase-catalyzed glycosylation to give a mixture of linear and cyclic oligomers (Figure 3).70 Similarly, 63-iodo-63-deoxy-R-maltotriosyl fluoride produces 6A,6D-diiodo-6A,6D-dideoxy-RCD in 38% yield,71 a derivative that is useful for the preparation of other substituted CDs (Figure 4). Despite the demonstrated utility of this approach, little has been done in this area, perhaps due to the relative difficulty in the preparation of

the acyclic precursors, which often requires several protection-deprotection steps.

4. Cyclodextrins in the Biocatalytic Transformation of Organic Compounds through Enzyme-Catalyzed Reactions In recent decades the chemical industry has been very concerned with the use of enzymes to transform organic compounds. Enzyme biocatalysts are currently applied in the production of fine chemicals, pharmaceuticals, and agricultural chemicals. The attractiveness of this approach undoubtedly comes from the high levels of chemoselectivity, regioselectivity, and stereoselectivity, cleanness, ease of disposal,


Figure 4. CGTase-catalyzed synthesis of 6A,6D-diiodo-6A,6Ddideoxy-RCD from a substituted R-maltotriosyl fluoride.

and the fact that biocatalytic processes generate far fewer residues than chemical processes. It is for these reasons that biocatalysis is often referred to as “green chemistry”.73 There are several intrinsic difficulties encountered in enzyme biocatalysis, and these include product and substrate inhibition, low solubility of reactants in the reaction media, slower reaction rates, and the fact that the enzymes are prone to be inactivated under various operational conditions.74 CDs have emerged as powerful tools for solving some of these problems, mainly due to their ability to form stable inclusion complexes with a wide variety of hydrophobic compounds. In fact, use of CDs in enzyme biocatalysis is an extensive and well-documented topic,75 and the effects that these materials have can be summarized as illustrated in Figure 5.

4.1. Increased Substrate Solubility and Availability with Cyclodextrins Under physiological conditions, enzymes work in aqueous media at pH values near neutral. It should be expected

Figure 5. Effects of CDs on enzyme biocatalysis.


that many biocatalytic transformations could be better performed under similar conditions. However, a large number of organic compounds have poor water solubility, which reduces their availability for transformation by enzyme-catalyzed reactions. On the other hand, enzymatic reactions in heterogeneous systems are characterized by slow rates of conversion resulting from the reduced transfer of substrates between the phases and to diffusion restrictions. Inclusion within CD cavities can improve the water solubility and availability of organic compounds, as substrates, in biocatalytic reactions (Figure 6). HydroxypropylβCD has been used in supramolecular carrier systems to facilitate the enzymatic synthesis of 7R-hydroxycholest-4en-3-one by oxidation of 3β,7R-cholest-5-ene-3,7-diol with cholesterol oxidase (EC 1.1.3.6).76 This route was significantly improved (more than 90% conversion) thanks to the participation of CDs in the solubilization of the substrate. This change enabled the transformation to 4-hydroxyestradiol with phenol oxidase (EC 1.10.3.1) from Mucuna pruriens with a 40% conversion yield. Mushroom tyrosinase (EC 1.14.18.1) converted 17β-estradiol, as a βCD complex, into 2-hydroxyestradiol in a selective way with a maximum yield of 30%. Significantly, the uncomplexed estradiol was not converted at all in either of these biocatalytic systems.77 These results indicate that βCD not only solubilizes the substrate but also favors its reactivity by acting as a catalyst. Complexation of fatty acids (both saturated and unsaturated) with CDs also increased their solubility in polar systems.78 The hydrolytic activity of lipases (EC 3.1.1.3) is, in general, markedly improved in the presence of CDs and their derivatives.79 Several natural and chemically modified CDs were evaluated as host compounds to improve substrate solubility and availability in the lipase-catalyzed esterification of oleic acid with n-butanol, and an increase in the conversion rate was observed in most of cases.80 The highest degree of transformation was achieved in the reaction performed in the presence of γCD. The biocatalytic activity of lipoxygenase (EC 1.13.11.12) has also been modulated by association of the substrates with CDs.81 Oxygenation of linoleic or arachidonic acid, entrapped in CDs (R, β, and γ), by soy bean lipoxygenase was higher


Villalonga et al.

also been complexed with methylated βCD and further oligomerized using this enzymatic method.88 In such cases, partial association between the CDs and the synthesized polymers has been described. These approaches undoubtedly constitute valuable contributions to “green chemistry” by reducing the levels of chemical and toxic waste generally produced in polymer synthesis.89

4.2. Reduced Substrate and Product Inhibition by Association with Cyclodextrins Figure 6. Solubilization of organic compounds by association with CDs.

by a factor of about five than that obtained on using Tween 20 as a solubilizing agent.82 The enzyme immobilized on Eupergit C also displayed better catalytic properties in the production of hydroperoxides from linoleic acid in the presence of βCD.83 In this regard, the yield of the enzymatic reaction performed with βCD was seven times higher than those without the oligosaccharide. Other interesting studies concerned the enzymatic polymerization of several organic compounds, which were solubilized by association with CDs (Figure 7). Horseradish

Figure 7. Enzymatic polymerization of phenols included in CDs.

peroxidase (EC 1.11.1.7) has been employed as a catalyst in aqueous media for oxidative polymerization of phenol84 and hydrophobic bifunctional phenols85 included in 2,6-diO-methyl derivatives of RCD and βCD. This enzyme was also used to polymerize inclusion complexes of 2,4-dihydroxyphenyl-4′-hydroxybenzylketone/2,6-dimethyl-βCD86 and ethyl 1-[(4-hydroxyphenyl)aminocarbonyl]-2-vinylcyclopropane carboxylate/2,6-dimethyl-βCD.87 Several acrylates have

Many enzymatic reactions are prone to substrate or product inhibition, a process that deactivates the enzyme at high concentrations of these compounds and leads to a decrease in the reaction rate. This problem can be solved by keeping the concentrations of these adducts at low levels either by continuous addition (or delivery) of the substrate or by gradual removal of the product by physical or chemical methods. When the substrate or products have structural properties that favor association with CDs, these oligosaccharides can be effectively employed as additives to increase biocatalytic reaction rates.75a The substrate inhibition exhibited by carboxypeptidase A (EC 3.4.173.1) in catalyzing the hydrolysis of (S)-2-O-(Nbenzoylglycyl)-β-phenyl lactate was limited by addition of βCD and hydroxypropyl-βCD (Figure 8). The CDs did not significantly change the maximum rate of reaction but increased the concentration of the substrate at which it was observed, an important result.90 βCD and its partially methylated derivative were used as molecular receptors during the lipase-mediated hydrolysis of triolein in aqueous solutions. The reaction rate of this enzymatic process was increased by up to a factor of 7 in the presence of the CDs, mainly by increasing substrate solubility and reducing product inhibition.91 Peracetylated βCD has been employed as a macrocyclic additive to enhance the enantiomeric ratio (E) and reaction rate in Pseudomonas cepacia lipase-catalyzed enantioselective transesterification of 1-(2-furyl)ethanol in organic solvents. The beneficial action of the CD used as a regulator of lipase was interpreted in terms of its host-guest complexation with the product, thereby preventing product inhibition.92

Figure 8. Effect of CD association on the inhibition of carboxypeptidase A by (S)-2-O-(N-benzoylglycyl)-β-phenyl lactate.



4.3. Increased Enantioselective Transformation of Substrates by Association with Cyclodextrins Due to their steric effects, CDs can play an important role in the enzymatic enantioselective transformation of organic compounds. When the prochiral guest molecule is included in the CD cavity, preferential enzymatic attack takes place only from one of the enantioselective faces, thus yielding higher enantioselectivity.75b,93 βCD has been used as an additive for the enzymatic stereoselective hydrolysis of (R,S)-1-phenylethyl propionate with Pseudomonas sp. Lipase. The enantioselective conversion of this substrate was improved in the presence of the oligosaccharide.94 Lipase was also employed for enantioselective hydrolysis of (R,S)-ketoprofen ethyl ester to the optically active (S)ketoprofen using hydroxypropyl-βCD as a chiral receptor (Figure 9). This strategy gave an enantiomeric excess of 99% Figure 10. Effect of hydroxypropyl-βCD on the lipase-catalyzed enantioselective hydrolysis of (R,S)-O-butyryl propanolol.

noindane as an amino donor in water-saturated ethyl acetate. The enantioselectivity of ω-transaminase for (2R)-1-amino2-indanol was increased to 22.1 in the presence of 5% γCD.100

4.4. Other Supramolecular-Based Effects

Figure 9. Effect of hydroxypropyl-βCD on the enantioselective hydrolysis of (R,S)-ketoprofen ethyl ester by lipase.

after conversion of 49% of the initial substrate.95 Use of native and methylated CDs as additives for the co-lyophilization of Pseudomonas cepacia lipase has been reported. These lyophilized adducts were subsequently employed as biocatalysts for the successful enantioselective transesterifications of racemic 6-methyl-5-hepten-2-ol and racemic 2,2-dimethyl1,3-dioxolane-4-methanol with a series of enol esters in cyclopentyl methyl ether solutions.96 The same strategy was also employed for Candida rugosa lipase, with increases in enantiomeric excess (S/R) of up to 2.3 times, on using dimethyl-βCD as an additive.97 The enzymatic preparation of (S)-propanolol by the hydrolytic action of Rhizopus niVeus lipase toward (R,S)-Obutyryl propranolol in the presence of hydroxypropylβCD has been described with a 90% enantiomeric excess achieved for the (S)-enantiomer (Figure 10).98 The same authors also reported the novel and promising intramolecular transacylation of (R,S)-O-butyryl propanolol catalyzed by Candida rugosa lipase in the presence of hydroxypropylβCD.99 The syntheses of trans-(1R,2R)- and cis-(1S,2R)-1-amino2-indanol have recently been described and involved a series of enantioselective enzymatic reactions using lipase and transaminase (EC 2.6.1.18) to catalyze the enantioselective hydrolysis of 2-acetoxyindanone to give (R)-2-hydroxy indanone. trans-1-Amino-2-indanol was produced from (R)2-hydroxy indanone using ω-transaminase and (S)-1-ami-

In addition to the uses described above, there are other examples in which CDs are employed as tools in different biocatalytic transformations. There are several interesting examples in which CDs have been employed as enzyme activators in organic media, an approach that does not involve an increase in solubility. In this type of reaction the catalytic effect of the CD involves host-guest interactions through H-bond formation. Co-lyophilization of subtilisin Carlsberg (EC 3.4.21.14) with methyl βCD increased the activity and enantioselectivity of the enzyme suspended in dry THF and acetonitrile in the transesterification of N-acetyl-L-phenylalanine ethyl ether with 1-propanol and transesterification of vinyl butyrate with sec-phenethyl alcohol. The mechanism proposed for this effect is based on the ability of the CD derivative to act as a “molecular lubricant” for the protein, probably after supramolecular association, which increases the structural mobility of the enzyme in organic solvents. The beneficial effect of this “molecular lubrication” is an increase in the catalytic activity and enantioselectivity.101 This interesting co-lyophilization strategy improved the long-term stability of this enzyme in THF.102 A similar increase in the catalytic activity of this protease was achieved by co-lyophilization with R-, β-, and γCD as well as with tri-O-methylβCD, while hydroxypropyl-βCD diminished the activity of the enzyme.103 In the same report, the effect of colyophilization with hydroxypropyl-βCD on the catalytic activity of R-chymotrypsin was studied using the transesterification of N-acetyl-L-tyrosine ethyl ester and methanol in an acetonitrile/water system. The results show that colyophilization with the CD derivatives led to a remarkable improvement in the catalytic activity of the enzyme, suggesting that this effect was mediated by the structural stabilization of the protein conformation in the acetonitrile/ water system.103 Co-lyophilization with βCD also increased the catalytic activity of a lipolytic extract of Penicillum coryophilum


toward p-nitrophenyl palmitate in n-heptane. In contrast, the same approach significantly reduced the performance of the enzyme in the synthesis of n-butyl oleate.104 In several applications, especially pharmacological ones, stabilization of the target molecule against enzymatic degradation is highly desired. In some cases, this goal can be achieved by association with CDs, which mask the potential cleavage sites as illustrated in Figure 11.

Figure 11. Reduction of enzyme-catalyzed transformation of substrates by association with CDs.

Desmopressin [1-(mercaptopropanoic acid)-8-D-arginine vasopressin], a synthetic analogue of the antidiuretic hormone vasopressin, is rapidly hydrolyzed by treatment with R-chymotrypsin (Figure 12). In the presence of hydroxypropyl CDs

Figure 12. Target sites for enzymatic cleavage (marked with arrows) of desmopressin (up) and furamide (down).

(β and γ), which can form inclusion complexes with the aromatic amino side chains of Tyr and Phe, the drug was nine times more stable against proteolytic degradation. On the other hand, the hydroxypropyl derivative of RCD only led to a protection factor of 3 under similar conditions.105 Similarly, other peptide drugs, such as deslorelin and buserelin acetate, have been protected against degradation by proteases with the aid of CD derivatives.106,107

Villalonga et al.

βCD and two of its methyl derivatives, 2,6-di-O-methylβCD and 2,3,6-tri-O-methyl-βCD, have been examined as protective agents for Furamide (2-furoyl 2,2-dichloro-4hydroxy-N-methylacetanilide) (Figure 12). The hydrolysis rate of this antiparasitaric drug by Candida cylindracea lipase was significantly reduced in the presence of the methylated CDs, not only by formation of inclusion complexes but also by partial inhibition of the enzyme.108 The biotransformation of several metabolites can give rise to undesirable effects. This situation is commonly observed in agricultural and food products, which progressively lose organoleptic and health qualities upon storage. It has been suggested that CDs may moderate the enzymatic browning of different fruits and vegetables because they form inclusion complexes with the substrates of polyphenol oxidase (EC 1.10.3.1), thereby preventing their oxidation to quinones and subsequent polymerization to brown pigments (Figure 13).109 This protecting effect has also been reported for the oxidation of phenols by lipoxygenase (EC 1.13.11.12) in which CDs act as secondary antioxidants in synergy with ascorbic acid.81,110 In a similar way, the slowing of the biocatalytic transformation of L-tyrosine with tyrosine phenol lyase (EC 4.1.99.2) due to its association with permethylated CDs (R and β) has been reported (Figure 14). In contrast, a higher conversion rate was found for this enzyme-catalyzed reaction in the presence of permethylated γCD, mainly due to the improved substrate recognition mechanism.111 CDs can serve as molecular receptors for changing the selective transformation of a mixture of substrates by enzymes. For example, RCD altered the substrate selectivity of R-chymotrypsin in catalyzing the hydrolysis of (S)-Nacetylleucine methyl ester and (S)-N-acetylphenylalanine methyl ester (Figure 15). It was suggested that this effect was mediated by complexation of the substrates by the CD.90

5. Cyclodextrin−Enzyme Neoglycoconjugates with Improved Functional Properties Enzymes are useful tools for the design of new and more efficient industrial processes, preparing valuable cosmetic and laundry products, and construction of selective and sensitive analytical devices.112 These catalytically active

Figure 13. CD-mediated protection of catechol against enzymatic oxidation.


Figure 14. CD-mediated modulation of the reaction rate for the enzymatic transformation of tyrosine with tyrosine phenol.

proteins are also effective drugs in enzyme replacement therapies against several illnesses.113 However, the practical uses of enzymes are often limited by their rapid inactivation upon exposure to elevated temperatures, extreme pH values, proteolytic attack, or the presence of surfactant or chaotropic agents.114 Given these drawbacks preparation of highly stable enzymes that are able to work under harsh conditions is required for biomedical, industrial, and biotechnological applications. Among the various approaches described to stabilize enzymes in aqueous media, chemical modification using water-soluble molecules constitutes one of the most promising. In general, the success of this method depends on choosing appropriate conditions based on the following factors: (1) the type, size and structure of the enzyme, (2) the structure and size of the modifying agent, and (3) the type of reaction and the conditions involved in the modification procedure.115 In particular, covalent conjugation of commercially available enzymes with carbohydrate derivatives has recently received considerable attention in enzyme technology.116 This strategy is based on the structural and functional stability that oligosaccharide chains confer on naturally occurring glycoenzymes.117 The unique structural and functional properties of carbohydrates, such as water


solubility, biocompatibility, and absence of toxicity, have also favored use of these compounds in the preparation of artificially modified enzymes, called “neoglycoenzymes”. Due to the complexity of this type of reaction it is impossible at present to predict which method will give the best results. Only one rule stands for all good methods: mild conditions. The stabilization conferred on enzymes by covalent glycosidation with carbohydrate compounds occurs through a combination of several factors. Among these, covalent cross-linking of the protein structure with sugar derivatives should be considered the most important approach in neoglycoenzymes prepared by multipoint attachment of ionic or chemically polyactivated carbohydrate derivatives.116a-c Other factors, such as hydrophilization of the enzyme surface, formation of new hydrogen bonds, and creation of salt bridges when charged polysaccharides are used, are also involved in maintaining the active neoglycoenzyme forms. In the case of enzymes with potential applications in drug formulations, the steric hindrance caused by the attached sugar moieties improves the protection against proteolytic degradation and recognition by the immune system.118 CDs have exceptional supramolecular host properties that allow their evaluation as unique stabilizers of the catalytically active conformations of enzymes. CDs are capable of forming stable inclusion complexes with hydrophobic guest compounds, such as the aromatic amino acid side chains located at the surface of enzymes.4,8 This kind of interaction enables stable intramolecular cross-links at the surface of the neoglycoenzymes formed by the conjugation of CDs moieties. In the intramolecular cross-linking of R-amylase (EC 3.2.1.1) with a polyaldehyde derivative of βCD using periodate oxidation the unavoidably extreme chemical conditions required to activate the oligosaccharide destroyed the structure of the βCD derivative, and consequently, the resulting modified enzyme cannot be considered as a true CD-based neoglycoenzyme.119

Figure 15. CD-mediated selective transformation of a mixture of (S)-N-acetylleucine methyl ester and (S)-N-acetylphenylalanine methyl ester with R-chymotrypsin.


Villalonga et al.

Figure 16. Synthesis of neoglycoenzymes by the interaction of commercially available enzymes with monoactivated CD derivatives.

5.1. Chemical Modification of Enzymes with Monoactivated Cyclodextrin Derivatives CDs can be modified at one or several hydroxyl groups with a wide variety of substituents, and acidic and basic derivatives can be prepared by chemical21 or enzymatic6 methods. An interesting family of CD derivatives that can be used for enzyme modification consists of compounds bearing a reactive group at a C-6 position. Attachment of a monoaldehyde derivative of βCD to the -amino groups of lysine residues of two different serine proteases, trypsin (EC 3.4.21.4) and R-chymotrypsin (EC 3.4.21.1), by reductive alkylation with NaBH4 has been reported (Figure 16).120-121 In this transformation the hydrolytic enzymes acquired remarkable stabilization against autolytic degradation in alkaline conditions (Figure 17), which is a desired property for those proteases used for laundry and industrial detergent formulations. In addition, both CD-modified enzymes showed

Figure 17. Kinetics of autolytic degradation for native (dashed line) and βCD-modified (solid line) trypsin (9) and R-chymotrypsin (B) at pH 9.0.

a noticeable improvement in their thermal stability properties, retaining high catalytic activity after incubation at elevated temperatures.121 Several monoactivated CDs with a carboxylate group at a C-6 position have also been employed to modify proteolytic and redox enzymes. Conjugation of R-chymotrypsin with mono-6-succinyl-6-deoxy-βCD was carried out using 1-ethyl3-(3-dimethylaminopropyl)carbodiimide (EDAC) as the coupling agent.121b The esterolytic activity of the enzyme was increased to about 122% after attachment of five CD moieties (Figure 17), suggesting that the active conformation of the enzyme was not affected by this glycosidation. High catalytic activity was also retained by Bacillus badius phenylalanine dehydrogenase (EC 1.4.1.20) after glycosidation with mono6-(5-carboxypentane-1-carboxamidoyl)-6-deoxy-βCD using a similar coupling strategy.122 In the case of this homooctameric enzyme this transformation yielded an improvement in both the catalytic efficiency and the affinity of the enzyme for its substrate. In a more detailed study trypsin was modified with monocarboxylated βCD derivatives that incorporated spacer arms of different lengths.123 Mono-6-(4-carboxybutane-1carboxamidoyl)-6-deoxy-βCD, mono-6-(5-carboxypentane1-carboxamidoyl)-6-deoxy-βCD, and mono-6-(10-carboxydecane-1-carboxamidoyl)- βCD were used as modifying agents for this serine protease. The CD-trypsin conjugates showed relevant increases in their hydrolytic activity (Figure 18), especially the adduct prepared with mono-6-(4-carboxybutane-1-carboxamidoyl)-6-deoxy-βCD. In comparison to the native trypsin, the esterolytic activity of this conjugate was higher by a factor of 2. This result can be considered relevant, especially given that a decrease in the enzymatic activity is generally observed when chemical conjugation is used. On the other hand, it is difficult to design new enzyme forms with such an increase in catalytic activity either by genetic engineering or covalent transformation. The observed catalytic efficiencies were 245%, 185%,


Figure 18. Specific activity of trypsin (Tryp) and its conjugates with mono-6-(4-carboxybutane-1-carboxamidoyl)-6-deoxy-βCD (Tryp-CD1), mono-6-(5-carboxypentane-1-carboxamidoyl)-6-deoxyβCD (Tryp-CD2), and mono-6-(10-carboxydecane-1-carboxamidoyl)-βCD (Tryp-CD3).

and 185% higher for the protease modified with mono-6(4-carboxybutane-1-carboxamidoyl)-6-deoxy-βCD, mono-6(5-carboxypentane-1-carboxamidoyl)-6-deoxy-βCD, and mono6-(10-carboxydecane-1-carboxamidoyl)-β-CD, respectively. In addition, these CD-trypsin complexes were more efficient biocatalysts in the synthesis of the benzoyl-arginine-phenylalaninamide dipeptide when compared to the native counterpart. All conjugates prepared by attaching monocarboxylated βCD derivatives to the surface of enzymes were significantly more resistant to thermal inactivation. From a practical point of view, these results are very important bearing in mind that enzymatic reactions conducted at elevated temperatures are desired in order to improve substrate conversion rates and reduce microbial contamination. Mono-6-amino-6-deoxy derivatives of R-, β-, and γCD were linked to the aspartic and glutamic acid residues located at the surface of bovine pancreatic trypsin through a reaction catalyzed by EDAC.124 In addition to the improved thermostability shown by the resulting neoglycoconjugate, the esterolytic activity of this protease was improved after glycosidation with CDs and the enzymatic activity increased when the ring size of the oligosaccharide increased. Mono6-amino-6-deoxy-βCD, mono-6-ethylenediamino-6-deoxyβCD, mono-6-propylenediamino-6-deoxy-βCD, and mono6-butylenediamino-6-deoxy-βCD derivatives were also used to modify the protease.125 The catalytic and thermal stability properties of trypsin were improved by attachment of CD residues, and these effects were particularly marked for CD derivatives having an even number of carbon atoms in the spacer arms (Figure 19). On the basis of these results it was suggested that both the functional and the stability properties of some enzymes could be modulated by controlling the size of the CD ring or the length of the spacer arms between the protein and the oligosaccharide residues. The mono-6-amino-6-deoxy-βCD derivative was also successfully employed in the preparation of thermostable neoglycoconjugates of phenylalanine dehydrogenase122 and R-chymotrypsin.126 Again, improved catalytic properties were observed in comparison to the non-glycosidated forms. A possible explanation for the observed increase in the affinity for the substrate exhibited by several of the CD-modified enzyme forms could be formation of inclusion complexes


Figure 19. Half-life times at 60 °C for trypsin (Tryp) and its conjugates with mono-6-amino-6-deoxy-βCD (Tryp-CD1), mono6-ethylenediamino-6-deoxy-βCD (Tryp-CD2), mono-6-propylenediamino-6-deoxy-βCD (Tryp-CD3), and mono-6-butylenediamino6-deoxy-βCD (Tryp-CD4).

between the hydrophobic substrates (BAEE, ATEE, and Phe) and the CD moieties conjugated near the active site. This kind of inclusion complex could increase the concentration of the substrate in the microenvironment of the active site, shifting the equilibrium to formation of the Michaelis complex. The thermal stabilization conferred on the enzymes by conjugation with all of the monoactivated CD derivatives studied was partially mediated by the occurrence of supramolecular associations between the attached CDs and the hydrophobic amino acid residues located at the surface of each neoglycoenzyme. In this way a more compact enzyme structure is formed, and this reinforces the folding of the protein when exposed to elevated temperatures, which in turn enables the protein to retain its high catalytic activity (Figure 20). This fact was proved by enzymatic and fluorescence experiments.121,122

Figure 20. Supramolecular-based mechanism proposed for the thermal stabilization of the CD-enzyme neoglycoconjugates.

5.2. Enzymatic Glycosidation of Enzymes with Monoaminated Cyclodextrins Chemical modification of enzymes with carbohydrate derivatives is particularly promising since this procedure is simple, inexpensive, and allows preparation of water-soluble biocatalysts with high functional stability.127 However, several disadvantages are associated with the toxicity of the reagents commonly used to modify enzymes by chemical


procedures, and these compounds are often unsuitable for preparing catalysts to be used in biomedical and food applications.128 The logical alternative to overcome these problems is use of enzymes to catalyze the attachment of the modifying sugar moieties to selected amino acid residues at the protein surface of the target biocatalyst. In this regard, transglutaminases (TGase, EC 2.3.2.13) constitute a powerful tool. Transaminases catalyze formation of intra- and intermolecular isopeptide bonds using the γ-carboxamide group of endoprotein glutamine residues as acyl-donor substrates and the -amino group of endoprotein lysine residues as acyl acceptors.129 In addition, several low molecular weight compounds containing primary amino groups can also be used as amino-donor substrates, giving rise to a variety of protein-(γ-glutamyl) derivatives.130 Another advantage associated with use of TGase as a catalyst for preparation of neoglycoconjugates is modification of glutamine units, which are uncommon target points for chemical transformation and not associated with enzymatic activity. From a practical point of view, this enzymatic approach seems to be highly effective considering that (1) microbial TGases are cheap products that are already in the market and (2) the enzymatic coupling process could be optimized using immobilized TGase forms. An intrinsic inconvenience of this enzymatic approach is that the number of proteins acting as glutaminyl substrates for TGases is restricted as both the primary and threedimensional protein structure determine whether or not a glutamine residue reacts as an acyl-donor substrate for the enzyme.129 For this reason, the ability of the target enzyme to act as a TGase amino acceptor should first be evaluated. Bovine pancreatic trypsin can be recognized as a substrate for StreptoVerticillum sp. TGase.131 The enzymatic glycosidation of trypsin with the mono-6amino-6-deoxy derivatives of R-, β-, and γCD has been described (Figure 21) and yielded neoglycoenzyme forms

Figure 21. Enzymatic preparation of trypsin-CD neoglycoconjugates by a TGase-catalyzed reaction.

with an average of 3 mol of oligosaccharide attached to each mol of protein.131 These conjugates showed improved catalytic performance, as evidenced by the values obtained for Km, kcat, and kcat/Km. These CD-trypsin complexes were also significantly more resistant to both autolytic degradation and incubation at elevated temperatures. Similar results were achieved on using several monoactivated βCD derivatives with different spacer armssmono-6-ethylenediamino-6deoxy-βCD, mono-6-propylenediamino-6-deoxy-βCD, mono6-butylenediamino-6-deoxy-βCD, and mono-6-hexylenediamino-6-deoxy-βCD.132 However, the esterolytic activity of trypsin was only increased after modification with the CD

Villalonga et al.

derivatives bearing an even number of carbon atoms in the spacer arms. This improvement in the catalytic hydrolysis of BAEE decreased when the length of the spacer arms in these modifying CD derivatives was increased.

5.3. Covalent Cross-Linking of Enzymes with Polymeric Cyclodextrin Derivatives Multipoint attachment of polymeric structures onto an enzyme surface is a well-known strategy to improve the stability of biocatalysts.127,133 When using carbohydrate-based macromolecules, formation of new stabilizing hydrogen bonds between the polypeptide and the modifying agent also contributes to the maintenance of the active enzyme conformation. On this basis and considering that supramolecular associations are reinforced when multivalent complexes are formed134 it could be expected that CD-based polymers can be successfully employed as glycosidation agents for stabilizing enzymes. Conjugation of carboxymethylcellulose (MW ) 30 kDa, 70% carboxymethylation) with βCD moieties yielded a water-soluble macromolecular structure with an average of 28 mol of βCD linked to each mol of polymer. This polysaccharide could be easily linked to the -amino groups of lysine residues in proteins through a carbodiimidecatalyzed reaction. This approach was employed to prepare a neoglycoform of Bacillus subtilis R-amylase, which retained about 90% of the initial amylolytic activity.135 The resulting neoglycoenzyme showed improved thermal stability with a 5-fold increase in its half-life time after incubation at 75 °C. An improvement in the functional behavior and stability was also achieved for other enzymes by its cross-linking with the aforementioned βCD-carboxymethylcellulose derivative.136a The trypsin conjugate retained about 110% and 95% of the initial esterolytic and proteolytic activity, respectively. Increases of about 8 and 16 °C in the optimum and melting temperatures were observed, respectively, for trypsin after conjugation. The neoglycoenzyme was also more stable against thermal incubation and also more resistant to autolytic degradation at pH 9.0. βCD-carboxymethylcellulose polymer was also conjugated to Cu,Zn-superoxide dismutase (SOD) (EC 1.15.1.1), its anti-inflammatory activity (carrageenaninduced paw edema) was increased 2.2 times after conjugation, and its plasma half-life time was prolonged from 4.8 min to 7.2 h.136b When catalase (EC 1.11.1.6), another antioxidant enzyme, was linked to the SOD neoglycoconjugate the anti-inflammatory activity increased 4.5 times. This result is due to protection catalase offers against inactivation of SOD by H2O2.136c A useful glycosidation agent was synthesized by crosslinking βCD with epichlorohydrin and further activation by introducing carboxylate groups into the macromolecular structure. This O-carboxymethyl-poly-βCD derivative (MW ) 13 kDa, 40% carboxymethylation) was employed to prepare neoglycoforms of trypsin and R-chymotrypsin (Figure 22), which retained high proteolytic and esterolytic activity.137a,b Glycosidation with this βCD-based polymer conferred resistance to both proteases against autolytic degradation at alkaline pH. These neoglycoenzymes were also markedly more stable than their native counterparts. The thermal stabilization conferred on both proteolytic enzymes after glycosidation with O-carboxymethyl-poly-βCD is an important result, especially considering all of the advantages associated with the use of enzymes at high temperatures.



Figure 22. Synthesis of neoglycoenzymes with O-carboxymethyl-poly-βCD. L-Phenylalanine

dehydrogenase (EC 1.4.1.20) was also conjugated with O-carboxymethyl-poly-βCD. Fluorescence studies revealed that a more compact protein structure was formed after glycosidation of the enzyme, and this conformational effect was justified by the occurrence of supramolecular cross-links between the protein and the attached CDbased polymer.137c The conformational stabilization observed for the enzymes modified with the CD polymers can be considered as the result of the cooperative contribution of several factors. Among these, covalent cross-linking with the macromolecular structures and formation of new hydrogen bonds and electrostatic interactions at the surface of the glycosidated proteins should be involved in the improved stabilization shown by these neoglycoconjugates. However, it was demonstrated that the occurrence of host-guest interactions between the attached CD moieties and the amino acid residues at the surface of the modified proteins are also associated with the thermal stabilization shown by the neoglycoenzymes prepared with the CD-based polymers.135-137

6. Cyclodextrins As Chaperones for Enzymes For some denatured proteins refolding constitutes a complex process that requires highly specific external assistance. In these cases refolding has received considerable attention at both the fundamental and the practical levels.138 From an experimental point of view, one of the key factors to be considered for the refolding of proteins is the occurrence of protein aggregation processes between the unfolded polypeptide chains.139 Protein aggregation is an important phenomenon in biotechnology in which the critical

step in the recovery of an active recombinant protein from solubilized inclusion bodies is protein refolding.140 The thermal inactivation mechanism of enzymes and other active proteins also involves formation of protein aggregates from unfolded intermediates with exposed hydrophobic residues. Similarly, protein aggregation constitutes an obstacle to the proper refolding of enzymes after treatment with denaturant agents such as guanidine hydrochloride and urea. The assistants in protein refolding are called chaperones. One such chaperone is GroEL, which is obtained from E. coli and is the best known protein that plays the role of assisting the refolding of denatured proteins. Some small molecules, called artificial chaperones, can serve a similar purpose. Most of the artificial chaperones studied to date are water-soluble additives. Several compounds, such as alcohols,141 detergents,142 polyethylene glycols,143 anions,144 and polyamines,145 have been successfully employed as folding aids for enzymes and other proteins, preventing aggregation by interfering with intermolecular hydrophobic interactions. Sensitive microcalorimetric studies on CD interactions with amino acid groups in unfolded proteins revealed that they may enhance the solubility of the denatured polypeptides. This can be achieved by masking the exposed hydrophobic residues and then assisting the refolding of the protein.146 Several approaches using CDs as potential chaperone mimics in protein refolding have been described. This renaturing process is inhibited by poorly reversible aggregation mechanisms. The simplest method for refolding enzymes and other proteins with CDs involves diluting the unfolded polypep-


Villalonga et al.

Figure 23. Direct refolding of denatured enzymes with CDs.

Figure 24. Two-step renaturing method for enzymes with CDs.

tides in solutions that contain either native CDs or their derivatives (Figure 23). This refolding strategy for enzymes is strongly modulated by the size and chemical structure of the CD derivatives. A high degree of esterase activity for carbonic anhydrase B (EC 4.2.1.1), previously denatured with guanidium chloride, was recovered by using RCD as a chaperone mimic.147 This result can be explained by the stronger association complexes formed between the aromatic rings located in the side chains of several amino acid residues and RCD in comparison with those formed with the β- and γ- counterparts. Substitution of hydroxy groups in CDs also leads to enhanced or decreased refolding ability for these oligosaccharides. A high level of refolding was achieved for carbonic anhydrase with acetyl-βCD and acetyl-γCD.131 Hydroxypropyl-βCD also exhibited good chaperone-like activity for this enzyme, but its enzymatic activity was completely lost when carboxymethylated CDs were employed as artificial chaperones.148 These interesting results suggest the possibility of designing specific CD derivatives with the desired chaperonelike activity. Alkaline phosphatase (EC 3.1.3.1) has also been refolded using this approach.149 Inspired by the GroEL chaperone system, a two-step refolding approach based on the sequential use of detergents and CDs has also been described.150 In the first step, a detergent forms a complex with the non-native protein and prevents aggregation, something very important. In the second step, CDs strip the detergent away from the protein, allowing proper refolding (Figure 24). This strategy has been

successfully employed to renature several enzymes, such as carbonic anhydrase, lysozyme (EC 3.2.1.17), citrate synthase (EC 2.3.3.1), xylanase (EC 3.2.1.8), and R-amylase.84,150 Enzymes (such as lysozyme) have also been refolded by size exclusion chromatography with incorporation of this artificial chaperone system.151 A similar approach for refolding enzymes has recently been described but uses high molecular weight CDs. In combination with surfactants these high molecular weight R-1,4-glucans, which have a degree of polymerization of 22 and 50 (referred to as cycloamyloses), exhibited artificial chaperone properties toward three enzymes in different categories: citrate synthase, carbonic anhydrase B, and lysozyme.152 A simplified variation of the two-step artificial chaperone method has recently been reported for lysozyme.153 This strategy is based on the direct addition of the surfactant to the denatured solution, which is then diluted directly into a refolding buffer containing CD. This simplified approach was used to refold lysozyme in a higher yield than those achieved by the classical two-step method. A different and more interesting refolding method has been described for lysozyme and carbonic anhydrase B and is based on the initial inclusion of the unfolded enzyme in spontaneously formed hydrogel nanoparticles of cholesterolbearing pullulans (Figure 25). Aggregation processes in the thermally denatured enzymes are avoided. In the second step the hydrogels are disrupted by association with CDs, allowing renaturation of the enzyme polypeptides.154



Figure 25. Refolding of enzymes with CD and hydrogel nanoparticles of cholesterol-bearing pullulan. (Reprinted with permission from ref 154a. Copyright 1999 American Chemical Society.)

Figure 26. Proposed supramolecular-mediated thermal stabilization mechanism for enzymes with CD-branched polymers.

CD derivatives have been proposed as thermoprotectant agents for enzymes with the aim of preventing unfolding and aggregation processes when the proteins are exposed to elevated temperatures. Polysaccharides, such as dextran and polymerized sucrose, have been conjugated with monoactivated CD derivatives and further used as additives for trypsin.155 The protection conferred by the polymeric CD derivatives to the active enzyme conformation was directly related to the amount of oligosaccharide moieties attached to the macromolecular backbone. Addition of substances that are capable of disrupting associations between the CD groups and the hydrophobic amino acid residues located at the

protein surface of the protease markedly reduced the thermoprotectant effect. These results support a supramolecularbased mechanism by which the enzyme surface is covered by the CD-branched polymer, preventing both unfolding of the catalytically active conformation and formation of protein aggregates (Figure 26). A great deal of work is still required in studying the role of CD derivatives as mimetic refolding chaperones. However, it seems that such systems will be indispensable. The main question concerns the type of derivative that could serve as a “universal” mimetic chaperonesif indeed this goal is possible.


7. Supramolecular Methods for Immobilizing Enzymes on Macro- and Nanosized Supports Immobilization of enzymes on solid supports is an issue of great importance in the context of industrial applications despite the fact that several of the existing biocatalytic processes are homogeneous.156,157 The immobilized enzyme must maintain high levels of catalytic activitysexpressed in terms of turnover number and maximum specificity constant (kcat/KM). On the other hand, the thermostability of the enzyme must be increased in order to support the new environment and reaction conditions. In most cases the catalytic activity of an enzyme is partially lost upon immobilization because it is highly sensitive when outside its natural environment, i.e., the cell. When the immobilization is physical in nature and takes place on a charged surface, there is a high probability that the enzymatic activity will be affected due to alterations in the conformation of the enzyme. For example, the electrostatic immobilization of carbonic anhydrase (EC 4.2.1.1) on charged surfaces was studied by AFM. The enzyme was oriented in such a way that the majority of active sites faced upward on a positively charged surface and downward on a negatively charge surface (Figure 27).158

Figure 27. Schematic representation of carbonic anhydrase (a) and the enzyme immobilized on a negatively charged surface (b) and a positively charged surface (c). (Reprinted in part with permission from ref 158. Copyright 2006 American Chemical Society.)

In cases where a protein is to be immobilized chemically, the reaction must take place under mild conditions in order to ensure that the conformation is not affected. This negative effect on its catalytic properties can be observed even when the variation in the conformation of the enzyme may seem insignificant. The expected loss in catalytic activity must not affect the specificity of the enzyme. This means that the variations in the conformation of the enzyme must not influence the steric characteristics of the active site channel. Iimmobilization of an enzyme on a solid surface should lead to an increase in its thermal stability in order to be considered efficient. This prerequisite is met if the immobilization takes place through several functional groups on the biomolecule. Such multipoint anchoring of an enzyme onto a surface helps in retaining the conformation. When this process is supramolecular in nature it is called “supramolecular multivalency”.134 This principal was applied in the development of “molecular printboards” that permits immobilization of stable and highly organized and correctly oriented proteins.159

Villalonga et al.

The immobilization process may appear simple at first sight, but in practical terms it is far from straightforward. The two most common side effects of enzyme immobilization, namely, loss of activity and increased thermostability, may have opposite effects on the enzymatic activity. Therefore, the changes in both factors upon immobilization must be analyzed as a whole. If the observed increase in thermal stability of an immobilized enzyme can be considered to offset the loss in catalytic activity, the method can be considered as efficientsa consideration that must always be made from an economic point of view. Different methods have been used to immobilize enzymes, and the approach chosen depends on the specific application required. Those methods in which CDs participate can be summarized as follows: (1) CD-containing polymers, (2) dendrimers with terminal CD moieties, and (3) macro-, micro-, or nanosized metallic (and oxide) surfaces capped with CDs. CD-containing polymers constitute excellent supports for immobilization of enzymes since they have the following important properties: the polymer matrix itself serves to entrap the enzyme; the cavities of the CDs act as hosts for aromatic moieties present in amino acid side chains; the CD moieties serve as pores that confer the required permeability on the polymer. The main drawback concerning CDcontaining polymers is the limited reproducibility of their synthesis. Dendrimers with terminal CD moieties constitute excellent nanodevices for immobilization of enzymes, but such systems are extremely expensive at present. Nevertheless, since dendrimers are increasingly used in different fields it is expected that synthetic methods for these materials will improve and become more economically viable in the future. Macro-, micro-, or nanosized metallic surfaces previously modified with CDs can be obtained by several different approaches. Electrodes, biochips, and nanoparticles have all been prepared in this way. Protein biochips have been widely studied in the search for new diagnosis methods.160 These systems currently play an important role in the development of nanobiotechnology and nanomedicine.161 The host-guest chemistry of CDs makes them ideal candidates for the supramolecular attachment of proteins and other molecules, but despite this fact, very little work has been done on protein biochips modified with CDs. The only reference found on this issue concerns a gold microsurface modified with aminopolymers of βCD. A second monolayer of carboxylated dextran containing adamantane moieties was associated in a supramolecular manner through inclusion of the latter in the βCD cavities. These biochips were used to bind antibodies.162 Biochips containing enzymes have received very little attention to date, probably because determination of the activity of an enzyme immobilized on a biochip is not a simple issue.163 Enzymes can be immobilized on a surface using plastics, and such systems can be used as microreactors. Use of this approach makes the device much cheaper than when other surfaces, particularly glass and silicon, are used.164 Such microreactors are of analytical importance but could also find use in industrial applications. A CD-containing polymer was used to immobilize lipase for the hydrolysis and transesterification of triglycerides. This reactor was highly economical.165



Figure 28. Schematic representation of a haemolysin mutant pore, with Met 113 substituted with Arg (upper schemes) with its interaction with heptakis-(6-deoxy-6-amino)-β-cyclodextrin (lower schemes). (Reprinted in part with permission from ref 166c. Copyright 2006 American Chemical Society.)

A completely novel approach to the use of CDs with proteins involves CDs that are able to modulate protein pores for stochastic sensing of organic analytes. R-Haemolysin, an exotoxin secreted by Staphylococcus aureus, can selfassemble on a lipid bilayer to form a heptameric pore with a seven-fold axis, similar to that in βCD. The latter compound can be introduced into the pore and partially block it, reducing the conductivity of the system by a factor of 3 (Figure 28). It appears that βCD is fixed within the pore by seven leucine residues and retained in that position as a molecular adaptor even at temperatures approaching 100 °C.166 This so-called “pore engineering” could serve in the future to regulate the active site channel of different enzymes as required.

7.1. Macro- and Micrometric Surfaces Modified with Cyclodextrins Native and modified CDs have been associated with different types of surfaces for a wide range of applications, but their main use is for analytical purposes. In different chromatographic techniques (GC, HPLC, CZE, and MEKC) CDs have been introduced into the composition of the column (stationary phase) in order to enhance enantiomeric separations.167 Such modifications have been useful in the purification and identification of products that have enzymatic activity. In this regard an important step forward was made when enzymes were also introduced into the chromatographic column in order to develop “in-line” techniques. In this approach, capillary zone electrophoresis (CZE) has been applied and given rise to a relatively new technique named “electrophoretically mediated microanalysis” (EMMA).168 This technique has recently been modified by introduction of CDs as part of the capillary column.169 The combination of CDs and enzymes in different devices has been introduced in the field of HPLC. Introduction of

CDs into the mobile phase was reported for the resolution of steroid stereoisomers. Since the necessary specificity could not be achieved, a “postcolumn reactor” containing hydroxysteroid dehydrogenases (EC 1.1.1.176) was added. These enzymes oxidize the hydroxysteroids, and this modification led to an effective resolution.170

7.2. Nanodevices Containing Cyclodextrins Nanometric devices are gaining great importance in different areas of the Nanosciences. Nanodevices can serve to transport drugs and biomolecules into the cells since the membrane is only permeable to species with diameters less than 50 nm.171 In nanomedicine the main goal is probably to obtain smart nanodevices that are able to target, with high specificity, damaged cells and repair or destroy them.171,172 Biomolecules should constitute the best targeting species for this mission, and this is especially the case for proteins. It is therefore important to prepare nanodevices that contain proteins, including enzymes. From a supramolecular point of view, CD-containing nanodevices could serve such a purpose. Nanodevices are being successfully used in bioanalysis.173 In this sense they have a similar purpose as biochips but also take advantage of the special properties that characterize nanodevices. Three main types of CD-containing nanodevices have been developed to date: CD termini dendrimers, nanosized CDcontaining polymers, and metal nanoparticles capped with perthiolated CD. Nevertheless, a combination of the three approaches (composites) has also been studied and will probably prove to be the best alternative.174 Silica nanoparticles capped with βCD heptamine have also been reported.175

7.2.1. Soft Nanodevices “Soft” nanodevices are those that do not contain metals, metal oxides, or semiconductor materials, and these are


gaining in importance.176 CD termini dendrimers and nanosized CD-containing polymers are good examples of soft nanodevices. Dendrimers, defined as “perfectly branched monodisperse macromolecules”, constitute excellent hosts in molecular recognition processes,177 and if they contain CD termini, then this property is significantly enhanced.178 The importance of carbohydrate-containing dendrimers was highlighted several years ago.179 Dendrimers are able to act not only as hosts but also as guests in supramolecular processes, which are very important when analyzing cell membranes (important targets) as hosts. Dendrimers mainly act as guests when they contain specific termini such as ferrocenyl, dansyl, or adamantyl units.180 All of these moieties are excellent guests of cyclodextrins. Dendrimers have been used as nanodevices with different applications, but they are most often employed in drug research.177,181 These applications are based on the unique architecture of dendrimers, which means they are able to interact with different types of species, including catalysts, chromophores, mesogens, and biomolecules. In these applications the dendrimer serves as a scaffold and is able to establish communication, serve in self-organization processes, or react with other components.177 Use of dendrimers as soft nanodevices that are able to supramolecularly associate and transport even the most dissimilar types of molecules has greatly increased in the past few years. At present, the main limitation in the application of dendrimers is the complexity of the synthetic methods used for their preparation. These methods involve complex convergent or divergent multistep procedures. Poly(amidoamine) dendrimers, PAMAM, are commercially available, albeit at relatively high prices. This type of dendrimer can be modified with an additional “generation” to introduce terminal cyclodextrins.181b PAMAM dendrimers of different generations modified with CD termini have become very effective in gene transfer, a new therapeutic approach (Figure 29).181b,182 CD-containing polymers have also been used in this way.183 Nevertheless, much work has still to be done in order achieve successful applications in this area. Glyco-dendrimers with a CD core have been developed, and carbohydrate substituents have received particular attention. These systems are represented schematically in Figure 30. It appears that the inclusion properties of glyco-dendrimers with a βCD core do not increase significantly with respect to βCD itself.184 Furthermore, in hyperbranched (tetradecaantennated C-6-branched mannopyranosyl and glucopyrannosyl) βCDs (Figure 31) the βCD core loses its recognition properties completely.185 Better results were obtained by Garcıá Fernańdez on studying a glycodendron.186 The persubstitution of βCD with different monosaccharides produces glycoclusters, which can act as sensors to delineate topological differences between two dimeric prototype proteins.187 The different types of glycodendrimers and mono- and polysubstituted glycoclusters based on βCD cores were compared with one another. Polysubstituted derivatives bearing multiple biorecognizable saccharide moieties show better lectin-binding properties due to the so-called “cluster effect”, and this is the basis of many lectin-carbohydrate interactions.188 On the other hand, monosubstituted conjugates exhibit superior inclusion capabilities, while βCDglycodendrimers combine both of these favorable features.189

Villalonga et al.

Figure 29. PAMAM G2 (A), G3 (B), and G4 (C) conjugated with βCD. (Reprinted with permission from ref 182a. Copyright 2002 American Chemical Society.)

Heteroglycodendrimers based on βCD cores apparently give the best results of all.190 Lectins are carbohydrate-binding proteins that bind to sugar moieties in cell surfaces or membranes and therefore change their physiology to cause agglutination, mitosis, or other biological modifications in the cell. Lectins show a high specificity toward a particular type or sequence of carbohydrate.191 CD derivatives can inhibit lectins to prevent proteins from attaching to the cell surface, as occurs in carbohydrate vaccines.192 The advantage of using CD dendrimers in this approach is that they can also act as drug carriers that are able to target a cell-bound lectin and allow site-directed drug delivery.193 A schematic representation of a simple CD derivative as a targeting drug (included in the CD cavity)



Figure 30. Modes of attachment of saccharides to a CD core.

carrier is presented in Figure 32. Drugs have also been conjugated to CD dendrimers and glycoclusters.194-196 Dendrimers have yet to find applications in enzyme association. These systems can be easily degraded by hydrolytic enzymes, and this constitutes a great limitation.181a Nevertheless, considering the increasing importance of glycoenzymes and neoglycoenzymes,116,117 dendrimers containing CDs should be considered as a promising research area in the near future. PAMAM dendrimers with low generations (1-3) do not offer good gene transfer, while higher generation dendrimers are reported to be cytotoxic.197 If these dendrimers are conjugated to R-, β-, and γCDs an enhanced gene transfer effect can be observed, especially in the case of RCD conjugated to PAMAM.181b The effect of the generation was studied, and G3 PAMAM conjugated with RCD was reported to show the most marked effect.182a CD-containing polymers with nanometric dimensions can be used as enzyme receptors, as reported in the literature.198 In particular, nanogels obtained by the self-assembly of cholesteryl-bearing pullulan and βCD are promising when associated with enzymes. These nanogels prevented the thermal aggregation of carbonic anhydrase B by selective trapping of the heat-denatured protein. After the complex formed between the nanogels and the studied enzyme was cooled, the activity of the latter spontaneously recovered upon release from the complex.199

7.2.2. Hard Nanodevices Proteins can denature when they interact with nanodevices that are able to access the most diverse sites of these biomolecules. This process is frequent when the nanoparticle has a charged surface.200 Nevertheless, several reports

indicate that this is by no means a rule.201 Furthermore, metal nanoparticles have been formed in vitro through bioreduction of the appropriate reagent, and morphological changes in the cells were not observed.202 Nanoparticles, mainly quantum dots and metallic nanoparticles, are already used in bioanalysis, and positive results have been obtained.173 For example, gold nanoparticles have been used to study protein-protein interactions.203 Apparently, the possibility of denaturation of a protein depends on the nature of the protein, the size of the nanoparticle, and its charge. The presence and characteristics of the capping molecules that form an interface between the hard nanodevice and the immobilized enzyme are very important.204 For example, denaturation was not observed on using BSA-capped citrate-coated gold nanoparticles, whereas this process did take place when the protein-capped bare nanoparticles were used.205 The conformation of cytochrome c was studied when immobilized on gold nanoparticles capped with negative, positive, and neutral species. With a neutral capping agent denaturation was not observed on cytochrome c, whereas a major effect was observed with positively charged gold nanoparticles.206 Neutrally charged CDs as capping agents should serve to form interfaces that are able to avoid any undesirable denaturation process of an immobilized enzyme on hard nanoparticles. This immobilization process should be supramolecular in nature and have a high specificity in terms of how the enzyme is associated with the particle. CD-containing nanoparticles of noble metals were first reported by Kaifer. Initially gold nanoparticles were studied (Figure 33),207 followed by platinum,208 palladium,208,209 and silver.210 In all these cases perthiolated CDs were used to cap the formed nanoparticles.


Villalonga et al.

Figure 31. Schematic representation of hyperbranched tetradeca-antennated C-6-branched mannopyranosyl- and glucopyrannosyl-βCDs. (Reprinted in part with permission from ref 185. Copyright 2001 American Chemical Society.)

Figure 32. Cyclodextrin drug carrier systems based on lectincarbohydrate interactions at cell surfaces.

Gold nanoparticles capped with perthiolated CDs have been studied as hosts for different enzymesseither native or modified. In all cases the enzyme is supramolecularly associated through multivalence inclusion within the CD

cavities and without direct interaction with the gold nanoparticle. The supramolecular association of a native enzyme with a nanoparticle capped with CDs should involve the aromatic hydrophobic moieties of the former being included in the CD cavities.171 Aliphatic moieties can also be included, but these have very low association constants.211 Nevertheless, aliphatic loops could achieve stable supramolecular interactions. An increase in the thermal stability of trypsin when supramolecularly associated with gold nanoparticles capped with perthiolated β- and γCDs has been reported (Figure 34) without a loss in catalytic activity. The best results were obtained with gold nanoparticles capped with perthiolated γCD. In this case a more compact structure for trypsin was observed by fluorescence determinations after supramolecular immobilization.212 L-Phenylalanine dehydrogenase was supramolecularly associated with gold nanoparticles capped with perthiolated βCD in its native form and also modified by conjugation of L-adamantane carboxylic acid. The enzyme immobilized in



remained free served as guests of βCD cavities of the modified SOD. The presence of catalase in the bienzymatic nanodevice increased its optimum pH range from 7.0-7.5 to 6.5-7.5 and its thermal stability by 7 °C. On the other hand, SOD in the resulting nanodevice was 90 times more resistant to inactivation by H2O2 after its supramolecular immobilization. The reports outlined above may constitute an important step forward in the design of bioactive nanoparticles, but studies on the stability and toxicity are still required.

8. Enzyme Biosensors with Supramolecular Architecture

Figure 33. Schematic representation of a gold nanoparticle capped with perthiolated βCD interacting with ferrocenyl and adamantanyl moieties. (Reprinted with permission from ref 207b. Copyright 2000 American Chemical Society.)

One of the main applications of CDs in enzyme immobilization is in the preparation of biosensors. Enzymes play a determinant role in biosensors, and one way in which they can be immobilized on electrode surfaces is through supramolecular interactions. The emergence of this approach means that CDs are gaining in importance as enzyme hosts. Biosensors can be electrochemical215 or optical in nature,216 although the former systems have been studied more extensively. Biosensors have a very important analytical advantage over other types of sensors: their high enantioselectivity. This factor makes them the best option for chiral drug determination.217

8.1. Electrochemical Biosensors

Figure 34. Native trypsin supramolecularly associated with a gold nanoparticle capped with perthiolated CD.

both forms retained its activity, and an increase in thermal stability was observed.213 Cu,Zn-superoxide dismutase (SOD) and catalase were modified in order to prepare a bienzymatic antioxidant nanodevice.214 In this study SOD was modified with βCD units and catalase with adamantane. Modified catalase was supramolecularly associated with the βCD-capped gold nanoparticles through inclusion of the adamantane moieties within the βCD cavities. The adamantane moieties that

CD-containing polymers have been widely used in the preparation of biosensors based on enzymes. The enzyme can be either trapped within the polymer matrix or covalently linked (Table 2). The CD cavities can serve to include ferrocene (and its derivatives), pyrroloquinoline quinine, phenoxazines, phenothiazines, tetrahydrofulvalene (TTF), or any other molecule that can act as electron-transfer (ET) mediator between the associated enzyme and the electrode (Figure 35). All of these redox mediators are water-insoluble compounds, and the inclusion process in the CD cavities overcomes this limitation. Another alternative, which gives similar results, is to covalently bind the mediator to the CD or cross-link it to the polymer.215,218 A CD-containing polymer can strongly entrap and retain the enzyme near the electrode surface and, at the same time, protect it from the environment. These conditions are of great importance in obtaining a good electrochemical response. The ET rate depends on the donor-acceptor distance (enzyme-electrode). For this reason, the ideal biosensor should consist of an electrode modified with only a monolayer containing the enzyme. When a polymer is used to entrap the enzyme this condition cannot be completely fulfilled. However, an acceptable approximation involves preparing a thin film. The thickness of the polymer matrix and the position at which the enzyme is entrapped attempt against the reproducibility of most of the reported biosensors. This factor is not mentioned in most of the reports. The sensitivity, linearity, and response time of the biosensor greatly depend on this property. It is interesting to note that the response time generally corresponds to high values (∼1 min), which constitute the main limitation of most of the reports. The active site of an enzyme must be accessible to its analyte, and the polymer should not significantly affect its diffusion from the environment. The permeability conferred by the CD cavities therefore represents a distinct advantage


Villalonga et al.

Table 2. Different Biosensors Reported Where the Enzyme Is Entrapped in a CD Polymer enzyme horseradish peroxidase horseradish peroxidase GOx GOx GOx GOx GOx GOx GOx GOx GOx + β-galactosidase + mutarotase horseradish peroxidase + GOx + β-galactosidase galactose oxidase laccase polyphenol oxidase horseradish peroxidase + choline oxidase + acetylcholine esterease β-amylase

comment

mediator

βCD polymer was prepared with glutaric dialdehyde βCD polymer was prepared with glutaric dialdehyde RCD polymer on glassy carbon, gold, or platinum disk that had covalently associated GOx RCD polymer in a similar system dextrin βCD-containing polymer enzyme was immobilized in a organic conducting carbon layer in the presence of βCD carboxymethylated βCD polymer carboxymethylated βCD polymer cross-linked βCD polymer 2-hydroxypropyl-βCD + preactivated Immunodyne nylon membrane enzymes cross-linked with βCD polymer Eastman-AQ polymer cross-linked with the enzymes and mediator; determination of hydrogen peroxide, glucose, and lactose βCD polymer on Pt disk electrode “cross-linked enzyme crystals” in 30% PVP gel cross-linked βCD polymer film. Dopamine: 1.0 nM to 1.0 µM polyurethane + plasticizer + lipophilic anion + perethylated. βCD screen-printed biosensor R-CD-R,ω-dicarboxylated poly(ethylene glycol) polymer

in the preparation of biosensors. The polymer itself generally contains micro- and nanochannels formed within the network, and the analyte can access the active site of the enzyme through these. Biological samples are characterized by a background of reducing agents (ascorbate, ureate, etc.) and other interfering species that are generally ionic in nature.219 CDs do not recognize these ions, and their interference can therefore be avoided.

ref

toluidine blue methylene blue 1,4-benzoquinone or TTF

220 221 222

ferrocene ferrocene Cr and Mn cyclopentadienyl half-sandwich complexes poly(pyridine) ruthenium complexes TTF ferrocene dimethylferrocene

223 224 225

227 238-239 230, 231

ferrocene

232

N-Me phenazine methosulfate

233

ferrocenemethanol

234 235 236

tetramethylbenzidine and ferrocene ferrocene ferrocene

226

237, 238 239

The properties of the CD-containing polymers allow the use of a diverse range of materials for the preparation of the electrodes. Not only can noble metals be used, such as platinum, gold, or silver, but also metal oxides, glassy carbon, and graphite. Glucose oxidase (GOx, EC 1.1.3.4) has been the enzyme most widely studied since it is relatively simple to handle and allows detection of an important substrate: glucose. Most of the reported methods are very similar, and the disposable information is not sufficient to define the best

Figure 35. CD-containing polymer entrapping GOx and ferrocene (mediator).


approach. The most representative reported biosensors are listed in Table 2. Biosensors based on CDs have been made without preparation of polymers and, of course, without the advantages and disadvantages associated with polymeric materials. The first report in this sense was based on supramolecular interactions. For this, cytochrome c modified with adamantane units was supramolecularly associated with the surface of an electrode modified with a monolayer of perthiolated βCD through inclusion of several of the conjugated units (Figure 36). A good electrochemical response was observed,


tected xanthine concentrations in the range from 300 µM to 10.4 mM at pH 7.0.243 The combined use of polymers with formation of CD monolayers for the preparation of biosensors has been reported.244 A monolayer of 11-mercaptoundecanoic acid was initially self-assembled on a gold surface. Monoamino-βCD units were conjugated onto the terminal carboxylic acid groups of the resulting monolayer, and these served to supramolecularly associate adamantane groups bearing PEGcontaining terminal carboxyl groups. These groups, in turn, served to covalently couple β-lactoglobulin.244a Another approach that also combines polymer properties with self-assembly processes was recently reported.244b For this, βCD was polymerized with epichlorohydrin and then thiolated. The thiol groups served to self-assemble the polymer on a gold electrode and also associate gold nanoparticles that apparently served as mediators. Xanthine oxidase modified with adamantane units was then supramolecularly associated with the βCD cavities. This sensor showed a decrease in its response in only 7% after 21 days of storage at 4 °C.

8.2. Optical Biosensors

Figure 36. Schematic representation of a biosensor in which adamantane-modified cytochrome c was supramolecularly immobilized on an electrode surface modified with a self-assembled monolayer of perthiolated βCD. (Reprinted with permission from ref 240a. Copyright 2002 American Chemical Society.)

and the intensity of the signal was not affected by overnight immersion of the electrode in a buffer solution.240a A similar procedure was used to immobilize L-phenylalanine dehydrogenase on gold electrodes that showed a linear amperometric response up to 3 mM L-phenylalanine with a lower detection limit of 15 µM when Meldola Blue was used as mediator.240b This biosensor retained about 97% of its initial electrocatalytic response toward 2.5 mM L-Phe after 21 days of incubation at 4 °C. These observations indicate that the conformation of enzymes modified with adamantane derivatives was strongly retained through a multivalent supramolecular association to the electrode surface.241 In a similar way, a gold electrode was modified with perthiolated CDs, forming a monolayer on the surface. Octanothiol was used to seal the remaining empty space, done despite the fact that it reduces the efficiency of the ET process. This modified gold electrode was used to selfassemble laccase with methylene blue included in the CD cavities as a mediator.242 A similar approach was also applied but without sealing the empty surface of the gold electrode and using a modified enzyme. In this report the authors did not discuss the stability of the biosensor. An amperometric xanthine biosensor was built using a gold electrode but modified with a monolayer of adamantane units, and the enzyme, xanthine oxidase (EC 1.1.3.22), was conjugated with βCD moieties. In this way, the modified enzyme was associated with the electrode through adamantane-βCD supramolecular interactions. The biosensor de-

Biosensors based on optical properties have received much less attention than the electrochemical biosensors discussed above. An optical biosensor was prepared using a complicated self-assembly process on a surface. A quencher-dyelabeled biotin-linked E. coli maltose binding protein was bound with a specific orientation to a NeutrAvidin-coated surface and employed as a bioreceptor (Figure 37). In addition, a flexible biotinylated DNA oligonucleotide was used as a tether to a fluorescence resonance electron-donor dye, and a distal βCD unit was bound in an equimolar amount to the same surface of DNA and directed its immobilization.216 The dye was included in the βCD cavity and substituted by maltose (the analyte). The biosensor is regenerated by washing the analyte away. A simpler opto-biosensor based on IR-ATR determinations has been prepared. GOx was used for glucose determination, for which it was immobilized on surfaces of glass beads, and these were introduced into a column to function as an enzyme reactor. RCD was associated with a PVBC polymer to coat IRE (internal reflection elements) in order to increase the sensitivity of the method. The presence of the RCD moieties enables the supramolecular association of the enzymatic product: gluconic acid.245 An apparently more promising approach to prepare an opto-biosensor was based on the luminescent properties of quantum dots (QD) as hybrid inorganic-bioreceptor sensing materials. Multiple copies of Escherichia coli maltosebinding protein (sugar receptors) were coordinated to each QD by a C-terminal oligohistidine segment. In one configuration, a βCD-QSY9 dark quencher conjugate was bound in the maltose-binding protein saccharide binding site, resulting in fluorescence resonance energy-transfer quenching of QD photoluminescence.246

9. Outlook Cyclodextrins and their derivatives constitute excellent hosts for both native and modified enzymes. This important property has encouraged use of CDs in a range of applications related to enzymes, as described in this review. Since the topic selected is relatively young it is more important at


Villalonga et al.

Figure 37. Schematic representation of an optobiosensor based on biotin-linked E. coli maltose binding protein that was bound with a specific orientation to a NeutrAvidin-coated surface and employed as a bioreceptor. A flexible biotinylated DNA oligonucleotide was used as a tether to a fluorescence resonance energy transfer, while a distal βCD unit was bound to the same surface of DNA. (Reprinted with permission from ref 216. Copyright 2004 American Chemical Society.)

this moment to consider the prospects for CD-enzyme interactions. Probably one of the earliest areas studied concerns use of CD derivatives in the preparation of biosensors. The new contributions to nanoscience research are expected to lead to new approaches for the design of biosensors. The dynamic nature of host-guest interactions between CD derivatives and enzymes can be exploited to increase the sensitivity of biosensing devices through signal amplification. For example, cyclodextrin-modified liposomes loaded with enzymatic transducers can be supramolecularly attached to antibodies and nucleic acid probes, thus allowing a signal increase in the vicinity of the biosensor surface. Similarly, recycling of modified surfaces could also be possible in devices containing complementary pairs of host/guest molecules (i.e., cyclodextrin/adamantane) due the reversible nature of the supramolecular interactions. The fact the CDs can stabilize proteins and assist in their refolding speaks about the prospects for CD-containing devices associated with proteinssor more specifically enzymes. As mentioned in this review, CD-containing devices have been successfully used in gene transfer. Now that gene synthesis is being studied on microchips, one may expect that chips modified with CDs could provide similar results.247 Use of molecular imprinting technology in CD-containing polymers should lead to improved results in this direction. One novel approach consists of formation of self-assembled monolayers of CDs using microcontact printing or nanoprint lithography.248 As pointed out in this review, nanodevices for use as smart carriers able to target affected cells are currently being developed. One important goal to be achieved by a smart nanodevice is to send a signal to the outside in order to provide information on where the detected cell is located. In this sense, polymeric CD-containing nanocapsules, with photoactive metal complexes (or dyes) included in the CD cavities, could serve such a purpose.249 Another approach could be based on use of CdSe or CdS quantum dots capped with cyclodextrins, which are able to supramolecularly associate drugs and target biomolecules while the semiconductor emits its intense fluorescent signal.250 An area where CD-enzyme interactions should find an important application is in biotechnological processes. The chaperone-like activity of CDs could be more extensively

exploited for development of downstream processes, in order to favor the easy recovery of valuable proteins produced by fermentation and cell culture technology. In this sense, foam fractionation is a low-cost protein separation process that suffers from the inconvenience of protein denaturation. CDs have been used to reduce the impact of this drawback, and positive results have been obtained.251 The design and synthesis of CD-based neoglycoenzymes appears to be a useful approach for preparation of new biocatalysts that are able to resist harsh operating conditions in order to catalyze organic reactions with improved efficiency. As expected, within only a few decades an important revolution in the biocatalytic transformation of organic compounds through enzyme-catalyzed reactions should change the current face of the chemical industry. Undoubtedly, the unique host properties of CDs should be taken into account when these novel enzymatic processes are designed. Substrate-assisted catalysis can also be supported by CDmodified enzymes when using substrates that can be supramolecularly recognized by the attached oligosaccharides. These versatile oligosaccharides should also be protagonists in the design and construction of novel enzyme biosensors as well as in the formulation of effective and biodegradable drug delivery systems for enzyme replacement therapy.

10. Abbreviations AFM ATEE BSA BAEE CD CGTase Con A CZE ET Gal GC GOx Glu GluNAc Mal Man MEKC PAMAM

atomic force microscopy N-R-acetyl-L-tyrosine ethyl ester hydrochloride bovine serum albumin N-R-benzoyl-L-arginine ethyl ester hydrochloride cyclodextrin cyclodextrin glucosyltransferase concanavalin A capillary zone electrophoresis electron transfer galactose gas chromatography glucose oxidase glucose N-acetylglucosamine maltose mannose micellar electrokinetic chromatography poly(amidoamine) dendrimer

Supramolecular Chemistry of Cyclodextrins PEG QD SOD SPR TGase TTF

poly(ethylene glycol) quantum dot Cu,Zn-superoxide dismutase surface plasmon resonante transglutaminase tetrahydrofulvalene

11. References (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22)

(23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (36) (37) (38) (39)

Saenger, W. Angew. Chem., Int. Ed. Engl. 1980, 19, 344. Wenz, G. Angew. Chem., Int. Ed. Engl. 1994, 33, 803. Szejtli, J. Chem. ReV. 1998, 98, 1743. Harper, J. B.; Easton, C. J.; Lincoln. S. F. Curr. Org. Chem. 2000, 4, 429. Davis, M. E.; Brewster, M. E. Nat. ReV. Drug DiscoV. 2004, 3, 1023. Rowan, A. S.; Hamilton, C. J. Nat. Prod. Rep. 2006, 23, 412-443. Bender, M. L.; Komiyama, M. Cyclodextrin Chemistry; SpringerVerlag: Berlin, 1978. Szejtli, J. Cyclodextrins and Their Inclusion Complexes; Acade´miai Kiado´: Budapest, 1982. Szejtli, J. Cyclodextrin Technology; Kluwer Academic Publishers: Dordrecht, 1988. ComprehensiVe Supramolecular Chemistry; Szejtli, J., Osa, T., Eds.; Elsevier: Oxford, 1996; Vol. 3. Cyclodextrins and Their Complexes. Chemistry, Analytical Methods, Applications; Dodziuk, H., Ed.; Wiley-VCH: New York, 2006. D’Souza, V. T.; Lipkowitz, K. B. Chem. ReV. 1998, 98, 1741. Saenger, W.; Jacob, J.; Gessler, K.; Steiner, T.; Hoffman, D.; Sanbe, H.; Koizumi, K.; Smith, S. M.; Tahaka, T. Chem. ReV. 1998, 98, 1787. Connors, K. A. Chem. ReV. 1997, 97, 1325. Scho¨nherr, H.; Beulen, M. W. J.; Bu¨gler, J.; Huskens, J.; van Veggel, F. C. J. M.; Reinhoudt, D. N.; Vancso, G. J. J. Am. Chem. Soc. 2000, 122, 4963. Zapotoczny, S.; Auletta, T.; de Jong, M. R.; Scho¨nherr, H.; Huskens, J.; van Veggel, F. C. J. M.; Reinhoudt, D. N.; Vancso, G. J. Langmuir 2002, 18, 6988. Auletta, T.; de Jong, M. R.; Mulder, A.; van Veggel, F. C. J. M.; Huskens, J.; Reinhoudt, D. N.; Zou, S.; Zapotoczny, S.; Scho¨nherr, H.; Vancso, G. J.; Kuipers, L. J. Am. Chem. Soc. 2004, 126, 1577. Schmid, G. Trends Biotechnol. 1989, 7, 244. van der Veen, B. A.; Uitdehaag, J. C. M.; Dijkstra, B. W.; Dijkhuizen, L. Biochim. Biophys. Acta 2000, 1543, 336. Croft, A. P.; Bartsch, R. A. Tetrahedron 1983, 39, 1417. Khan, A. R.; Forgo, P.; Stine, K. J.; D’Souza, V. T. Chem. ReV. 1998, 98, 1977. Jicsinszky, L.; Hashimoto, H.; Fenyvesi, EÄ .; Ueno, A. Cyclodextrin Derivatives. In ComprehensiVe Supramolecular Chemistry; Szejtli, J., Osa, T., Eds.; Pergamon: Oxford, 1996; Vol. 3 (Cyclodextrins), p 57. Easton, C. J.; Lincoln, S. F. Modified Cyclodextrins; Imperial College Press: London, 1999. Yamamoto, M.; Yoshida, A.; Hirayama, F.; Uekama, K. Int. J. Pharm. 1989, 49, 163. Okada, Y.; Kubota, Y.; Koizumi, K.; Hizukuri, S.; Ohfuji, T.; Ogata, K. Chem. Pharm. Bull. 1988, 36, 2176. Uekama, K.; Hirayama, F.; Irie, T. Chem. ReV. 1998, 98, 2045. Sakano, Y.; Sano, M.; Kobayashi, T. Agric. Biol. Chem. 1985, 49, 3391. Hisamatsu, M.; Yamada, T. Starch 1989, 41, 239. Shiraishi, T.; Kusano, S.; Tsumuyara, Y.; Sakano, Y. Agric. Biol. Chem. 1989, 53, 2181. Kobayashi, S.; Kainuma, K.; French, D. In Proceedings of the 1st International Symposium on Cyclodextrins; Szejtli, J., Ed.; Riedel: Dordrecht, 1982; p 51. Kobayashi, S.; Shibuya, N.; Young, B. M.; French, D. Carbohydr. Res. 1984, 126, 215. Suzuki, H.; Uede, H.; Kobayashi, S.; Nagai, T. Eur. J. Pharm. Sci. 1993, 1, 159. Koizumi, K.; Utamura, T.; Sato, M.; Yagi, Y. Carbohydr. Res. 1986, 153, 55. Kitahata, S.; Yoshimura, Y.; Okada, S. Carbohydr. Res. 1987, 159, 303. Yoshimura, Y.; Kitahata, S.; Okada, S. Agric. Biol. Chem. 1988, 52, 1655. Koizumi, K.; Okada, Y.; Fujimoto, E.; Takagi, Y.; Ishigami, H.; Hara, K.; Hashimoto, H. Chem. Pharm. Bull. 1991, 39, 2143. Okada, Y.; Koizumi, K.; Kitahata, S. Carbohydr. Res. 1994, 254, 1. Okada, Y.; Koizumi, K. Chem. Pharm. Bull. 1998, 46, 319. Hizukuri, S.; Abe, J.-i.; Koizumi, K.; Okada, Y.; Kubota, Y.; Sakai, S.; Mandai, T. Carbohydr. Res. 1989, 185, 191.

Chemical Reviews, 2007, Vol. 107, No. 7 3113 (40) Shiraishi, T.; Fujimoto, D.; Sakano, Y. Agric. Biol. Chem. 1989, 53, 3093. (41) Kubota, Y.; Fukuda, M.; Murogushi, M.; Koizumi, K. Biol. Pharm. Bull. 1996, 19, 1068. (42) Okata, K.; Koikumi, K. Chem. Pharm. Bull. 1998, 46, 319. (43) Apparu, C.; Driguez, H.; Williamson, G.; Svensson, B. Carbohydr. Res. 1995, 277, 313. (44) Jørgensen, C. T.; Svendsen, A.; Brask, J. Carbohydr. Res. 2005, 340, 1233. (45) Ishiguro, T.; Fuse, T.; Oka, M.; Kurasawa, T.; Nakamishi, M.; Yasumura, Y.; Tsuda, M.; Yamagushi, T.; Nogami, I. Carbohydr. Res. 2001, 331, 423. (46) Tavornvipas, S.; Hirayama, F.; Arima, H.; Uekama, K.; Ishiguro, T.; Oka, M.; Hamayasu, K.; Hashimoto, H. Int. J. Pharm. 2002, 249, 199. (47) Koikumi, K.; Kitahata, S.; Hashimoto, H. Methods Enzymol. 1994, 247, 64. (48) Kitahata, S.; Hara, K.; Fujita, K.; Kuwahara, N.; Koikumi, K. Biosci. Biotechnol. Biochem. 1992, 56, 1518. (49) Koikumi, K.; Tanimoto, T.; Okada, Y.; Hara, K.; Fujita, K.; Hashimoto, H.; Kitahata, S. Carbohydr. Res. 1995, 278, 129. (50) Hamayasu, K.; Hara, K.; Kondo, Y.; Tanimoto, T.; Koikumi, K.; Nakano, H.; Matsuda, K.; Biosci. Biotechnol. Biochem. 1997, 61, 825. (51) Okada, Y.; Matsuda, K.; Koikumi, K.; Hamayasu, K.; Hashimoto, H.; Kitahata, S. Carbohydr. Res. 1998, 310, 229. (52) Hamayasu, K.; Fujita, K.; Hara, K.; Hashimoto, H.; Tanimoto, T.; Koikumi, K.; Nakano, H.; Kitahata, S. Biosci. Biotechnol. Biochem. 1999, 63, 1677. (53) Masanori, I.; Hamayasu, K.; Fujita, K.; Hashimoto, H.; Koikumi, K.; Kitahata, S. Carbohydr. Res. 2001, 336, 203. (54) Hara, K.; Fujita, K.; Kuwahara, N.; Tanimoto, T.; Hashimoto, H.; Koikumi, K. Biosci. Biotechnol. Biochem. 1994, 58, 652. (55) Kitahata, S.; Fujita, K.; Takagi, Y.; Hara, K.; Hashimoto, H.; Tanimoto, T.; Koizumi, K. Biosci. Biotechnol. Biochem. 1992, 56, 242. (56) Tanimoto, T.; Omatsu, M.; Ikuta, A.; Nishi, H.; Murakami, H.; Nakano, H.; Kitahata, S. Biosci. Biotechnol. Biochem. 2005, 69, 732. (57) Matsuda, K.; Inazu, T.; Haneda, K.; Mizuno, M.; Yamanoi, T.; Hattori, K.; Yamamoto, K.; Kumagai, H. Bioorg. Med. Chem. Lett. 1997, 7, 2353. (58) Yamanoi, T.; Yoshida, N.; Oda, Y.; Akaike, E.; Tsutsumida, M.; Kobayashi, N.; Osumi, K.; Yamamoto, K.; Fujita, K.; Takahashi, K.; Hattori, K. Bioorg. Med. Chem. Lett. 2005, 15, 1009. (59) Hamayasu, K.; Fujita, K.; Hara, K.; Hashimoto, H.; Tanimoto, T.; Koizumi, K. J. Appl. Glycosci. 1999, 46, 445. (60) Kitahata, S.; Tanimoto, T.; Ikuta, A.; Tanaka, K.; Fujita, K.; Hashimoto, H.; Murakami, H.; Nakano, H.; Koizumi, K. Biosci. Biotechnol. Biochem. 2000, 64, 1223. (61) Kitahata, S.; Tanimoto, T.; Okada, Y.; Ikuta, A.; Tanaka, K.; Murakami, H.; Nakano, H.; Koizumi, K. Biosci. Biotechnol. Biochem. 2000, 64, 2406. (62) Bonnet, V.; Boyer, C.; Langlois, V.; Duval, R.; Rabiller, C. Tetrahedron Lett. 2003, 44, 8987. (63) Furuike, T.; Sadamoto, R.; Niikura, K.; Monde, K.; Sakairi, N.; Nishimura, S.-I. Tetrahedron 2005, 61, 1737. (64) Klohr, E. A.; Koch, W.; Klemm, D.; Dicke, R. Regioselectively substituted esters of oligosaccharides and polysaccharides and process for their preparation, European Patent EP1035135, 2000. (65) Akkara, J. A.; Kaplan D. L.; Ferdinando, F.; Jonathan, D. S. Transesterification of insoluble polysaccharides, U.S. Patent US6063916, 2000. (66) Pattekan, H. H.; Divagar, S. Indian J. Chem. B 2002, 41, 1025. (67) Pedersen, N. R.; Kristensen, J. B.; Bauw, G.; Ravoo, B. J.; Darcy, R.; Larsen, K. L.; Pedersen, L. H. Tetrahedron: Asymmetry 2005, 16, 615. (68) Xiao, Y.; Wu, Q.; Wang, N.; Lin, X. Carbohydr. Res. 2004, 339, 1279. (69) Wang, N.; Wu, Q.; Xiao, Y.; Chen, C.; Lin, X. Bioorg. Med. Chem. 2005, 13, 3667. (70) Cottaz, S.; Apparu, C.; Driguez, H. J. Chem. Soc., Perkin Trans. 1991, 1, 2235. (71) Apparu, C.; Cottaz, S.; Bosso, C.; Driguez, H. Carbohydr. Lett. 1994, 1, 349. (72) Bornaghi, L.; Utille, J.-P.; Penninga, D.; Schmidt, A. K.; Dijkhuizen, L.; Schulz, G. E.; Driguez, H. Chem. Commun. 1996, 2451. (73) (a) Schmid, A.; Dordick, J. S.; Hauer, B.; Kiener, A.; Wubbolts, M.; Witholt, B. Nature 2001, 409, 258. (b) Krieger, N.; Steiner, W.; Mitchell, D. Food Technol. Biotechnol. 2004, 42, 219. (74) Schoemaker, H. E.; Mink, D.; Wubbolts, M. G. Science 2003, 299, 1694. (75) (a) Harper, J. B.; Easton, C. J.; Lincoln, S. F. Curr. Org. Chem. 2000, 4, 429. (b) Ghanem, A. Org. Biomol. Chem. 2003, 1, 1282.

3114 Chemical Reviews, 2007, Vol. 107, No. 7 (76) Alexander, D. L.; Fisher, J. F. Steroids 1995, 60, 290. (77) Woerdenbag, H. J.; Pras, N.; Frijlink, H. W.; Lerk, C. F.; Malingre´, T. M. Phytochemistry 1990, 29, 1551. (78) Szente, L.; Szejtli, J.; Szemań, J.; Kato´, L. J. Inclusion Phenom. Mol. Recognit. Chem. 1993, 16, 339. (79) (a) Kolossva´ry, G. J.; Bańky-El, E. Biotechnol. Tech. 1996, 10, 115. (b) Chen, J. P. Biotechnol. Lett. 1989, 11, 633. (c) Commenil, P.; Belingheri, L.; Sancholle, M.; Dehorter, B. Lipids 1995, 30, 351. (80) Shin, H. D.; Kim, J. H.; Kim, T. K.; Kim, S. H.; Lee, Y. H. Enzyme Microb. Technol. 2002, 30, 835. (81) Lo´pez-Nicola´s, J. M.; Bru, R.; Garcıá-Carmona, F. Biochim. Biophys. Acta 1997, 1347, 140. (82) Jyothirmayi, N.; Ramadoss, C. S. Biochim. Biophys. Acta 1991, 1083, 193. (83) Pe´rez-Gilabert, M.; Garcıá-Carmona, F. Biotechnol. Prog. 2005, 21, 1742. (84) Mita, N.; Tawaki, S.; Uyama, H.; Kobayashi, S. Macromol. Biosci. 2002, 2, 127. (85) Reihmann, M. H.; Ritter, H. Macromol. Chem. Phys. 2000, 201, 798. (86) Mejias, L.; Schollmeyer, D.; Sepulveda-Boza, S.; Ritter, H. Macromol. Biosci. 2003, 3, 395. (87) Pang, Y.; Ritter, H.; Tabatabai, M. Macromolecules 2003, 36, 7090. (88) Bernhardt, S.; Glo¨ckner, P.; Ritter, H. Polym. Bull. 2001, 46, 153. (89) Ritter, H.; Tabatabai, M. Prog. Polym. Sci. 2002, 27, 1713. (90) Easton, C. J.; Harper, J. B.; Head, S. J.; Lee, K.; Lincoln, S. F. J. Chem. Soc., Perkin Trans. 2001, 1, 584. (91) Kolossvary, G. J.; Banky-Elod, E. Biotechnol. Tech. 1996, 10, 115. (92) Ghanem, A.; Schurig, V. Tetrahedron: Asymmetry 2001, 12, 2761. (93) Singh, M.; Sharma, R.; Banerjee, U. C. Biotechnol. AdV. 2002, 20, 341. (94) Ceynowa, J.; Koter, I. Acta Biotechnol. 1997, 17, 253. (95) Kim, S. H.; Kim, T. K.; Shin, G. S.; Lee, K. W.; Shin, H. D.; Lee, Y. H. Biotechnol. Lett. 2004, 26, 965. (96) Mine, Y.; Zhang, L.; Fukunaga, K.; Sugimura, Y. Biotechnol. Lett. 2005, 27, 383. (97) Mine, Y.; Fukunaga, K.; Itoh, K.; Yoshimoto, M.; Nakao, K.; Sugimura, Y. J. Biosci. Bioeng. 2003, 95, 441. (98) Avila-Gonza´lez, R.; Pe´rez-Gilabert, M.; Garcıá-Carmona, F. J. Biosci. Bioeng. 2005, 100, 423. (99) Avila-Gonza´lez, R.; Pe´rez-Gilabert, M.; Lo´pez-Lo´pez, M. A.; GarcıáCarmona, F. Biotechnol. Prog. 2005, 21, 338. (100) Yun, H.; Kim, J.; Kinnera, K.; Kim, B. G. Biotechnol. Bioeng. 2006, 93, 391. (101) Santos, A. M.; Montan˜ez Clemente, I.; Barletta, G.; Griebenow, K. Biotechnol. Lett. 1999, 21, 1113. (102) Gonza´lez Martıńez, S.; Alvira, E.; Vergara Cordero, L.; Ferrer, A.; Montan˜e´s-Clemente, I.; Barletta, G. Biotechnol. Prog. 2002, 18, 1462. (103) Hasegawa, M.; Yamamoto, S.; Kobayashi, M.; Kise, H. Enzyme Microb. Technol. 2003, 32, 356. (104) Baron, A. M.; Sarquis, M. I. M.; Baigori, M.; Mitchell, D. A.; Krieger, N. J. Mol. Catal. B: Enzym. 2005, 34, 25. (105) Fredholt, K.; Østergaard, J.; Savolainen, J.; Friis, G. J. Int. J. Pharm. 1999, 178, 223. (106) Koushik, K. N.; Bandi, N.; Kompella, U. B. Pharm. DeV. Technol. 2001, 6, 595. (107) Matsubara, K.; Ando, Y.; Irie, T.; Uekama, K. Pharm. Res. 1997, 14, 1401. (108) Monteiro, J. B.; Chiaradia, L. D.; Brandaõ, T. A. S.; Magro, J. D.; Yunes, R. A. Int. J. Pharm. 2003, 267, 93. (109) (a) Hicks, K. B.; Sapers, G. M.; Seib, P. A. U.S. Patent 4,975,293, 1990. (b) Fayad, N.; Marchal, L.; Billaud, C.; Nicolas, J. J. Agric. Food Chem. 1997, 45, 2442. (c) Sojo, M. M.; Nun˜ez-Delicado, E.; Garcıá-Carmona, F.; Sańchez-Ferrer, A. J. Agric. Food Chem. 1999, 47, 518. (d) Nun˜ez-Delicado, E.; Sojo, M. M.; Garcıá-Carmona, F.; Sańchez-Ferrer, A. J. Agric. Food Chem. 2003, 51, 2058. (e) Gacche, R. N.; Zore, G. B.; Ghole, V. S. J. Enzyme Inhib. Med. Chem. 2004, 19, 175. (110) (a) Nun˜ez-Delicado, E.; Sańchez-Ferrer, A.; Garcıá-Carmona, F. J. Agric. Food Chem. 1997, 45, 2830. (b) Nun˜ez-Delicado, E.; Sojo, M. M.; Sańchez-Ferrer, A.; Garcıá-Carmona, F. Arch. Biochem. Biophys. 1999, 367, 274. (111) Koralewska, A.; Augustyniak, W.; Temeriusz, A.; Kanska, M. J. Inclusion Phenom. Macrocycl. Chem. 2004, 49, 193. (112) (a) Kirk, O.; Borchert, T. V.; Fuglsang, C. C. Curr. Opin. Biotechnol. 2002, 13, 345. (b) Galante, Y. M.; Formantici, C. Curr. Org. Chem. 2003, 7, 1399. (c) Tombelli, S.; Mascini, M.; Sacco, C.; Turner, A. P. F. Anal. Chim. Acta 2000, 418, 1. (113) (a) Schiffmann, R.; Kopp, J. B.; Austin, H. A.; Sabnis, S.; Moore, D. F.; Weibel, T.; Balow, J. E.; Brady, R. O. JAMA 2001, 285, 2743. (b) Valdivia, A.; Perez, Y.; Dominguez, A.; Caballero, J.; Gomez, L.; Schacht, E. H.; Villalonga, R. Macromol. Biosci. 2005, 5, 118. (c) Dominguez, A.; Valdivia, A.; Caballero, J.; Martıńez, G.; Schacht, E.; Villalonga, R. J. Bioact. Compat. Polym. 2005, 20, 557. (d) Pe´rez,

Villalonga et al.

(114) (115) (116)

(117) (118) (119) (120) (121) (122) (123) (124) (125) (126) (127)

(128) (129) (130) (131) (132) (133) (134) (135) (136)

(137)

(138) (139) (140)

(141) (142) (143) (144) (145)

Y.; Valdivia, A.; Go´mez, L.; Simpson, B. K.; Villalonga, R. Macromol. Biosci. 2005, 5, 1220. (e) Gilgun-Sherki, Y.; Rosenbaum, Z.; Melamed, E.; Offen, D. Pharmacol. ReV. 2002, 54, 271. Industrial Enzymology, 2nd ed.; Godfrey, T., West, S. I., Eds.; Macmillan Press: London, 1996. Venkatesh, R.; Sundaram, P. V. Protein Eng. 1998, 11, 691. (a) Villalonga, R.; Villalonga, M. L.; Go´mez, L. J. Mol. Catal. B: Enzym. 2000, 10, 483. (b) Villalonga, M. L.; Reyes, G.; Villalonga, R. Biotechnol. Lett. 2004, 26, 209. (c) Masa´rova´, J.; Mislovicˇova´, D.; Gemeiner, P.; Michalkova´, E. Biotechnol. Appl. Biochem. 2001, 34, 127. (d) Villalonga, R.; Tachibana, S.; Pe´rez, Y.; Asano, Y. Biotechnol. Lett. 2005, 27, 1311. (e) Go´mez, L.; Villalonga, R. Biotechnol. Lett. 2000, 22, 1191. Wang C.; Eufemi, M.; Turano, C.; Giartosio, A. Biochemistry 1996, 35, 7299. (a) Fujita, T.; Yasuda, Y.; Takakura, Y.; Hashida, M.; Sezaki, H. J. Controlled Release 1990, 11, 149. (b) Duncan, R.; Spreafico, F. Clin. Pharmacokinet. 1994, 27, 290. Morand, P.; Biellman, J. F. FEBS Lett. 1991, 289, 148. Fernańdez, M.; Fragoso, A.; Cao, R.; Banõs, M.; Villalonga, M. L.; Villalonga, R. Biotechnol. Lett. 2002, 24, 1455. Fernańdez, M.; Villalonga, M. L.; Fragoso, A.; Cao, R.; Villalonga, R. Biotechnol. Appl. Biochem. 2002, 36, 235. Villalonga, R.; Tachibana, S.; Cao, R.; Ramirez, H. L.; Asano, Y. Biochem. Eng. J. 2006, 30, 26. Fernańdez, M.; Fragoso, A.; Cao, R.; Banõs, M.; Ansorge-Schumacher, M.; Hartmeier, W.; Villalonga, R. J. Mol. Catal. B: Enzym. 2004, 31, 47. Fernańdez, M.; Fragoso, A.; Cao, R.; Banõs, M.; Villalonga, R. Enzyme Microb. Technol. 2002, 31, 543. Fernańdez, M.; Fragoso, A.; Cao, R.; Villalonga, R. J. Mol. Catal. B: Enzym. 2003, 21, 133. Fernańdez, M.; Fragoso, A.; Cao, R.; Villalonga, R. Process Biochem. 2005, 40, 2091. (a) Villalonga, R.; Go´mez, L.; Ramı´rez, H. L.; Villalonga, M. L. J. Chem. Technol. Biotechnol. 1999, 74, 635. (b) Go´mez, L.; Ramı´rez, H. L.; Villalonga, R. Biotechnol. Lett. 2000, 22, 347. (c) Darias, R.; Villalonga, R. J. Chem. Technol. Biotechnol. 2001, 76, 489. (d) Go´mez, L.; Ramı´rez, H. L.; Villalonga, R. Acta. Biotechnol. 2001, 21, 265. Valdivia, A.; Villalonga, R.; Sommella, M. G.; Pe´rez, Y.; Mariniello, L.; Porta, R. J. Biotechnol. 2006, 122, 326. Aeschlimann, D.; Paulsson, M. Thromb. Haemost. 1994, 71, 402. (a) Mancuso, F.; Porta, R.; Calignano, A.; Di Pierro, P.; Sommella, M. C.; Esposito, C. Peptides 2001, 22, 1453. (b) Folk, J. E.; Chung, S. I. Methods Enzymol. 1995, 11, 358. Villalonga, R.; Fernańdez, M.; Fragoso, A.; Cao, R.; Di Pierro, P.; Mariniello, L.; Porta, R. Biotechnol. Bioeng. 2003, 81, 732. Villalonga, R.; Fernańdez, M.; Fragoso, A.; Cao, R.; Mariniello, L.; Porta, R. Biotechnol. Appl. Biochem. 2003, 38, 53. Ramı´rez, H. L.; Chico, B.; Hoste, K.; Schacht, E. H.; Villalonga, R. J. Bioact. Compat. Polym. 2002, 17, 161. Fulton, D. A.; Stoddart, J. F. Bioconjugate Chem. 2001, 12, 655. Darias, R.; Herrera, I.; Fragoso, A.; Cao, R.; Villalonga, R. Biotechnol. Lett. 2002, 24, 1665. (a) Villalonga, M. L.; Fernańdez, M.; Fragoso, A.; Cao, R.; Villalonga, R. Prep. Biochem. Biotechnol. 2003, 33, 53. (b) Valdivia, A.; Pe´rez, Y.; Ramı´rez, H. L.; Cao, R.; Villalonga, R. Biotechnol. Lett. 2006, 28, 1465. (c) Valdivia, A.; Pe´rez, Y.; Cao, R.; Banõs, M.; Garcıá, A.; Villalonga, R. Macromol. Biosci. 2007, 7, 70. (a) Villalonga, M. L.; Reyes, G.; Fragoso, A.; Cao, R.; Villalonga, R. Biotechnol. Appl. Biochem. 2005, 41, 217. (b) Fernańdez, M.; Villalonga, M. L.; Fragoso, A.; Cao, R.; Banõs, M.; Villalonga, R. Process Biochem. 2004, 39, 535. (c) Villalonga, R.; Tachibana, S.; Cao, R.; Matos, M.; Asano, Y. Enzyme Microb. Technol. 2007, 40, 471. Fitter, J. Biophys. J. 2003, 84, 3924. Rozema, D.; Gellman, S. H. Biochemistry 1996, 35, 15760. (a) Thatcher, D. R.; Hitchock, A. Protein folding in biotechnology. In Mechanisms of Protein Folding; Pain, R. H., Ed.; IRL Press: Oxford, 1994; p 229. (b) Cleland, J. L. Impact of protein folding on biotechnology. In Protein Folding in ViVo and in Vitro; Cleland, J. L., Ed.; ACS Symposium Series 526; American Chemical Society: Washington, DC, 1993; p 1. Kumar, Y.; Tayyab, S.; Muzammil, S. Arch. Biochem. Biophys. 2004, 426, 3. Wetlaufer, D. B.; Xie, Y. Protein Sci. 1995, 4, 1535. Cleland, J. L.; Builder, S. E.; Swartz, J. R.; Winkler, M.; Chang, J. Y.; Wang, D. I. Biotechnology 1992, 10, 1013. Muzammil, S.; Kumar, Y.; Tayyab, S. Biochem. Biophys. Acta 2000, 1476, 139. Kudou, M.; Shiraki, K.; Fujiwara, S.; Imanaka, T.; Takagi, M. Eur. J. Biochem. 2003, 270, 4547.

Supramolecular Chemistry of Cyclodextrins (146) Cooper, A.; Lovatt, M.; Nutley, M. A. J. Inclusion Phenom. Mol. Recognit. Chem. 1996, 25, 85. (147) (a) Karuppiah, N.; Sharma, A. Biochem. Biophys. Res. Commun. 1995, 211, 60. (b) Sharma, A.; Karuppiah, N. U.S. Patent 5,728,804, 1998. (148) Sharma, L.; Sharma, A. Eur. J. Biochem. 2001, 268, 2456. (149) Yazdanparast, R.; Khodagholi, F. Arch. Biochem. Biophys. 2006, 446, 11. (150) (a) Rozema, D.; Gellman, S. H. J. Am. Chem. Soc. 1995, 117, 2373. (b) Gellman, S. H.; Rozema, D. Method for refolding misfolded enzymes with detergent and cyclodextrin, U.S. Patent 5,563,057, 1996. (151) Dong, X. Y.; Wang, Y.; Shi, J. H.; Sun, Y. Enzyme Microb. Technol. 2002, 30, 792. (152) Machida, S.; Ogawa, S.; Xiaohua, S.; Takaha, T.; Fujii, K.; Hayashi, K. FEBS Lett. 2000, 486, 131. (153) Lanckriet, H.; Middelberg, A. P. J. Biotechnol. Prog. 2004, 20, 1861. (154) (a) Akiyoshi, K.; Sasaki, Y.; Sunamoto, J. Bioconjugate Chem. 1999, 10, 321. (b) Nomura, Y.; Ikeda, M.; Yamaguchi, N.; Aoyama, Y.; Akiyoshi, K. FEBS Lett. 2003, 553, 271. (c) Nomura, Y.; Sasaki, Y.; Takagi, M.; Narita, T.; Aoyama, Y.; Akiyoshi, K. Biomacromolecules 2005, 6, 447. (155) (a) Fernańdez, M.; Villalonga, M. L.; Fragoso, A.; Cao, R.; Villalonga, R. Enzyme Microb. Technol. 2004, 34, 78. (b) Fernańdez, M.; Villalonga, M. L.; Caballero, J.; Fragoso, A.; Cao, R.; Villalonga, R. Biotechnol. Bioeng. 2003, 83, 743. (156) (a) Burton, S. G.; Cowan, D. A.; Woodley, J. M. Nat. Biotechnol. 2004, 20, 37. (b) Schmid, A.; Dordick, J. S.; Kiener, A.; Wubbolts, M.; Wtholt, B. Nature 2001, 409, 258. (157) End, N.; Schoening, K.-U. Top. Curr. Chem. 2004, 242 (Immob. Cat.), 273. (158) Wang, X.; Zhou, D.; Sinniah, K.; Clarke, C.; Birch, L.; Li, H.; Rayment, T.; Abell, C. Langmuir 2006, 22, 887. (159) Ludden, M. J. W.; Mulder, A.; Tampe´, R.; Reinhoudt, D. N.; Huskens, J. Angew. Chem., Int. Ed. 2007, 46, 1. (160) (a) Niemeyer, C. M. Angew. Chem., Int. Ed. 2003, 42, 5796. (b) Whitesides, G. M. Nat. Biotechnol. 2003, 21, 1161. (c) Wilson, D. S.; Nock, S. Angew. Chem., Int. Ed. 2003, 42, 494. (161) (a) Whitesides, G. M. Small 2005, 1, 172. (b) Whitesides, G. M. Nat. Biotechnol. 2003, 21, 1161. (c) La Van, D. A.; McGuire, T.; Langer, R. Nat. Biotechnol. 2003, 21, 1184. (162) David, C.; Millot, M. C.; Renard, E.; Sebille, B. J. Inclusion Phenom. Macrocycl. Chem. 2002, 44, 369. (163) (a) Grace, Y. J.; Chen, G. Y. Y.; Uttamchandani, M.; Zhu, Q.; Wang, G.; Yao, S. Q. ChemBioChem. 2003, 4, 336. (b) Eppinger, J.; Funeriu, D. P.; Miyake, M.; Denizot, L.; Miyake, Y. Angew. Chem., Int. Ed. 2004, 43, 3806. (164) Qu, H.; Wang, H.; Huang, Y.; Zhong, W.; Lu, H.; Kong, J.; Yang, P.; Liu, B. Anal. Chem. 2004, 76, 6426. (165) Temesvari, J.; Banky, E.; Biacs, P. A. Acta Alimentaria 1994, 23, 215. (166) (a) Kang, X.-F.; Gu, L.-Q.; Cheley, S.; Bayley, H. Angew. Chem., Int. Ed. 2005, 44, 1495. (b) Bayley, H.; Cremer, P. S. Nature 2001, 413, 226. (c) Astier, Y.; Braha, O.; Bayley, H. J. Am. Chem. Soc. 2006, 128, 1705. (167) (a) Easton, C. J.; Lincoln, S. F. Chem. Soc. ReV. 1996, 25, 163. (b) Wolf, C. Chem. Soc. ReV. 2005, 34, 595. (c) Schurig, V. Chirality 2005, 17, S205. (d) Mikusˇ, P.; Vala´sˇkova´, I.; Havrańek, E. J. Pharm. Biomed. Anal. 2003, 33, 157. (168) Bao, J.; Regnier, F. E. J. Chromatogr. A 1992, 608, 217. (169) Van Dyck, S.; Van Schepdael, A.; Hoogmartens, J. Electrophoresis 2002, 23, 1341. (170) Lam, S.; Malikin, G. Chirality 1992, 4, 395. (171) Cao, R.; Villalonga, R.; Fragoso, A. IEE Proc.-Nanobiotechnol. 2005, 152, 159. (172) Sukhorukov, G. B.; Rogach, A. L.; Zebli, B.; Liedl, T.; Skirtach, A. G.; Ko¨hler, K.; Antipov, A. A.; Gaponik, N.; Susha, A. S.; Winterhalter, M.; Parak, W. J. Small 2005, 1, 194. (173) Penn, S. G.; He, L.; Natan, M. J. Curr. Opin. Chem. Biol. 2003, 7, 609. (174) Skitach, A. G.; Dejugnat, C.; Braun, D.; Susha, A. S.; Rogach, A. L.; Parak, W. J.; Mo¨hwald, H.; Sukhorukov, G. B. Nano Lett. 2005, 5, 1371. (175) Mahalingam, V.; Onclin, S.; Meter, M.; Ravoo, B. J.; Huskens, J.; Reinhoudt, D. N. Langmuir 2004, 20, 11756. (176) Nayak, S.; Lyon, L. A. Angew. Chem., Int. Ed. 2005, 44, 7686. (177) (a) Ambade, A. V.; Savariar, E. N.; Thayumanavan, S. Mol. Pharmaceutics 2005, 2, 264. (b) Paleos, C. M.; Tsiourvas, D.; Sideratou, Z. Mol. Pharmaceutics 2007, 4, 169. (178) Michels, J. J.; Huskens, J.; Reinhoudt, D. N. J. Chem. Soc., Perkin Trans. 2002, 2, 102.

Chemical Reviews, 2007, Vol. 107, No. 7 3115 (179) (a) Jayaraman, N.; Nepogodiev, S. A.; Stoddart, J. F. Chem. Eur. J. 1997, 3, 1193. (b) Wang, Q.; Dordick, J. S.; Linhardt, R. J. Chem. Mater. 2002, 14, 3232. (180) Ong, W.; Go´mez-Kaifer, M.; Kaifer, A. E. Chem. Commun. 2004, 1677. (181) (a) Boas, U.; Heegaard, P. M. H. Chem. Soc. ReV. 2004, 33, 43. (b) Arima, H.; Kihara, F.; Hirayama, F.; Uekama, K. Bioconjugate Chem. 2001, 12, 476. (182) (a) Kihara, F.; Arima, H.; Tsutsumi, T.; Hirayama, F.; Uekama, K. Bioconjugate Chem. 2002, 13, 1211. (b) Kihara, F.; Arima, H.; Tsutsumi, T.; Hirayama, F.; Uekama, K. Bioconjugate Chem. 2003, 14, 342. (c) Wada, K.; Arima, H.; Tsutsumi, T.; Hirayama, F.; Uekama, K. Biol. Pharm. Bull. 2005, 28, 500. (d) Wada, K.; Arima, H.; Tsutsumi, T.; Chihara, Y.; Hattori, K.; Hirayama, F.; Uekama, K. J. Controlled Release 2005, 104, 397. (e) Arima, H. Yakugaku Kenkyu no Shinpo 2003, 19, 1. (183) (a) Popielarski, S. R.; Mishra, S.; Davis, M. E. Bioconjugate Chem. 2003, 14, 672. (b) Reineke, T. M.; Davis, M. E. Bioconjugate Chem. 2003, 14, 247. (c) Reineke, T. M.; Davis, M. E. Bioconjugate Chem. 2003, 14, 255. (d) Cheng, J.; Khin, K. T.; Jensen, G. S.; Liu, A.; Davis, M. E. Bioconjugate Chem. 2003, 14, 1007. (e) Shuai, X.; Merdan, T.; Unger, F.; Kissel, T. Bioconjugate Chem. 2005, 16, 322. (f) Bellocq, N. C.; Kang, D. W.; Wang, X.; Jensen, G. S.; Pun, S. H.; Schluep, T.; Zepeda, M. L.; Davis, M. E. Bioconjugate Chem. 2004, 15, 1201. (g) Pun, S. H.; Davis, M. E. Bioconjugate Chem. 2002, 13, 630. (h) Bellocq, N. C.; Pun, S. H.; Jensen, G. S.; Davis, M. E. Bioconjugate Chem. 2003, 14, 1122. (184) Vargas-Verengel, A.; Ortega-Caballero, F.; Santoyo-Gonza´lez, F.; Garcıá-Lo´pez, J. J.; Jimeńez-Martinez, J. J.; Garcıá-Fuentes, L.; OrtizSalmeron, E. Chem. Eur. J. 2002, 8, 812. (185) Ortega-Caballero, F.; Gimeńez-Martıńez, J. J.; Garcıá-Fuentes, L.; Ortiz-Salmeroń, E.; Santoyo-Gonza´lez, F.; Vargas-Berenguel, A. J. Org. Chem. 2001, 66, 7786. (186) Baussanne, I.; Benito, J. M.; Ortiz-Mellet, C.; Garcıá-Fernańdez, J. M.; Law, H.; Defaye, J. Chem. Commun. 2000, 1489. (187) Andre´, S.; Kaltner, H.; Furuike, T.; Nishimura, S.-i.; Gabius, H.-J. Bioconjugate Chem. 2004, 15, 87. (188) Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321. (189) Benito, J. M.; Go´mez-Garcıá, M.; Ortiz-Mellet, C.; Baussanne, I.; Defaye, J.; Garcıá-Fernańdez, J. M. J. Am. Chem. Soc. 2004, 126, 10355. (190) Go´mez-Garcıá, M.; Benito, J. M.; Rodrı´guez-Lucena, D.; Yu, J.; Chmurski, K.; Ortiz-Mellet, C.; Gutie´rrez-Gallego, R.; Maestre, A.; Defaye, J.; Garcıá-Fernańdez, J. M. J. Am. Chem. Soc. 2005, 127, 7970. (191) Lis, H.; Sharon, N. Chem. ReV. 1998, 98, 637. (192) Toyokuni, T.; Singhal, A. K. Chem. Soc. ReV. 1995, 231. (193) Fransen, E. J. F.; Jansen, R. W.; Vaalburg, M.; Meijer, D. K. F. Biochem. Pharm. 1993, 45, 1215. (194) Parrot-Lopez, H.; Galons, H.; Coleman, A. W.; Mahuteau, J.; Miocque, M. Tetrahedron Lett. 1992, 33, 209. (195) Parrot-Lopez, H.; Leray, E.; Coleman, A. W. Supramol. Chem. 1993, 3, 37. (196) Kassab, R.; Fe´lix, C.; Parrot-Lopez, H.; Bonaly, R. Bioorg. Med. Chem. Lett. 1997, 7, 7555. (197) Kukowska-Latallo, J. F.; Bielinska, A. U.; Johnson, J.; Splindler, R.; Tomalia, D. A.; Baker, J. R., Jr. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4897. (198) Rusa, C. C.; Wei, M.; Bullions, T. A.; Shuai, X.; Tamer Uyar, T.; Tonelli, A. E. Polym. AdV. Technol. 2005, 16, 269. (199) (a) Nomura, Y.; Sasaki, Y.; Takagi, M.; Narita, T.; Aoyama, Y.; Akiyoshi, K. Biomarcomolecules 2005, 6, 447. (b) Morimoto, N.; Endo, T.; Ohtomi, M.; Iwasaki, Y.; Akiyoshi, K. Macromol. Biosci. 2005, 5, 710. (200) Verma, A.; Simard, J. M.; Worrall, J. W. E.; Rotello, V. M. J. Am. Chem. Soc. 2004, 126, 13987. (201) (a) Gole, A.; Dash, C.; Soman, C.; Sainkar, S. R.; Rao, M.; Sastry, M. Bioconjugate Chem. 2001, 12, 684. (b) MacDonald, I. D. G.; Smith, W. E. Langmuir 1996, 12, 706. (202) Anshup Venkataraman, J. S.; Subramaniam, C.; Kumar, R. R.; Priya, S.; Kumar, T. R. S.; Omkumar, R. V.; John, A.; Pradeep, T. Langmuir 2005, 21, 11562. (203) Tsai, C.-S.; Yu, T.-B.; Chen, C.-T. Chem. Commun. 2005, 4273. (204) Senaratne, W.; Andruzzi, L.; Ober, C. K. Biomacromolecules 2005, 6, 2427. (205) Brewer, S. H.; Glomm, W. R.; Johnson, M. C.; Knag, M. K.; Franzen, S. Langmuir 2005, 21, 9303. (206) Aubin-Tam, M.-E.; Hamad-Schifferli, K. Langmuir 2005, 21, 12080. (207) (a) Liu, J.; Ong, W.; Roman, E.; Lynn, M. J.; Kaifer, A. E. J. Am. Chem. Soc. 1999, 121, 4304. (b) Liu, J.; Mendoza, S.; Roman, E.; Lynn, M. J.; Xu, R.; Kaifer, A. E. Langmuir 2000, 16, 3000. (c) Liu, J.; Alva´rez, J.; Ong, W.; Roman, E.; Kaifer, A. E.; Peinador, C. J. Am. Chem. Soc. 2001, 123, 11148.

3116 Chemical Reviews, 2007, Vol. 107, No. 7 (208) Alvarez, J.; Liu, J.; Roman, E.; Kaifer, A. E. Chem. Commun. 2000, 1151. (209) (a) Liu, J.; Alvarez, J.; Ong, W.; Roman, E.; Kaifer, A. E. Langmuir 2001, 17, 6762. (b) Strimbu, L.; Liu, J.; Kaifer, A. E. Langmuir 2003, 19, 483. (210) Liu, J.; Ong, W.; Kaifer, A. E.; Peinador, C. Langmuir 2002, 18, 5981. (211) Ve´gvari, A Ä .; Larsson, A.-K.; Hjerteń, S.; Mannervik, B. ChemBioChem 2002, 3, 1117. (212) Villalonga, R.; Cao, R.; Fragoso, A.; Ortiz, P. D.; Damiao, A. E.; Villalonga, M. L. Supramol. Chem. 2005, 17, 387. (213) Villalonga, R.; Tachibana, S.; Cao, R.; Ortiz, P. D.; Go´mez, L.; Asano, Y. J. Exp. Nanosci. 2006, 1, 249. (214) Villalonga, R.; Cao, R.; Fragoso, A.; Damiao, A. E.; Ortiz, P. D.; Caballero, J. J. Mol. Catal. B: Enzym. 2005, 35, 79. (215) Amstrong, F. A.; Wilson, G. S. Electrochim. Acta 2000, 45, 2623. (216) Medintz, I. L.; Anderson, G. P.; Lassman, M. E.; Goldman, E. R.; Betancourt, L. A.; Maurd, J. M. Anal. Chem. 2004, 76, 5620. (217) Stefan, R.-I.; van Staden, J. F.; Aboul-Enein, H. Y. Electroanalysis 1999, 11, 1233. (218) (a) Luong, J. H. T.; Brown, R. S.; Male, K. B.; Cattaneo, M. V.; Zhao, S. Trends Biotechnol. 1995, 13, 457. (b) Luong, J. H. T.; Brown, R. S.; Schmidt, P. M. J. Mol. Recognit. 1995, 8, 132. (c) Luong, J. H.; Masson, C.; Brown, R. S.; Male, K. B.; Nguyen, A. L. Biosens. Bioelectron. 1994, 9, 577. (219) Pearson, J. E.; Gill, A.; Vadgama, P. Ann. Clin. Biochem. 2000, 37, 119. (220) Liu, H.; Wang, Y.; Zhang, G. Huaxue Chuanganqi 2002, 22, 40. (221) Han, S.-B.; Zhu, M.; Yuan, Z.-B. Gaodeng Xuexiao Huaxue Xuebao 1999, 20, 1036. (222) (a) Kutner, W.; Wu, H.; Kadish, K. M. Electroanalysis 1994, 6, 934. (b) Wu, B.-Z.; Wu, H.-H. Huaxue Xuebao 1998, 56, 364. (223) Sun, K.; Li, Z.; Chen, X. Huaxue Yanjiu Yu Yingyong 2001, 13, 376. (224) Liu, S.; Mo, W.; Chen, F. Fenxi Ceshi Xuebao 1999, 18, 42. (225) Forrow, N. J; Walters, S. J. Biosens. Bioelectron. 2004, 19, 763. (226) Kosela, E.; Elzanowska, H.; Kutner, W. Anal. Bioanal. Chem. 2002, 373, 724. (227) Chen, Q.; Pamidi, P. V. A.; Wang, J.; Kutner, W. Anal. Chim. Acta 1995, 306, 201. (228) Liu, S.; Mo, W.; Fenxi, C. Ceshi Xuebao 1999, 18, 42. (229) (a) Zhu, B.-S.; Ying, T.-L.; Zhang, X.-L.; Qi, D.-Y. Shanghai Daxue Xuebao, Ziran Kexueban 1999, 5, 353. (b) Ying, T.; Sun, K.; Wang, C.; Qi, D. Huaxue Chuanganqi 1998, 18, 9. (230) (a) Groom, C. A.; Luong, J. H. T. Biosens. Bioelectron. 1994, 9, 305. (b) Luong, J. H. T.; Male, K. B.; Groom, C. A.; Zhao, S. NATO ASI Ser., Ser. E: Appl. Sci. 1993, 252, 211.

Villalonga et al. (231) Bu, H.-Z.; Mikkelsen, S. R.; English, A. M. Anal. Chem. 1995, 67, 4071. (232) Liu, H.; Li, H.; Ying, T.; Sun, K.; Qin, Y.; Qi, D. Anal. Chim. Acta 1998, 358, 137. (233) Li, H.-H.; Yan, S.-H.; Qi, D.-Y.; Liu, H.-Y. Shengwu Huaxue Yu Shengwu Wuli Jinzhan 1998, 25, 162. (234) Jia, N.; Jiang, L.; Zhu, Y.; Wu, X.; Zhang, Z. Huaxue Yanjiu Yu Yingyong 2003, 15, 341. (235) Roy, J. J.; Abraham, T. E.; Abhijith, A. S.; Kumar, P. V. S.; Thakur, M. S. Biosens. Bioelectron. 2005, 21, 206. (236) (a) Tu, Y.-F.; Chen, H.-Y. Biosens. Bioelectron. 2002, 17, 19. (b) Tu, Y.-F.; Chen, H.-Y. Anal. Biochem. 2001, 299, 71. (237) Kataky, R.; Parker, D. Analyst 1996, 121, 1829. (238) Kataky, R.; Dell, R.; Senanayake, P. K. Analyst 2001, 126, 2015. (239) Kitano, H.; Miyamoto, T.; Kawasaki, H. J. Colloid Interface Sci. 2004, 279, 425. (240) (a) Fragoso, A.; Caballero, J.; Almirall, E.; Villalonga, R.; Cao, R. Langmuir 2002, 18, 5051. (b) Villalonga, R.; Fujii, A.; Shinohara, H.; Asano, Y.; Cao, R.; Tachibana, S.; Ortiz, P. Biotechnol. Lett. 2007, 29, 447. (241) Mulder, A.; Huskens, J.; Reinhoudt, D. N. Org. Biomol. Chem. 2004, 2, 3409. (242) Chmurski, K.; Koralewska, A.; Temeriusz, A.; Bilewicz, R. Electroanalysis 2004, 16, 1407. (243) Villalonga, R.; Matos, M.; Cao, R. Electrochem. Commun. 2007, 9, 454. (244) (a) Guerrouache, M.; Karakasyan, C.; Gaillet, C.; Canva, M.; Millot, M. C. J. Appl. Polym. Sci. 2006, 100, 2362. (b) Villalonga, R.; Camacho, C.; Cao, R.; Hernańdez, J.; Matıás, J. C. Chem. Commun. 2007, 942. (245) Yang, J.; Liang, S.-C. Anal. Chim. Acta 2005, 537, 385. (246) Medintz, I. L.; Clapp, A. R.; Mattoussi, H.; Goldman, E. R.; Fisher, B.; Mauro, J. M. Nat. Mater. 2003, 2, 630. (247) Engels, J. W. Angew. Chem., Int. Ed. 2005, 44, 7166. (248) Crepo-Biel, O.; Dordi, B.; Maury, P.; Pe´ter, M.; Reinhoudt, D. N.; Huskens, J. Chem. Mater. 2006, 18, 2545. (249) Haider, J. M.; Pikramenou, Z. Chem. Soc. ReV. 2005, 34, 120. (250) Palaniappan, K.; Xue, C.; Arumugam, G.; Hackley, S. A.; Liu, J. Chem. Mater. 2006, 18, 1275. (251) Burapatana, V.; Prokop, A.; Tanner, R. D. Appl. Biochem. Biotechnol. 2006, 129-132, 247.

CR050253G

Supramolecular Chemistry of Cyclodextrins in Enzyme Technology

Recommend Documents