A Synthetic Cysteine Oxidase Based on a Ferrocene-Cyclodextrin

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Bioconjugate Chem. 1999, 10, 464−469

A Synthetic Cysteine Oxidase Based on a Ferrocene-Cyclodextrin Conjugate Suzanne K. Schreyer and Susan R. Mikkelsen* Department

of

Chemistry,

University

of

Waterloo,

Waterloo,

Ontario,

Canada

N2L

3G1. Received

November 5, 1998; Revised Manuscript Received January 22, 1999

We report a novel synthetic cysteine oxidase consisting of a ferrocene-β-cyclodextrin conjugate in which the ferrocene moiety is bound to the secondary hydroxyl side of the cyclodextrin cavity through an ethylenediamine linker. Cysteine oxidation occurs after the ferrocene group is electrochemically oxidized to the ferricinium form, and this generates a voltammetric electrocatalytic wave, the magnitude of which is related to the rate constant for cysteine oxidation. Comparison of cysteine oxidation rates for the primary and secondary β-cyclodextrin derivatives (105 and 1470 M-1 s-1, respectively) shows that the secondary derivatives are more effective synthetic enzymes. Substrate selectivity of the secondary derivative is demonstrated by comparison of oxidation rates for cysteine (1470 M-1 s-1) and glutathione (260 M-1 s-1) at pH 7.0. The rate constant for cysteine oxidation was 3-fold higher at pH 8.0. With a constant synthetic enzyme concentration, electrocatalytic limiting currents increased linearly with increasing cysteine concentration to a maximum at 6 mM cysteine; above this concentration, the current decreased significantly. These and other results suggest that product inhibition of the catalytic cycle occurs as a result of cystine binding more strongly to the cyclodextrin than cysteine.

INTRODUCTION

Synthetic enzymes have been interesting to chemists and biochemists for several decades. Fundamentally, they are relatively simple models of more complicated biochemical catalysts, possessing both selectivity and enhanced reaction rates in structurally well-defined and relatively small molecules. Their potential applications have encouraged research, since they are expected to be stable under conditions of temperature, pH, and solvent composition that would denature or inactivate natural, protein-based biocatalysts. In essence, an enzyme may be thought of as a selective substrate-binding site attached rigidly or flexibly to a nearby catalytic site. This simple definition has been realized by various researchers in molecules that selectively catalyze hydrolyses (1, 2), isomerizations (3-5), addition (6), and decarboxylation (7). Examples of synthetic oxidoreductases that use cyclodextrins or selective substrate recognition have used attached inorganic catalytic groups [iron-sulfur complexes (8), an oxodiperoxomolybdenum complex (9), polyamine complexes of various divalent metal ions (10), and ferrocene (11)] or organic catalysts [porphyrins (12, 13) and flavin analogues (1418)] to mimic biological redox processes. The first synthetic flavoenzyme was reported by Tabushi and Kodera and consisted of riboflavin bound to one of the primary β-cyclodextrin hydroxyl groups (14). They reported a 240-fold rate enhancement for the oxidation of n-hexyldihydronicotinamide by the primary flavocyclodextrin in comparison with free riboflavin possessing an equivalent but inert substituent. Ye et al. have reported several studies of β-cyclodextrin derivatized with isoalloxazines (the redox-active part of * To whom correspondence should be addressed. Former address: Concordia University, Montreal, Canada. E-mail: [email protected]. Fax (519) 746-0435.

riboflavin) on either the primary or secondary hydroxyl side of the cavity (16, 17). In this work, the catalysts were compared with free riboflavin for their abilities to oxidize nicotinamides and substituted benzyl acohols (16) and to oxidize alcohols or thiols and reduce aldehydes (17). Early results showed that p-tert-butylbenzyl alcohol was oxidized to its corresponding aldehyde by the secondary flavocyclodextrin 650 times faster than by riboflavin (16a). Subsequent studies with the same flavocyclodextrin revealed that thiol oxidation was effectively catalyzed, and the authors concluded that although the secondary cyclodextrin derivative had a dissociation constant for phenylmethanethiol binding similar to the primary derivative (1.89 mM vs 2.38 mM, respectively), its overall rate (as kcat/Kdiss) was twice as fast as the primary derivative (0.587 vs 0.246 M s-1) because orientation and alignment of the reacting atoms produced a higher kcat value in the secondary derivative. Suzuki et al. reported catalytic alcohol oxidation to aldehydes by a primary β-cyclodextrin derivative of ferrocenecarboxylic acid (11). In this 1:1 ferrocene:cyclodextrin derivative, all of the remaining hydroxyl groups (14 secondary and six primary hydroxyls) are acetylated, and this results in a lipophilic catalyst. The reactive ferricinium form was generated electrochemically during controlled-potential coulometry experiments using acetonitrile as a solvent. Although kinetic constants are not reported, the authors showed that after 12 h, each catalyst molecule had converted an average of 22 benzyl alcohol molecules and 59 1-naphthalenemethanol molecules to their corresponding aldehyde products; these values are more than 10-fold higher than those obtained after 24 h using methylferrocenecarboxylate, a model catalyst in which substrate binding could not occur. The authors propose a catalytic cycle in which the attached ferrocene, initially bound through the narrower primary opening to the cyclodextrin cavity, is excluded from the

10.1021/bc9801326 CCC: $18.00 © 1999 American Chemical Society Published on Web 03/24/1999

A Synthetic Cysteine Oxidase

cavity upon oxidation, allowing substrate binding. Upon substrate oxidation, the attached ferrocene reassociates with the cavity while the oxidation product is excluded. In this paper, we report the synthesis and preliminary kinetic characterization of two new ferrocene-β-cyclodextrin derivatives. For both, ferrocenemonocarboxaldehyde was reacted with ethylenediamine under reducing conditions prior to reaction with the primary or secondary p-toluenesulfonyl derivative of β-cyclodextrin to produce a -CH2NHCH2CH2NH- spacer linking one of the cyclopentadienyl rings of the ferrocene moiety with the cyclodextrin. UV-vis spectrophotometry and electrospray mass spectrometry were used to confirm product structures, and voltammetric experiments were performed to kinetically characterize the two species as synthetic thiol oxidases. EXPERIMENTAL SECTION

Materials. β-Cyclodextrin hydrate (β-CD), ferrocene carboxaldehyde, dimethylaminomethyl ferrocene, sodium hydride (67% dispersion in mineral oil), p-toluenesulfonyl chloride, reduced glutathione, L-cysteine hydrochloride hydrate, sodium formate, N,N-dimethylformamide, 4A molecular seives, anhydrous pyridine, sodium cyanoborohydride, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were purchased from Aldrich. Oxidized glutathione, L-cystine dihydrochloride, sodium acetate, dibasic sodium phosphate, and ethylenediamine were purchased from Sigma. Glacial acetic acid, dimethyl sulfoxide, acetone, and HPLC-grade acetonitrile were obtained from Baker. Potassium hexacyanoferrate(II) and monobasic sodium phosphate were obtained from Fisher. Chemicals were of the highest available grade and were used as received. All solutions were made using distilled, deionized water from a Barnstead Nanopure ion-exchange system. Instrumentation. A Cary 1 double-beam UV-vis spectrophotometer was used to obtain absorbance spectra, with 1 cm quartz cuvettes and blank 0.1 M phosphate buffer, pH 7.0 reference solutions. Purification of β-CD derivatives was done using an HPLC system consisting of a reciprocating pump (Waters model 510), a Rheodyne 2500 injector with a 100 µL sample loop, a 150 × 4.6 mm C18 column (Phenomenex ODP-50 4D) and a refractive index detector (Waters model 410). Electrochemical experiments were performed in nitrogen-deaerated solutions at 25 °C using a BAS 100A potentiostat (Bioanalytical Systems) with glassy carbon or carbon paste working and silver/silver chloride reference electrodes (Bioanalytical Systems) and a coiled NiChrome wire auxiliary electrode. Mass spectra were obtained using an SSQ 700 electrospray mass spectrometer (Finnegan MAT) in the positive ion mode. Methods. Preparation of Cyclodextrin Derivatives. β-CD was initially converted to either the primary or secondary tosylate according to selective literature procedures (19, 20). Briefly, the primary tosylate was formed by combining 2.04 g (1.8 mmol) β-CD with an excess (8.22 g, 43 mmol) of p-toluenesulfonyl chloride in 400 mL of pyridine. After 48 h at room temperature, the solution was vacuum distilled to give a viscous yellow liquid. The secondary tosylate was prepared by combining 1.1 g of β-CD with 60 mg of 60% sodium hydride dispersion in 40 mL of DMF (dried over 4A molecular sieves) and stirred at room temperature under dry conditions for 12 h. p-Toluenesulfonyl chloride (0.68 g) dissolved in 5 mL of dry DMF was then added, and stirring was continued for 1 h. Water (30 mL) was then

Bioconjugate Chem., Vol. 10, No. 3, 1999 465

added dropwise, and the solution was combined with 500 mL of acetone. The product was collected by filtration as a white precipitate. Ferrocenecarboxaldehyde (2.14 g, 10.0 mmol) was dissolved in 100 mL of 0.1 M HEPES buffer, 0.60 g (10.0 mmol) of ethylenediamine was added, and the pH was adjusted to 7.5. Sodium cyanoborohydride (6.28 g) was then added, and the solution was stirred at room temperature for 12 h. A 1:1 molar ratio of either the primary or secondary β-CD tosylate was then added to the filtered solution, and the reaction proceeded with stirring at room temperature for 5 days. Products were purified by reversed-phase HPLC, using a mobile phase consisting of 10% aqueous acetonitrile. Peaks were identified by injecting standard solutions of the starting materials and intermediate product mixtures. The primary ferrocene-β-CD reaction product exhibited a unique redox-active component that eluted at 5.5 min, in comparison with the primary tosylate (13.6 min) and the ferrocenylmethylethylenediamine product mixture (2.3 and 19.5 min). The secondary ferrocene-βCD product showed two unique components under these conditions (5.9 and 9.8 min), one of which was redoxactive with a formal potential of +220 mV vs Ag/AgCl (9.8 min); the component that eluted at 5.9 min is probably a secondary ethylenediamine-β-CD conjugate with no ferrocene attached. The secondary β-CD tosylate eluted at 12.1 min under these conditions. Electrochemical studies by Matsue et al. have shown that noncovalent ferrocene-cyclodextrin association products associate and dissociate so rapidly that the resulting voltammograms display averaged features (22); thus, only covalently bound ferrocene-β-CD species would elute under our slower chromatographic conditions. Purified samples of the primary and secondary β-CD tosylates as well as their ferrocenylmethylethylenediamine-β-CD products (1°-Fc-β-CD and 2°-Fc-β-CD, respectively) were collected for mass spectrometric analysis. Larger quantities of the two ferrocene derivatives were purified and concentrated with a rotary evaporator for electrochemical experiments. Electrochemical Studies. Glassy carbon working electrodes (0.071 cm2) were polished prior to each voltammogram using a slurry of 1 µm alumina (Buehler) in water on a polishing cloth (Bioanalytical Systems), and were sonicated in and rinsed with water prior to use. Unless otherwise stated, voltammograms were recorded from 0.0 to 0.7 V (vs Ag/AgCl) at a scan rate of 2 mV/s. At glassy carbon electrodes, thiol oxidation is kinetically slow, but is facilitated by an EC′ or electrocatalytic reaction mechanism in which an electrocatalyst, such as a ferrocene derivative or hexacyanoferrate (II), is readily oxidized at the electrode and subsequently oxidizes the thiol to the thiyl radical as it is reduced (eqs 1-3):

ferrocene f ferricinium + e-

(1)

k

ferricinium + R-SH 98 ferrocene + R-S‚ + H+ (2) 2R-S‚ f R-S-S-R

(3)

In this scheme, eqs 1 and 3 are expected to proceed rapidly, while the measured k values apply to eq 2 as the rate-determining step. Rate constants for thiol oxidation by electrocatalysts were determined using dilution experiments, with a 10:1 molar ratio of thiol to ferrocene-cyclodextrin conjugate. Under these conditions, the limiting electrocatalytic

466 Bioconjugate Chem., Vol. 10, No. 3, 1999

Schreyer and Mikkelsen

Figure 1. Electrospray mass spectrum of primary tosylate of β-CD dissolved in 10 mM sodium formate. Spectrum has been corrected by subtraction of the atomic weight of sodium.

and 6.50 × 10-6 cm2/s for hexacyanoferrate(II) (23), rate constants are readily calculated. RESULTS AND DISCUSSION

Figure 2. UV-vise spectra of (A) 42 µM dimethylaminomethylferrocene, (B) primary ferrocenylmethylethylenediamine-βCD, and (C) secondary ferrocenylmethylethylenediamine-β-CD. All species were dissolved in 10 mM phosphate buffer, pH 7.0.

current is described by eq 4, below (21):

ilim ) nFAC(Dk[thiol])1/2

(4)

where ilim is the limiting electrocatalytic oxidation current, n is the number of electrons in the electrochemical reaction, F is Faraday’s constant, A is the electrode area, C is the concentration of the electrocatalyst, D is the diffusion coefficient of the electrocatalyst, and k is the rate constant for thiol oxidation, shown in eq 2. Thus, a plot of limiting current against C[thiol]1/2 will have a slope equal to nFA(Dk)1/2. Using diffusion coefficients obtained by Matsue et al. of 6.4 × 10-6 and 2.6 × 10-6 cm2/s for free and CD-bound ferrocenecarboxylic acid at 27 °C (22)

Structural Characterization of CD Derivatives. Electrospray mass spectrometry was used to characterize the products of the primary and secondary CD tosylation reactions in 0.1 M sodium acetate buffer, pH 4.0, as described by Tinke et al. (24) and Lamcharfi et al. (25). Under these conditions, a positive ion is generated by the association of sodium ion with the CD. Figure 1 shows the sodium-corrected mass spectrum obtained for the primary tosylation reaction product, where a strong signal can be seen at the expected m/z value of 1289, which corresponds to singly tosylated β-CD. Smaller signals at 1134 and 1443 indicate the presence of small quantities of the starting CD and the ditosylated product, respectively. Similar results were observed for the secondary tosylation reaction product: prominent peaks were observed at m/z 1134, 1289, and 1443, with the m/z 1289 peak being about 5-fold more intense than the m/z 1443 peak. HPLC-purified primary and secondary ferrocenylmethylethylenediamine-β-CD were examined in 0.1 M phosphate buffer, pH 7.0, by UV-vis spectrophotometry and compared with a standard solution of dimethylaminomethylferrocene. The spectra, shown in Figure 2, all possess strong maxima near 205 nm. The molar absorptivity of dimethylaminomethylferrocene at 204 nm (2.71 × 104 M-1 cm-1) was used to determine the concentrations of the CD derivatives in subsequent electrochemical experiments. Electrospray mass spectrometry as described above was used to examine the ferrocene-CD conjugates. Under these conditions, no molecular ion peak was observed at the expected m/z value of 1561, but prominent peaks were observed at the much smaller m/z values of 403, 457, and 487 for both the primary and secondary conjugates, as shown in Figure 3. Varying the buffer and cone voltage conditions did not result in the expected molecular ion peaks. The most prominent peaks in the spectra shown in Figure 3, at m/z 403.2, can be attributed to the fragmen-

A Synthetic Cysteine Oxidase

Bioconjugate Chem., Vol. 10, No. 3, 1999 467

Figure 3. Electrospray mass spectrum of (A) primary ferrocenylmethylethylenediamine-β-CD, and (B) secondary ferrocenylmethylethylenediamine-β-CD, after purification by reversed-phase HPLC. CD derivatives were dissolved in 10 mM aqueous sodium formate. Spectra were not corrected for sodium.

tation products shown in Figure 4, in which the ferrocenylmethylethylenediamine moiety is bound to a single glucose residue through either a primary (Figure 4A) or secondary (Figure 4B) hydroxyl site. This is consistent with recent electrospray ion trap mass spectrometry fragmentation patterns for oligosaccharides observed by Sheeley and Reinhold (26). Electrocatalysis of Thiol Oxidation. Preliminary electrochemical studies were done with simple electrocatalyst species such as ferrocyanide, ferrocenecarboxaldehyde, and dimethylaminomethylferrocene to determine the thiol oxidation rates for their ferricyanide and ferricinium forms by electrocatalytic voltammetry as described in the Experimental Section. Table 1 shows the rates obtained for cysteine and glutathione oxidation in 0.10 M phosphate buffer, pH 7.0, in the presence and absence of free 10 mM β-CD. Thiol oxidation by ferricyanide has been studied by absorption spectrophotometry, where rate constants of 4.3 and 30 M-1 s-1 were obtained for the oxidation of mercaptoacetic acid at pH 1.4 and 1.9, respectively (27), and a value of 61 M-1 s-1 was obtained for the oxidation

Figure 4. Proposed fragments of (A) primary ferrocenylmethylethylenediamine-β-CD, and (B) secondary ferrocenylmethylethylenediamine-β-CD, each having a mass of 403.2 amu.

468 Bioconjugate Chem., Vol. 10, No. 3, 1999

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Table 1. Rate Constants for the Oxidation of Thiols by Electrocatalystsa electrocatalyst ferricyanide

thiol

[β-CD] (mM)

pH

k (M-1 s-1)

GSH

0 10 0 10 0 10 0 10 0 10 0 0 0 0 0

7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 8.0 8.0 7.0 7.0 7.0 7.0 8.0

48 38 90 38 2.1 21 103 134 30 23 143 105 260 1470 4120

Cys ferriciniumcarboxaldehyde dimethylaminomethylferricinium

GSH GSH Cys

1°-Fc-β-CD 2°-Fc-β-CD

GSH Cys GSH Cys

a Measured by linear-sweep voltammetry (2 mV/s) at glassy carbon electrodes in solutions containing a 10-fold excess of thiol and 10 mM phosphate. Abbreviations: GSH, glutathione; Cys, cysteine; 1°-Fc-β-CD, primary ferrocenylmethylethylenediamineβ-CD; 2°-Fc-β-CD, secondary ferrocenylmethylethylenediamineβ-CD. RSD values were less than 7%.

Figure 5. Limiting electrocatalytic current vs [ferricyanide][thiol]1/2, for (A) cysteine and (B) glutathione oxidation. Data were recorded at 500 mV vs Ag/AgCl following linear-sweep voltammetry at 2 mV/s at a glassy carbon working electrode in 0.10 M phosphate, pH 7.0.

of mercaptopropionic acid at pH 3.8 (28). This trend toward increased rates at higher pH values is continued in the values observed in this work. Figure 5 shows plots of limiting electrocatalytic current against [ferricyanide][thiol]1/2 for the determination of cysteine and glutathione oxidation rate constants. The rate constants compiled in Table 1 indicate not only that the rates of cysteine and glutathione oxidation by ferricyanide (90 and 48 M-1 s-1, respectively) are of the same order of magnitude as those reported earlier, but also that the rates are significantly lower when 10 mM β-CD is present. This indicates that both cysteine and glutathione bind β-CD while ferricyanide does not, so that the effective thiol concentration is lower in the presence of the CD. Results obtained for the two ferrocene derivatives, on the other hand, show increased thiol oxidation rates in the presence of 10 mM β-CD. Dimethylaminomethylferricinium oxidizes glutathione at a 1.3-fold higher rate, while ferriciniumcarboxaldehyde oxidizes glutathione at a 10-fold faster rate in the presence of 10 mM β-CD. These unexpected results are attributed to the simultaneous association of glutathione and ferricinium derivative with free CD. The formation of ternary complexes of CDs with two guest species has been observed previously by stopped-flow kinetic measurements, where association constants for complex formation were deter-

Figure 6. Limiting electrocatalytic current vs cysteine concentration, with [2°-Fc-β-CD] held constant at 0.80 mM. Data were recorded at 500 mV vs Ag/AgCl following linear-sweep voltammetry at 2 mV/s at a glassy carbon working electrode in 0.10 M phosphate, pH 7.0.

mined from CD-catalyzed ester hydrolysis rates that increased in the presence of the second guest species (29). Table 1 also shows thiol oxidation rates obtained for the primary and secondary ferrocene derivatives of β-CD. Clearly, the primary derivative does not facilitate thiol oxidation, since rates obtained with this species are not much different than those seen with dimethylaminomethylferrocene; thus, in this derivative, the ferrocene/ ferricicinium couple behaves as if it had no CD attached, as long as its lower diffusion coefficient is taken into account. However, the secondary derivative is a much better catalyst of thiol oxidation, especially with cysteine as a substrate: rate constants of 260 and 1470 M-1 s-1 were observed for glutathione and cysteine oxidation, respectively. Furthermore, if the pH is increased to 8.0 in 0.10 M phosphate, the rate of cysteine oxidation increases to 4120 M-1 s-1, while free dimethylaminomethylferrocene shows a rate of 30 M-1 s-1. These results suggest that sulfhydryl deprotonation is important during some stage of the reaction, since the pKa is 8.15 for the cysteine sulfhydryl (30). Possibly, the relatively low oxidation rate observed for free dimethylaminomethylferrocene at higher pH is due to the known instability of ferricinium toward nucleophiles (31). However, the 140fold acceleration factor due to covalently bound β-CD is of a similar magnitude to the 650-fold acceleration of p-tert-butylbenzyl alcohol oxidation by the secondary flavocyclodextrin derivative reported by Ye et al. (16a). Calibration curves of limiting current against cysteine concentration were obtained while holding the total concentration of secondary ferrocene-β-CD constant. A typical plot, obtained using a glassy carbon working electrode, is shown in Figure 6. A similar curve was observed using a carbon paste working electrode. A linear region can be seen in Figure 6 below 6 mM cysteine, while higher cysteine concentrations yield decreased electrocatalytic wave heights. The shape of this curve suggests product inhibition, in that tighter binding of the disulfide product to the β-CD cavity would effectively reduce the available catalyst concentration; this phenomenon has been documented extensively for enzymatic reactions (32). Electrochemical dilution experiments provided confirmation of this type of inhibition. Rate constants observed at pH 7.0 for the oxidation of glutathione by 2°-Fc-β-CD decreased 10-fold when the bulk solution initially contained oxidized glutathione (GS-SG) at half the glutathione concentration. Similarly, when cystine was initially present in the bulk solution at only 4% of the cysteine concentration (this was limited by cystine solubility), the observed rate constant for cysteine oxida-

A Synthetic Cysteine Oxidase

tion by 2°-Fc-β-CD decreased to less than 10% of the value observed in the absence of cystine. Thus, the disulfide oxidation products appear to associate with the CD cavity more strongly than the sulfhydryl substrates. Despite this, at cysteine concentrations below 6 mM (see Figure 6), 2°-Fc-β-CD appears to be a very effective synthetic cysteine oxidase. ACKNOWLEDGMENT

The authors are grateful to the Natural Sciences and Engineering Research Council of Canada (NSERC) and Merck Frosst Canada Inc. for financial support. LITERATURE CITED (1) Bender, M. L., and Komiyama, M. (1978) Cyclodextrin Chemistry, Chapters V, pp 33-50, and VII, pp 61-63, and references therein, Springer-Verlag, New York. (2) Breslow, R. (1995) Biomimetic Chemistry and Artificial Enzymes: Catalysis by Design Acc. Chem. Res. 28, 146-153, and references therein. (3) Murakami, Y., Hisaeda, Y., Kikuchi, J., Ohno, T., Suzuki, M., Matsuda, Y., and Matsuura, T. (1988) Hydrophobic Vitamin B12. Part 6. Carbon-Skeleton Rearrangement via Formation of Host-Guest Complexes Derived from an “Octopus” Azaparacyclophane and Hydrophobic Vitamin B12 Derivatives: A Novel Holoenzyme Model System J. Chem. Soc., Perkin Trans. 2, 1237-1246. (4) Murakami, Y., Hisaeda, Y., and Ohno, T. (1990) Hydrophobic Vitamin B12. VII. Ring-Expansion Reactions Catalyzed by Hydrophobic Vitamin B12 in Octopus Azaparacyclophane J. Coord. Chem. 21, 13-22. (5) Murakami, Y., Hisaeda, Y., and Ohno, T. (1991) Hydrophobic Vitamin B12. Part 9. An Artificial Holoenzyme Composed of Hydrophobic Vitamin B12 and Synthetic Bilayer Membrane for Carbon-Skeleton Rearrangements J. Chem. Soc., Perkin Trans. 2, 405-416. (6) Nomura, E., Taniguchi, H., and Otsuji, Y. (1994) CalixareneCatalyzed Generation of Dichlorocarbene and its Application to Organic Reactions: The Catalytic Action of Octopus-Type Calix[6]arene Bull. Chem. Soc. Jpn. 67, 792-799. (7) Ho, M. Y. K., and Rechnitz, G. A. (1987) Highly Stable Biosensor Using an Artificial Enzyme. Anal. Chem. 59, 536537. (8) Siegel, B. (1979) Preparation and Redox Properties of a Cyclodextrin-Based Ferredoxin Model J. Inorg. Nucl. Chem. 41, 609-610. (9) Bonchio, M., Carofiglio, T., Di Furia, F., and Fornasier, R. (1995) Supramolecular Catalysis: Enantioselective Oxidation of Thioanisole in Water by Hydrogen Peroxide Catalyzed by Mo(VI) in the Presence of β-Cyclodextrin J. Org. Chem. 60, 5986-5988. (10) Tabushi, I., Shimizu, N., Sugimoto, T., Shiozuka, M., and Yamamura, K. (1977) Cyclodextrin Flexibly Capped with Metal Ion. J. Am. Chem. Soc. 99, 7100-7103. (11) Suzuki, I., Chen, Q., Kashiwagi, Y., Osa, T., and Ueno, A. (1993) Electrochemical Conversion of Alcohols into Aldehydes Mediated by Lipophilic β-Cyclodextrin Bearing a Ferrocene Moiety. Chem. Lett., 1719-1722. (12) Kuroda, Y., Sera, T., and Ogoshi, H. (1991) Regioselectivities and Stereoselectivities of Singlet Oxygen Generated by Cyclodextrin Sandwiched Porphyrin Sensitization. Lipoxygenase-Like Activity J. Am. Chem. Soc. 113, 2793-2794. (13) Kuroda, Y., Ito, M., Sera, T., and Ogoshi, H. (1993) Controlled Electron Transfer between Cyclodextrin-Sandwiched Porphyrin and Quinones. J. Am. Chem. Soc. 115, 7003-7004. (14) Tabushi, I., and Kodera, M. (1987) Flavocyclodextrin as a Promising Flavoprotein Model. Efficient Electron-Transfer Catalysis by Flavocyclodextrin. J. Am. Chem. Soc. 109, 47344735.

Bioconjugate Chem., Vol. 10, No. 3, 1999 469 (15) Marzone, M., and Roda, C. (1990) β-Cyclodextrin-(7,8Dimethylalloxazine) Derivatives as Artificial Oxido-Reductase Agents. Proc. 5th Int. Symp. Cyclodextrins, 687-690. (16) (a) Ye, H., Tong, W., and D’Souza, V. T. (1992) Efficient Catalysis of a Redox Reaction by an Artificial Enzyme. J. Am. Chem. Soc. 114, 5470-5472. (b) Ye, H., Rong, D., Tong, W., and D’Souza, V. T. (1992) Artificial Redox Enzymes. Part 4. Structure and Properties. J. Chem Soc., Perkin Trans. 2, 2071-2076. (17) Ye, H., Tong, W., and D’Souza, V. T. (1994) Flavocyclodextrins as Artificial Redox Enzymes. Part 4. Catalytic Reactions of Alcohols, Aldehydes, and Thiols. J. Chem. Soc., Perkin Trans. 2, 2431-2437. (18) Rong, D., Ye, H., Boehlow, T. R., and D’Souza, V. T. (1992) Artificial Redox Enzymes. 1. Synthetic Strategies. J. Org. Chem. 57, 163-167. (19) Melton, L. D., and Slessor, K. N. (1971) Synthesis of Monosubstituted Cyclohexaamyloses. Carbohyd. Res. 18, 2937. (20) Rong, D., and D’Souza, V. T. (1990) A Convenient Method for Functionalization of the 2-Position of Cyclodextrins. Tetrahedron Lett. 31, 4275-4278. (21) Badia, A., Carlini, R., Fernandez, A., Battaglini, F., Mikkelsen, S. R., and English, A. M. (1993) Intramolecular Electron-Transfer Rates in Ferrocene-Derivatized Glucose Oxidase. J. Am. Chem. Soc. 115, 7053-7060. (22) Matsue, T., Evans, D. H., Osa, T., and Kobayashi, N. (1985) Electron-Transfer Reactions Associated with Host-Guest Complexation. Oxidation of Ferrocenecarboxylic Acid in the Presence of β-Cyclodextrin. J. Am. Chem. Soc. 107, 34113417. (23) Adams, R. N. (1969) Electrochemistry at Solid Electrodes, p 219, Marcel Dekker, New York. (24) Tinke, A. P., van der Hoeven, R. A. M., Niessen, W. M. A., van der Greef, J., Vincken, J.-P., and Schols, H. A. (1993) Electrospray Mass Spectrometry of Neutral and Acidic Oligosaccharides: Methylated Cyclodextrins and Identification of Unknowns Derived from Fruit Material J. Chromatogr. 647, 279-287. (25) Lamcharfi, E., Chuilon, S., Kerbal, A., Kunesch, G., Libot, F., and Virelizier, H. (1996) Electrospray Ionization Mass Spectrometry in Supramolecular Chemistry: Characterization of Non-Covalent Cyclodextrin Complexes J. Mass Spectrom. 31, 982-986. (26) Sheeley, D. M., and Reinhold, V. N. (1998) Structural Characterization of Carbohydrate Sequence, Linkage, and Branching in a Quadrupole Ion Trap Mass Spectrometer: Neutral Oligosaccharides and N-Linked Glycans. Anal. Chem. 70, 3053-3059. (27) Kapoor, R. C., Kachhwaha, O. P., and Sinha, B. P. (1969) Oxidation Kinetics of Thioglycolic Acid by Ferricyanide Ion in Acid Medium J. Phys. Chem. 73, 1627-1631. (28) Bohning, J. J., and Weiss, K. (1960) The Kinetics of Oxidation of 3-Mercaptopropionic Acid with Potassium Ferricyanide J. Am. Chem. Soc. 82, 4724-4728. (29) (a) Tee, O. S., Bozzi, M., Clement, N., and Gadosy, T. A. (1995) Catalysis of the Reaction of p-Nitrophenyl Alkanoates with Cyclodextrins by Potential Inhibitors: Simple Allosteric Activation. J. Org. Chem. 60, 3509-3517. (b) Tee, O. S., and Giorgi, J. B. (1997) The Effect of Alcohols on the Basic Cleavage of m-Nitrophenyl Hexanoate by β-Cyclodextrin: Allosteric Reaction Mode Switching. J. Chem. Soc., Perkin Trans. 2, 1013-1018. (30) Martell, A. E., and Smith, R. M. (1974) Critical Stability Constants, Vol. I, p 47, Plenum Press, New York. (31) Prins, R., Korswagen, A. R., and Kortbeek, A. G. T. G. (1972) Decomposition of the Ferricenium Cation by Nucleophilic Reagents. J. Organomet. Chem. 39, 335-344. (32) Rudolph, F. B. (1979) Product Inhibition and Abortive Complex Formation. Methods Enzymol. 63, 411-436.

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