Interaction of a Ferrocenoyl-Modified Peptide with Papain: Toward

effective competitive inhibitor to papain, with a Ki of 9 µM at pH 6.2. ... of the ferrocenoyl moiety is influenced by papain binding, resulting in a...
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Bioconjugate Chem. 2003, 14, 601−606

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Interaction of a Ferrocenoyl-Modified Peptide with Papain: Toward Protein-Sensitive Electrochemical Probes Kevin Plumb and Heinz-Bernhard Kraatz* Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK S7N 5C9, Canada . Received November 25, 2002; Revised Manuscript Received March 16, 2003

A new ferrocenoyl tetrapeptide, Fc-Gly-Gly-Tyr-Arg-OH, has been synthesized, which acts as an effective competitive inhibitor to papain, with a Ki of 9 µM at pH 6.2. The electrochemical potential of the ferrocenoyl moiety is influenced by papain binding, resulting in a small cathodic shift of 30 mV.

INTRODUCTION

Molecular recognition of molecules and their electrochemical detection using receptors equipped with electrophores is a rapidly expanding research area and has resulted in the development of receptors selective for cations, anions, and neutral molecules (1, 2). A number of studies have made use of ferrocene derivatives having a podant group that will selectively bind to a substrate and will allow the binding to be monitored electrochemically (3, 4). Recently, we have developed a novel class of ferrocene (Fc)-peptide conjugates, such as Fc-Leu-PheOMe (I), which allow the electrochemical monitoring of the interaction of organic substrates, such as 3-aminopyrazole derivatives, with the podant peptide chain (5, 6) to give the corresponding adducts (II).

Upon ligand coordination to the peptide, the redox potential of the ferrocene group shifts significantly to lower potential, making it easier to oxidize the ferrocene electrophore. The magnitude of the shift has been related to the strength of the interaction. This observation led us to speculate that Fc-peptides, having a suitable peptide sequence attached to the electrophore, which will strongly and selectively interact with a protein, may be useful as highly selective and sensitive electrochemical sensors for the detection of large proteins (7). As a first step in this direction, we decided to develop a Fc-peptide for the detection of papain, a 212 amino acid proteolytic enyzme isolated from the papaya fruit. The structure of several papain/inhibitor complexes were reported in the literature (8-10). On the basis of earlier experiments by Blumberg, Schechter, and Berger (11, 12), Kaiser showed * To whom correspondence should be addressed. Fax: +1-306-966-4730; Tel: +1-306-966-4660. E-mail: kraatz@ skyway.usask.ca.

that H-Gly-Gly-Tyr-Arg-OH has a high affinity for binding to papain at pH 6 and can be readily attached to a polymer resin without loosing its binding affinity for papain (13). Thus we chose the N-Fc derivative of this tetrapeptide, Fc-Gly-Gly-Tyr-Arg-OH (7), as our synthetic target for our study. Here we report its synthesis, the results of a study of its interaction with papain, and the results of solution electrochemical studies. MATERIALS AND METHODS

Materials and General Procedures. 1-Ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDAC‚ HCl) and 1-hydroxybenzotriazole hydrate (HOBt) were purchased from Quantum Technologies. Papain (2× crystallized), N-benzoylarginine ethyl ester hydrochloride (BAEE) and H-Gly-Gly-OH were purchased from SigmaAldrich. Boc-Tyr(OBzl)-OH and H-Arg(NO2)-OMe‚HCl were obtained from Advanced ChemTech. Ferrocene monocarboxylic acid (Fc-OH) was purchased from Strem Chemicals. Unless otherwise stated, all starting materials were obtained from commercial sources and were used as received. Only L-amino acids were used in this study. CH2Cl2 (ACS grade) was dried over CaH2 and freshly distilled prior to use. Fc-OBt (4)was prepared as reported in the literature (14). 1 H NMR spectra were recorded on a Bruker AMX-300, reported in ppm (δ) relative to tetramethylsilane, and referenced to the residual signals of either CHCl3 (δ 7.27), DMSO (δ 2.62), or MeOH (δ 4.87). 2D-COSY experiments were used to assist in the spectral assignments. Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Analytical thin-layer chromatography was carried out on silica gel 60 F254 aluminum oxide plates (Merck). Preparative column chromatography was performed on silica gel 60 (Merck, 230-400 mesh). All reactions were performed at room temperature and pressure unless otherwise stated. Synthesis. Preparation of Boc-Gly-Gly-OH (1). In a typical experiment, 1,4-dioxane (35 mL) and Et3N (11 mL) were added to a solution of H-Gly-Gly-OH (6.61 g, 50 mmol) in deionized water (35 mL), followed by Boc2O (55 mmol, 12.0 g). The solution was stirred for 16 h, followed by treatment with a solution of deionized water (75 mL) and EtOAc (120 mL). The aqueous layer was again washed with EtOAc (120 mL), followed by acidification with 10% citric acid (500 mL) to a pH of 2.0. The two EtOAc layers collected to this point were discarded.

10.1021/bc0256446 CCC: $25.00 © 2003 American Chemical Society Published on Web 05/07/2003

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The aqueous layer was extracted 3× with EtOAc (50 mL). The EtOAc layers were combined, dried over Na2SO4, and evaporated under reduced pressure, yielding a colorless wax. The product was dried under a reduced pressure for 3 days to give compound 1 as a white solid (yield: 9.01 g, 71%): mp 110-115 °C. FAB-MS 233.1 (M + 1)+. 1H NMR (DMSO, 300 MHz) δ 8.05 (1H, t, J ) 5.5 Hz, NH G2); 6.99 (1H, t, J ) 5.7 Hz, NH G1); 3.76 (2H, d, J ) 5.5 Hz, CH2 G2); 3.56 (2H, d, J ) 5.7 Hz, CH2 G1); 1.38 (9H, s, carbamate). Preparation of Boc-Tyr(Bzl)-Arg(NO2)-OMe (2). To a solution of Boc-Tyr(Bzl)-OBt, prepared in situ from BocTyr(Bzl)-OH (20 mmol, 7.43 g), HOBt (22 mmol, 3.37 g), and EDAC‚HCl (22 mmol, 4.22 g) in dry CH2Cl2 (200 mL, 0 °C) was added and stirred overnight a solution of H-Arg(NO2)-OMe, obtained by treatment of H-Arg(NO2)OMe‚HCl (22 mmol, 5.93 g) with Et3N (24 mL) in dry CH2Cl2 (120 mL). The reaction mixture was then treated consecutively with aqueous solutions of NaHCO3 (sat.), citric acid (10%), and water, dried over Na2SO4, and evaporated to dryness under reduced pressure. The crude product was purified by flash column chromatography (SiO2, 1:1 EtOAc-ACN) to give compound 2 as a white crystalline solid (yield: 10.39 g, 89%): mp 75-80 °C. FAB-MS 587.3 (M + 1)+. 1H NMR (CDCl3, 300 MHz) δ 8.72 (2H, br s, NHC(NH)NHNO2); 7.68 (1H, br s, NHC(NH)NHNO2); 7.41 (5H, m, m-, p-, o-CH Bzl); 7.11 (2H, d, J ) 8.6 Hz, o-CH Y); 6.91 (2H, d, J ) 8.6 Hz, m-CH Y); 6.77 (1H, br s, NH R); 5.18 (1H, d, NH Y); 5.04 (2H, s, CH2 Bzl); 4.57 (1H, m, CH R); 4.29 (1H, m, CH Y); 3.71 (3H, s, OCH3); 3.58 (1H, br s, 1H of the two diastereotopic CH2 groups of CH2CH2CH2NH R); 3.24 (1H, br s, 1H of the two diastereotopic CH2 groups of CH2CH2CH2NH R); 2.97 (2H, m, CH2 Y); 1.87 (1H, br m, 1H of the two diastereotopic CH2 groups of CH2CH2CH2NH); 1.65 (3H, br m, 2H of CH2CH2CH2NH and 1H of the two diastereotopic CH2 groups of CH2CH2CH2NH); 1.38 (9H, s, Boc group). Preparation of Boc-Gly-Gly-Tyr(Bzl)-Arg(NO2)-OMe (3). Boc-Tyr(Bzl)-Arg(NO2)-OMe (8.8 mmol, 5.16 g) was dissolved in CH2Cl2, (6 mL) and treated with TFA (6 mL) for 30 min. The CH2Cl2 and TFA were subsequently removed in vacuo. The resulting residue was redissolved in CH2Cl2 (10 mL) and cooled in an ice bath prior to the dropwise addition of Et3N (2 mL). To this was added a solution of Boc-Gly-Gly-OBt, prepared in situ from BocGly-Gly-OH (8 mmol, 1.86 g), HOBt (8.8 mmol, 1.35 g), and EDAC‚HCl (8.8 mmol, 1.69 g) in dry CH2Cl2 (25 mL, 0 °C). The reaction mixture was then warmed to room temperature and left to stir overnight. The resulting solution was then treated as per 2. The product was purified by flash column chromatography (SiO2, 90:10 CHCl3:MeOH) and recrystallized in CHCl3 to yield white crystalline compound 3 (yield: 3.47 g, 62%). IR (cm-1, KBr, film): 1656, 1740 (CdO); 861, 1250, 1511 NO2. FABMS 701.4 (M + 1)+. 1H NMR (MeOH, 300 MHz ) δ 7.36 (5H, m, CH Bzl); 7.14 (2H, d, J ) 8.6 Hz, o-CH Y); 6.88 (2H, d, J ) 8.7 Hz, m-CH Y); 5.02 (2H, s, CH2 Bzl); 4.52 (1H, m, R-CH R); 4.41 (1H, m, R-CH Y); 3.80 (2H, s, CH2 G2); 3.71 (2H, s, CH2 G1); 3.65 (3H, s, OCH3); 3.25 (2H, m, CH2 of CH2CH2CH2NH); 3.06 (1H, m, 1H of the two diastereotopic CH2 groups of Y); 2.90 (1H, m, 1H of the two diastereotopic CH2 of Y); 1.86 (1H, m, 1H of the two diastereotopic CH2 groups of CH2CH2CH2NH); 1.73 (3H, m, 2H of CH2 of CH2CH2CH2NH and 1H of the two diastereotopic CH2 groups of CH2CH2CH2NH); 1.42 (9H, s, C(CH3)3 Boc group). Preparation of Fc-Gly-Gly-Tyr(Bzl)-Arg(NO2)-OMe (5). After the removal of the Boc group from 3 (1.1 mmol, 0.77

Plumb and Kraatz

g) using TFA (1.5 mL), the excess acid was removed in vacuo and Et3N (1.0 mL) in CH2Cl2 (10 mL) was added. This solution was added to a stirring solution of Fc-OBt (14) (1.0 mmol, 0.33 g) in CH2Cl2 (20 mL) and allowed to react overnight. Purification was carried out by flash column chromatography (SiO2, 5:1 EtOAc:MeOH, Rf ) 0.5) to give 5 in 83% yield as an orange solid (0.67 g). Anal. Calcd for C38H44N8O9Fe: C, 56.16; H, 5.46; N, 13.79. Found: C, 56.35; H, 5.88; N, 13.42. IR (cm-1, KBr, film) 1653, 1741 (CdO); 1261, 1512 (NO2). FAB-MS 813.3 (M + 1)+. 1H NMR (MeOH, 300 MHz ) δ 7.38 (5H, m, CH Bzl); 7.17 (2H, d, J ) 8.6 Hz, o-CH Y); 6.87 (2H, d, J ) 8.6 Hz, m-CH Y); 5.01 (2H, s, CH2 Bzl); 4.78 (2H, m, o-CH substituted Cp ring); 4.38 (1H, m, R-CH R); 4.26 (3H, m, m-CH substituted Cp and R-CH Y); 4.22 (5H, m, CH unsubstituted Cp ring); 3.95 (2H, s, CH2 G2); 3.82 (2H, s, CH2 G1); 3.64 (3H, s, OCH3); 3.16 (3H, m, 2H of CH2CH2CH2NH and 1H of the two diastereotopic CH2 groups of Y); 2.94 (1H, m, 1H of the two diastereotopic CH2 groups of Y); 1.81 (1H, br m, 2H of CH2CH2CH2NH and 1H of the two diastereotopic CH2 of CH2CH2CH2NH); 1.60 (3H, br m, CH2 of CH2CH2CH2NH and CH2CH2CH2NH). Preparation of Fc-Gly-Gly-Tyr(Bzl)-Arg(NO2)-OH (6). To a solution of 5 (0.500 g, 0.62 mmol) in MeOH (2.0 mL) was added 1 N NaOH (2.2 mL) with stirring. The reaction was stored at room temperature for 3 h after which 1 N HCl (1.0 mL) was added. The MeOH was then removed in vacuo followed by cooling of the solution in a ice bath prior to the dropwise addition of 1 N HCl (2.0 mL). The solution was then stored in the fridge for 2 h after which the precipitate was filtered off and washed 3× with cold, distilled water (50 mL) and dried under reduced pressure overnight to give 6 as a yellow solid in 85% yield (426 mg). FAB-MS 799.3 (M + 1)+. 1H NMR (MeOH, 300 MHz ) δ 7.32 (5H, m, CH Bzl); 7.17 (2H, d, J ) 8.6 Hz, o-CH Y); 6.87 (2H, d, J ) 8.7 Hz, m-CH Y); 5.00 (2H, s, CH2 Bzl); 4.78 (2H, m, o-CH substituted Cp ring); 4.58 (1H, m, R-CH R) 4.35 (3H, m, m-CH substituted Cp and R-CH Y); 4.20 (5H, CH unsubstituted Cp ring); 3.95 (2H, s, CH2 G2); 3.82 (2H, s, CH2 G1); 3.20 (3H, m, 2H of CH2CH2CH2NH and 1H of the two diastereotopic CH2 groups of Y); 2.97 (1H, m, 1H of the two diastereotopic CH2 groups of Y); 1.87 (1H, m, 1H of the two diastereotopic CH2 groups of CH2CH2CH2NH); 1.65 (3H, m, 2H of CH2CH2CH2NH and 1H of the two diastereotopic CH2 groups of CH2CH2CH2NH). Preparation of Fc-Gly-Gly-Tyr-Arg-OH (7). A solution of Fc-Gly-Gly-Tyr(Bzl)-Arg(NO2)-OH (100 mg, 0.13 mmol) in a 3:1 mixture of MeOH and distilled water (20 mL) was hydrogenated using 5% palladium on powdered charcoal (65 mg) as catalyst. The reduction was performed at room temperature in a Parr Apparatus at 60 psi for 20 h. The progress of the reaction was followed by TLC (60:40 CHCl3-MeOH). The reaction mixture was then filtered and evaporated to dryness under reduced pressure. The crude product was purified by column chromatography (SiO2, 1:1 MeCN:MeOH, Rf ) 0.3) to give 7 as an orange solid (yield: 58 mg, 70%). Anal. Calcd for C30H37N7O7Fe: C, 54.31; H, 5.62; N, 14.78. Found: C, 53.98; H, 5.70; N, 15.05. FAB-MS 664.1 (M + 1)+. IR (cm-1, KBr, film): 1650 (CdO amide); 1742 (CdO ester); 1 H NMR (MeOH, 300 MHz) δ 7.09 (2H, d, J ) 8.5 Hz, o-CH Y); 6.69 (2H, d, J ) 8.5 Hz, m-CH Y); 4.82 (2H, s, o-CH substituted Cp ring); 4.55 (1H, m, R-CH R); 4.41 (2H, m, m-CH substituted Cp); 4.25 (5H, s, CH unsubstituted Cp ring); 3.98 (2H, s, CH2 G2); 3.87 (2H, q, CH2 G1); 3.13 (3H, m, 2H of CH2CH2CH2NH and 1H of the two diastereotopic CH2 groups of CH2 Y); 2.91 (1H, m, 1H of the two diastereotopic CH2 groups of Y); 1.87 (1H,

Ferrocenoyl Peptide Papain Interactions

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Chart 1. (a) enzyme diluent (activation buffer), 100 mL: (b) substrate solution, 50 mL: (c) substrate solution diluent, 50 mL: (d) inhibitor solution, 1.0 mL: (e) active enzyme solution, 5.0 mL: (f) 0.01 N NaOH, 50 mL

br m, 1H of the two diastereotopic CH2 groups of CH2CH2CH2NH); 1.70 (3H, m, 2H of CH2CH2CH2NH and CH2CH2CH2NH). Inhibitor Studies. In preparation of the inhibitor studies the following solutions were prepared as shown in Chart 1. Into a series of seven sterile polypropylene sample tubes labeled A1-A7, also defined as series A, were added the following ascending volumes of substrate solution and a magnetic stir bar: 100 µL, 130 µL, 190 µL, 280 µL, 400 µL, 550 µL, 910 µL. The volumes of the solutions were were made up to 1000 µL by adding the corresponding amounts of the substrate solution diluent. The same procedure was repeated to produce series B-F. A two-point calibration was performed on the pH electrode at the beginning of each set of experiments. The titrimetric experiments for all six series were run one series at a time. Just prior to running each series, additions of 1000 µL of 3.0 M NaCl and 1000 µL of Millipore water were made. Immediately prior to the addition of the enzyme, the inhibitor was added and the pH of the solution was adjusted to 6.240 ( 0.010 with 0.01 N NaOH. The following ratios of enzyme to inhibitor were used in determining the volume of inhibitor solution to add: 1:0, 1:50, 1:100, 1:250, 1:500, 1:750, 1:1000. Based on the desired ratio and the known concentration of the activated enzyme in solution determined by UV-vis spectrophotometry where mg papain/mL ) A280 × 0.4, the appropriate volume of inhibitor was added. The active enzyme was then pipetted into the magnetically stirred solution, and the pH electrode was added in order to monitor the change in pH of the solution. Only the initial rate of change is useful in these types of experiments. Thus once the enzyme is added it is crucial to wait only a few seconds for the enzyme to mix with the other components of the solution and to allow the pH meter to stabilize. This occurred fairly quickly and generally between pH 6.22 and 6.20. The time was then measured for the pH to drop an additional 0.05 from the pH at which the meter was observed to have stabilized. This was observed to take between 1 min and 30 min depending on the concentrations of substrate and inhibitor present during the trial. The pH electrode was rinsed thoroughly with Millipore water between each trial. Electrochemical Studies. All electrochemical experiments were carried out using a CV-50W Voltammetric Analyzer (BAS) at room temperature (20 ( 2 °C). No special precautions were taken to exclude oxygen. Borate buffer (pH 6.2) in Millipore water was used thoughout the electrochemical studies. Sodium perchlorate was used as supporting electrolyte (0.1 M). For the cyclic voltammetry studies, a glassy carbon working electrode (BAS, diameter 2 mm) and a platinum wire counter electrode were used. The glassy carbon working electrode was polished with 3 µm followed by 1 µm and then 0.5 µm alumina prior to use to remove any surface contaminants. The reference electrode was a Ag/AgCl electrode (BAS). iR compensation was applied. Backgrounds of the buffer containing 0.1 M NaClO4 were collected before each set of experiments and then subtracted from the spectra. The experiment was repeated at least 10 times to get reliable

1.0 mM EDTA, 0.06 mM mercaptoethanol, 5.0 mM cysteine‚HCl 41.4 mM BAEE, 0.38 mM disodium EDTA, 1.90 mM cysteine‚HCl 0.38 mM disodium EDTA, 1.90 mM cysteine‚HCl 1 mg Fc-GGYR/mL substrate solution diluent 0.05-1.0 mg papain/mL enzyme diluent

values for E1/2. The heterogeneous electron-transfer rate constant was obtained by simulations using the DigiSim 2.1 software (BAS). RESULTS AND DISCUSSION

Compound 7 was synthesized in serveral steps by standard solution-phase carbodiimide coupling (15) according to Scheme 1. After the deprotection of Boc-Tyr(Bzl)-Arg(NO2)-OMe (2) with trifluoroacetic acid in dichloromethane, the resulting free dipeptide ester was coupled with Boc-Gly2OH (1) using EDC/HOBt to give the fully protected tetrapeptide Boc-Gly2-Tyr(Bzl)-Arg(NO2)-OMe (3). After removal of the Boc group, the N-deprotected tetrapeptide was then added to a solution of Fc-OBt (14). The desired fully protected Fc-tetrapeptide Fc-Gly2-Tyr(Bzl)-Arg(NO2)-OMe (5) was obtained as an orange solid. The 1H NMR spectrum of compound 5 exhibited the expected 2:2:5 signal pattern for monosubstituted Fcpeptides (see Figure 1). Signals of the R-Hs of Arg and Tyr were observed at δ 4.51 and 4.22, respectively. Base hydrolysis of the methyl ester group resulted in formation of the free acid 6, which lacks the resonance due to the methoxy group at δ 3.67. As a last step, the desired fully deprotected Fc-tetrapeptide Fc-Gly-Gly-Tyr-Arg-OH (7) was obtained after hydrogenation of a methanol-water solution of 6 with 5% Pd on carbon. The progress of the Arg deprotection was conveniently followed by IR spectroscopy. Compound 7 lacks the strong absorption in the IR due to the NO2 group at 1512 cm-1. The orange product was isolated by column chromatography and fully characterized by 1H NMR, FT-IR, and FAB mass spectrometry. Its 1H NMR spectrum lacks the benzylic protons present in compounds 5 and 6. In addition, the aromatic region shows resonances only due to the Tyr aromatic. To evaluate the ability of compound 7 to inhibit substrate hydrolysis and thus its ability to interact with papain, we carried out inhibition studies. The MichaelisMenten constant (Km) for papain as well as the inhibition constant Ki for the ferrocenoyl tetrapeptide 7, were determined using N-benzoylarginine ethyl ester hydrochloride (BAEE‚HCl) as substrate (16). Initial hydrolysis rates were determined using a pH meter in a solution containing NaCl (1.0 mol L-1), Na2EDTA (0.1 mM), and cysteine‚HCl (0.6 mM), at 23 °C. The enzyme was activated prior to use in a solution of mercaptoethanol (0.06 mM), Na2EDTA (1.0 mM), and cysteine‚HCl (5.0 mM). Papain concentrations ranged between 0.33 and 0.66 µg/mL determined by UV-vis spectroscopy given by mg papain/mL ) A280 × 0.4. Figure 1 shows the Lineweaver-Burk plot indicating competitive inhibition, as was reported before for H-GlyGly-Tyr-Arg-OH (13). A Km of 15 mM was determined for the substrate at pH 6.2, which compares well with a Km reported by Blumberg et al. (11). The Dixon-Webb plot (Figure 2) clearly indicates competitive inhibition and thus indicates a direct interaction of the Fctetrapeptide with the substrate binding site of papain (17,

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Scheme 1: Synthesis of Fc-Gly2-Tyr-Arg-OH (7)a

a (i) TFA deprotection of 2, Et N; HOBt, EDC, RT, 12 h; (ii) TFA deprotection of 3, Et N; CH Cl , Fc-OBt, RT; (iii) MeOH, NaOH 3 3 2 2 (1 N); (iv) 5% Pd/C, MeOH:water 3:1, H2 (60 psi), 20 h.

1H

Figure 1. NMR spectra for compounds 3 and 5-7. Note the sharp singlet for compound 3 at δ 1.4 due to the Boc group and the appearance of doublets at δ 4.8 and δ 4.3 in compounds 5-7 corresponding to the protons of the substituted cyclopentadienyl ring and the strong singlet at δ 4.2 due to the unsubstituted Cp ring of the Fc conjugates. Stepwise deprotection of the fully protected Fc-Gly-Gly-Tyr(Bzl)-Arg(NO2)-OMe (5) leads to the free acid 6, which lacks the OMe resonance at δ 3.64, and finally to the target compound 7, which lacks the multiplet at δ 7.4 of the benzyl group from the tyrosine residue.

18). A Ki was derived at pH 6.2 from plots of νo/ν versus [I], while holding the substrate concentration constant, where νo is the initial, uninhibited rate of hydrolysis and ν is the initial rate in the presence of the inhibitor. The plot is linear, and the inhibition constant Ki was calculated from the slope (Figure 4) to be 9 µM. This is

Figure 2. Double-reciprocal (Lineweaver-Burk) plots showing that Fc-Gly-Gly-Tyr-Arg-OH (7) behaves as a competitive papain inhibitor for BAEE hydrolysis. Km calculated as 15 mM. Substrate:inhibitor ratio: 9 ) no inhibitor, + ) 1: 50, 2 ) 1:100, [ ) 1:250, /) 1:500, b ) 1:750.

significantly better than for the related benzyl-protected derivative H-Gly-Gly-Tyr(Bzl)-Arg-OH (123 µM at pH 6). Kaiser and co-workers noted that inhibition is enhanced by the presence of the aromatic group (13). Furthermore, N-conjugation increased the affinity for papain. The Fc group appears to enhance binding significantly. We are currently investigating this further and attempting to cocrystallize compound 7 with papain to get more insight into the nature of the interaction. The electrochemical properties of compound 7 were investigated using cyclic voltammetry (CV) in borate buffer using NaClO4 (0.1 M) as supporting electrolyte.

Ferrocenoyl Peptide Papain Interactions

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Figure 3. Dixon-Webb plot showing that Fc-Gly-Gly-Tyr-ArgOH (7) is a competitive inhibitor to papain. Substrate:inhibitor ratio: 9 ) 1:50, /) 1:100, 2 ) 1:250, + ) 1:750. Figure 5. Sucessive additions of papain to a 1 mM solution of Fc-Gly-Gly-Tyr-Arg-OH (7) in borate buffer (20 mM, pH 6.2). Glassy carbon, Pt counter, Ag/AgCl reference electrode, scan rate ) 100 mV s-1, 0.1 M NaClO4.

potential are modest but specific to the presence of papain in solution. No changes in the redox potential of compound 7 were observed in the presence of other enzymes, such as trypsin and pepsin. CONCLUSIONS Figure 4. Plot of υo/υ vs [I] where υo is the rate of hydrolysis in the absence of the inhibitor and υ is the rate in the presence of the inhibitor. The Ki for Fc-Gly-Gly-Tyr-Arg-OH (7) was determined to be 9 µM where 1/Ki ) slope (1 + [S]/Km).

Binding of papain to 7 is expected to be optimal at pH 6.2, thus making it necessary to control the pH of the solution during the electrochemical studies. Our experiments show that at pH e 7, the redox potential remains reversible and is experiencing reproducibly small anodic shifts of about 5 mV. At pH g 10, the oxidation wave irreversible due to the reaction of the OH- with the ferrocenium cation, compatible with earlier reports of the decomposition of ferrocene derivatives in basic solutions by an EC mechanism, in which the oxidized species reacts with OH- to give various decomposition products (19). A solution of 7 in borate buffer at pH 6.2 exhibits a fully reversible one-electron oxidation due to the ferrocene/ ferrocenium redox couple at 430 mV (vs Ag/AgCl; Epa Epc ) 66 mV). Figure 5 shows the changes in the CVs for compound 7 upon addition of small aliquots (0.06 equiv per addition) of a solution of papain up to a maximum of 0.7 equivalents at pH 6.2, the optimum pH for papain binding. The addition of stoichiometric amounts of papain to a buffered solution of 7 led to a reduction of the oxidative and reductive peak current, as expected for a papain-7 with a diffusion coefficient that is significantly smaller than that of 7. Addition to a full 1 equivalent mixture resulted in the formation of a gelatinous precipitate. In addition to the decrease in current intensity, a cathodic shift of the half-wave potential of the 7/7+-redox couple of 30 mV to 400 mV is observed. In addition, the peak separation increases in the presence of papain to 87 mV, indicating a slowing of the electron-transfer process from the 7‚papain complex to the electrode surface (ko ) 1.2 × 10-3 cm s-1 by simulation of the CV using DigiSim 2.1, BAS). The shift of the reduction peak potential is larger, indicating a stronger interaction of papain to 7+. No significant changes in the redox potential were observed outside the optimum pH for binding of papain to the tetrapeptide (7 e pH e 5). The changes in the redox

In conclusion, we have synthesized a ferrocenoyl tetrapeptide receptor specifically designed to bind to papain. Binding of papain to the receptor molecule causes an electrochemical response. At present the electrochemical response is very modest. However, the response is in the presence of an equimolar amount of papain. Although, higher papain concentrations could not be evaluated due to the insolubility of the papain under these conditions, our results may significantly expand the applications of ferrocene modified receptors to include biomolecules. The next step in our work will be to bind the receptor to a surface and to develop a surface-bound protein sensor platform using ferrocenoyl peptidylcystamines (20, 21). This will also solve the signal intensity since now the redox signal of the probe is not any longer diffusion dependent. ACKNOWLEDGMENT

We thank the Health Utilization and Research Commission (HSURC) of the Province of Saskatchewan for a Biomedical Establishment Grant to H.B.K. H.B.K. is Canada Research Chair in Biomaterials. LITERATURE CITED (1) Beer, P. D., Gale, P. A., and Chen, G. Z. (1999) Electrochemical molecular recognition: pathways between complexation and signaling. J. Chem. Soc., Dalton Trans. 1897-1909. (2) Beer, P. D., and Gale, P. A. (2001) Anion recognition and sensing: The state of the art and future perspectives. Angew. Chem., Int. Ed. 40, 486-516. (3) Carr, J. D., Lambert, L., Hibbs, D. E., Hursthouse, M. B., Abdul Malik, K. M., and Tucker, J. H. R. (1997) Novel electrochemical sensors for neutral molecules. Chem. Commun. 1649-1650. (4) Carr, J. D., Coles, S. J., Hassan, W. W., Hursthouse, M. B., Abdul Malik, K. M., and Tucker, J. H. R. (1999) The effect of protonation on the spectroscopic and redox properties of a series of ferrocenoyl derivatives. J. Chem. Soc., Dalton. Trans. 57-62. (5) Kraatz, H. B., Leek, D. M., Houmam, A., Enright, G. D., Lusztyk, J., and Wayner, D. D. M. (1999) The ferrocene moiety as a structural probe: redox and structural properties

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