Applications of Tailored Ferrocenyl Molecules as ... - ACS Publications

Bioconjugate Chem. 2007, 18, 199−208

199

Applications of Tailored Ferrocenyl Molecules as Electrochemical Probes of Biochemical Interactions Isabelle Tranchant,⊥,‡ Anne-Cećile Herve´,†,§ Stephen Carlisle,† Phillip Lowe,†,# Christopher J. Slevin,†,# Camilla Forssten,† John Dilleen,†,# Alethea B. Tabor,⊥ David E. Williams,*,†,| and Helen C. Hailes*,⊥ Department of Chemistry, University College London, 20 Gordon Street, London WC1H 0AJ, U.K., and Unipath Ltd, Priory Business Park, Bedford, MK44 3UP, U.K. Received February 17, 2006; Revised Manuscript Received August 31, 2006

The development of electrochemical probes useful for investigating the occupancy by other molecules of sites on complex proteins such as human serum albumin (HSA) is described. Ferrocenyl-(oxoethylene)-fatty acid compounds of different fatty acid chain length probed different binding sites on HSA. The interaction could be changed from one primarily with a drug binding site, when the probe was ferrocene methanol, to one predominantly with mediumchain fatty acid binding sites, by adding an (oxoethylene)-fatty acid substituents. Finally, the interaction could be changed to one interacting primarily with high-affinity long-chain fatty acid binding sites, as the fatty acid chain length in ferrocene-(oxoethylene)-fatty acid molecules increased. These results strongly implied that the binding could be further tailored by relatively simple modifications to the probe, for example, by changing the balance of hydrophobicity and hydrophilicity. The possibility of a procedure using mass-produced electrochemical cells to determine the fractional occupancy of different sites on HSA is demonstrated.

INTRODUCTION Serum albumin is of significance as a carrier for small molecules in blood (1, 2). Arguably, the fractional occupancy of different binding sites on albumin could be an important medical diagnostic indicator (1, 3). In particular, the binding of fatty acid to serum albumin is an important interaction in mammalian metabolism. It has previously been demonstrated that the binding of fatty acids to human serum albumin (HSA) influences the binding of a number of other drug and biomolecules in the body (4). The ability of serum albumin to bind a number of medium- and long-chain fatty acids simultaneously enables it to be an effective carrier for these molecules, which play a key role in cell functioning. The buffer capacity of albumin for fatty acid concentration in the blood has been proposed as a significant diagnostic indicator, for cardiac ischemia (5). Fatty acids are also known to bind to a number of other proteins, particularly the class of fatty acid binding proteins (FABP) (6). The work reported here has aimed to provide probes for fatty acid interactions with proteins, which are complementary to other methods such as the electronic spin resonance (ESR) spectroscopy method using binding of the spinprobe 16-doxyl stearate, described in ref 3. Our focus has been to develop electrochemical methods, in view of their instrumental convenience. Here we demonstrate the principle of electrochemical probes of site occupancy and that implementation in mass-produced electrochemical cells is feasible, while highlighting some of the challenges raised by such procedures. * Author to whom correspondence should be addressed: E-mail: [email protected] (D.E.W.); [email protected] (H.C.H.); fax: +44 (0)20 7679 7463. ⊥ University College London. † Unipath Ltd. § Current address: Diatos NV, Brusselsestraat 69, B-3000 Leuven, Belgium. ‡ Current address: Diatos SA, Therapeutic Chemistry Department, 3-5 Impasse Reille, 75014 Paris, France. # Current address: Sterling Medical Innovations, Unit 10, Scion House, Stirling University Innovation Park, Stirling, FK9 4NF. | Current address: Department of Chemistry, University of Auckland, Private Bag, Auckland, New Zealand.

Fatty acid-protein interactions and, in particular, fatty acidHSA interactions have been studied by various methods including X-ray crystallography (7), in which distinct binding sites for a range of fatty acids were identified. HSA has been specifically labeled with a fluorescent molecule and used to study oleate binding (8). The binding of a fluorescently labeled fatty acid such as 11-(dansylamino)undecanoic acid (DAUDA) to HSA (9) and to FABP (10) was studied by measuring the fluorescent signal response to the changing microenvironment around the fluorescent moiety on binding. DAUDA was also used to investigate the binding of other fatty acids as well as other molecules such as bilirubin in competitive studies with HSA (9). Equilibrium dialysis has been used with radiolabeled fatty acids to study the binding of a range of fatty acids to albumin (11); analysis was done in terms of a multiple noninteracting binding sites. Binding of a range of fatty acids to an acrylodan modified FABP (ADIFAB) has been characterized by fluorescence, and subsequently the binding characteristics of these fatty acids to HSA was determined by using the modified FABP to determine the free fatty acid concentration in the presence of HSA (12). We have previously demonstrated the design and synthesis of a range of molecules consisting of a functional group that confers some specific biochemical activity, linked to an electrochemical label, to enable the reporting of activity (13). The two groups were linked by a spacer group, which was chosen to influence the solubility and enhance the electrochemical properties of the molecule, and may also improve the biochemical functionality through structural or specific chemical interactions. We report here the use of a range of molecules based on the earlier design, consisting of a fatty acid group, linked to a solubilizing spacer group, which in turn is linked to a ferrocene based electrochemically active signaling moiety, of the general structure 1 shown in Figure 1. The ideal structural characteristics were identified in compound 2, as an amide at linker A, carbamate at linker B, and a short poly(ethylene glycol) (PEG) unit, for investigating fatty acid binding to a number of fatty acid binding molecules. In this paper, we illustrate how m, the length of the fatty acid, may be varied (compounds synthesized for m ) 2, 5, 7,

10.1021/bc060039e CCC: $37.00 © 2007 American Chemical Society Published on Web 11/11/2006

200 Bioconjugate Chem., Vol. 18, No. 1, 2007

Figure 1. Ferrocene multifunctional conjugates. Scheme 1. Synthesis of Ferrocene Conjugates with Chain Lengths (m + 1)a

a (i) DSC, CH3CN, 87%; (ii) amino acid, THF/H2O, 30-80%; (iii) potassium phthalimide, DMF, 58%; (iv) HCl, MeOH, 67%; (v) CrO3, H2SO4, 50%; (vi) N2H4, EtOH, reflux, 4 h, 80%; (vii) N2H4, EtOH, reflux, 3 h, then NaOH, 84%.

10, 13, and 15) to allow us to study specifically the interactions of different fatty acids with proteins, particularly albumin. The approach is a general one, so that other small molecules might be electrochemically labeled using similar methods. One of the key aspects is the use of carefully chosen signaling, linker, and spacer groups to deliver a compound that has fast electrochemistry at a potential accessible in biologically relevant media such as blood and plasma and which has the desired solubility, stability, and probe characteristics. This work is related in concept to the studies using fluorescently labeled fatty acids (9). However, the earlier studies on DAUDA (9) showed it interacting with the bilirubin site rather than the fatty acid binding sites of albumin. Hence, we have varied the length of the labeled fatty acid and to some extent altered the balance of aromatic, aliphatic, hydrophobic, and hydrophilic elements to provide a range of probes that might access different sites on HSA. Comparison with theory for noninteracting multiple binding sites (11, 12, 14, 15), using binding constants already determined for fatty acids to HSA, allowed investigation of the competitive binding of labeled fatty acids with natural fatty acids and demonstrated the feasibility of using instrumentally simple methods and mass-produced electrochemical cells to probe fractional occupancy of different sites on HSA.

EXPERIMENTAL PROCEDURES Syntheses of Compounds Based on the General Structure, 2, but with Different Numbers of Carbons in the Fatty Chain. The ferrocene PEG-amide 3 was activated (13) as the N-hydroxysuccinimidyl (NHS) ester, which was isolated, and then coupled with the commercially available amino acids β-alanine, 6-aminohexanoic acid, 8-aminooctanoic acid, to give 4, 5, and 6 in 40-80% yield (Scheme 1). Two longer chain analogues were prepared using 14-aminotetradecanoic acid and 16-aminohexadecanoic acid, which were synthesized via the phthalimides using established synthetic manipulations (16, 17). 14-Aminotetradecanoic acid (7) was prepared from 1-bromo14-(tetrahydropyran-2-yloxy)tetradecane via the addition of phthalimide, OTHP deprotection, oxidation using Jones’ reagent

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to the acid, and finally removal of the phthalimide group using hydrazine. 16-Aminohexadecanoic acid (8) was prepared in 84% overall yield from methyl 16-phthalimide hexadecanoate through removal of the phthalimide group and hydrolysis of the ester. 14-Tetradecanoic acid (7) and 16-aminohexadecanoic acid (8) were coupled with the NHS ester of 3 as before to give the PEG conjugates 9 and 10, respectively (Scheme 1). Materials. Unless otherwise noted, solvents and reagents were reagent grade from commercial suppliers and used without further purification. Tetrahydrofuran (THF) was dried by distillation from a sodium/benzophenone suspension under a dry N2 atmosphere. CH2Cl2 was dried by distillation from CaH2 under a dry N2 atmosphere. All moisture-sensitive reactions were performed under a nitrogen atmosphere using oven-dried glassware. Reactions were monitored by TLC on Kieselgel 60 F254 plates with detection by UV, or permanganate, ninhydrin, and phosphomolybdic acid stains. Flash column chromatography was carried out using silica gel (particle size 40-63 µm). Melting points are uncorrected. 1H NMR and 13C NMR spectra were recorded in CDCl3 at the field indicated unless otherwise indicated. 1-Bromo-14-(tetrahydropyran-2-yloxy)tetradecane was prepared as previously described (18). Methyl 16-phthalimide hexadecanoate was prepared as previously reported (19). 3-(Ferrocenoylamide-3-oxapentanyloxycarbonylamino)propanoic Acid (4). The reaction was carried out under anhydrous conditions. Compound 3 was activated as the N-hydroxysuccinimide ester (NHS) as previously described (13). The NHS intermediate (150 mg, 0.33 mmol) was dissolved in DMF (2 mL), and triethylamine (54 µL, 0.39 mmol) was added. To this solution was added β-alanine (30 mg, 0.33 mmol), dissolved in a mixture of water/DMF (3.3 mL/1.3 mL). The reaction was stirred at room temperature (rt) for 18 h, and the solvents were then evaporated in vacuo. The resulting oil was purified by flash silica chromatography (5% methanol in CH2Cl2) to afford 4 (102 mg, 73%) as an orange oil. 1H NMR (300 MHz; methanol-d4) 2.25 (t, 2H, J 6.1 Hz, CH2CO2H), 3.18 (m, 2H, CH2NH), 3.32 (dt, J 10.3 and 5.2 Hz, 2H, CH2NH), 3.44 (t, J 5.2 Hz, 2H, CH2O), 3.49 (t, J 5.2 Hz, 2H, m, CH2O), 3.99 (m, 2H, C5H2), 4.03 (m, 2H, CH2OCO), 4.17 (s, 5H, C5H5), 4.57 (m, 2H, C5H2); 13C NMR (75 MHz; methanol-d ) 34.2, 36.4, 39.3, 63.6, 68.0, 4 69.3, 69.6, 69.7, 70.5, 75.0, 156.7 (CdO carbamate), 171.7 (Cd O amide), 174.6 (CdO acid); m/z HRMS calculated for C19H24N2O6FeNa (MNa)+ 455.08760, found (ES+) 455.08592. 6-(Ferrocenoylamide-3-oxapentanyloxycarbonylamino)hexanoic Acid (5). The reaction was carried out under anhydrous conditions. Compound 3 was activated as the N-hydroxysuccinimide ester as previously described (13). The NHS intermediate (50 mg, 0.11 mmol) was dissolved in DMF (0.7 mL) and triethylamine (18 µL, 0.13 mmol) was added. To this solution was added 6-aminohexanoic acid (15 mg, 0.11 mmol), dissolved in a mixture of water/DMF (1.1 mL/0.4 mL). The reaction was stirred at rt for 18 h, and the solvents were then evaporated in vacuo. The resulting oil was purified by flash silica chromatography (5% methanol in CH2Cl2) to afford 5 (42 mg, 80%) as an orange oil. 1H NMR (300 MHz; CDCl3) 1.33 (quin, J 7.2 Hz, 2H), 1.47 (quin, J 7.2 Hz, 2H), 1.61 (quin, J 7.2 Hz, 2H), 2.33 (t, 2H, J 7.2 Hz, CH2CO2H), 3.12 (m, 2H, CH2NH), 3.54 (m, 2H, CH2NH), 3.65 (m, 4H, CH2O), 4.19 (s, 5H, C5H5), 4.22 (m, 2H, CH2OCO), 4.33 (m, 2H, C5H2), 4.73 (m, 2H, C5H2), 5.04 (m, 1H, NH), 6.59 (m, 1H, NH); 13C NMR (75 MHz; CDCl3) 25.8, 29.4, 29.7, 34.1, 39.3, 39.6, 63.9, 68.4, 69.7, 69.8, 70.5, 72.3, 75.7, 156.5 (CdO carbamate), 171.0 (Cd O amide), 177.9 (CdO acid); m/z HRMS calculated for C22H31N2O6Fe M+ 474.14532, found (ES+) 474.14527. 8-(Ferrocenoylamide-3-oxapentanyloxycarbonylamino)octanoic Acid (6). The reaction was carried out under anhydrous conditions. Compound 3 was activated as the N-hydroxysuc-

Ferrocenyl Molecules as Electrochemical Probes

cinimide ester as previously described (13). The NHS intermediate (150 mg, 0.33 mmol) was dissolved in DMF (2 mL) and triethylamine (55 µL, 0.39 mmol) was added. To this solution was added 6-aminooctanoic acid (15 mg, 0.11 mmol), dissolved in a mixture of water/DMF (3.3 mL/1.3 mL). The reaction was stirred at rt for 18 h and the solvents were then evaporated in vacuo. The resulting oil was purified by flash silica chromatography (5% methanol in CH2Cl2) to afford 6 (63 mg, 40%) as an orange oil. 1H NMR (300 MHz; CDCl3) 1.28 (m, 6H), 1.39 (quin, J 6.6 Hz, 2H), 1.60 (quin, J 5.8 Hz, 2H), 2.31 (t, 2H, J 6.6 Hz, CH2CO2H), 3.09 (dt, J 6.8 and 5.8 Hz, 2H, CH2NH), 3.55 (dt, J 5.3 and 4.9 Hz, 2H, CH2NH), 3.65 (m, 4H, CH2O), 4.19 (s, 5H, C5H5), 4.20 (t, J 4.5 Hz, 2H, CH2OCO), 4.31 (m, 2H, C5H2), 4.71 (m, 2H, C5H2), 4.90 (br m, NH), 6.59 (br m, 1H, NH); 13C NMR (75 MHz; CDCl3) 24.7, 26.5, 28.78, 28.79, 29.7, 34.0, 39.4, 41.0, 63.7, 68.3, 69.7, 69.8, 70.1, 70.5, 75.8, 156.5 (CdO carbamate), 171.0 (CdO amide), 177.9 (Cd O acid); m/z HRMS calculated for C24H34N2O6Fe M+ 502.17661, found (FAB+) 502.17918. 14-Aminotetradecanoic Acid (7). The reaction was carried out under anhydrous conditions. 1-Bromo-14-(tetrahydropyran2-yloxy)tetradecane (63 mg, 0.17 mmol) and potassium phthalimide (48 mg, 0.26 mmol) in DMF (1 mL) were heated at 40 °C for 4 h. Water (10 mL) was added, and the product was extracted into CH2Cl2 (3 × 10 mL). The organic phase was dried (MgSO4) and evaporated to give 2-(14-tetrahydropyran-2-yloxy-tetradecyl)-isoindole-1,3-dione (64 mg, 58%) which was used without further purification. 1H NMR (300 MHz; CDCl3) 1.25 (m, 20H), 1.51 (m, 6H), 1.62 (m, 4H), 3.33 (m, 2H), 3.45 (m, 2H), 3.68 (m, 2H, CH2O), 4.52 (m, 1H, CHO), 7.68 (m, 2H, ArH), 7.78 (m, 2H, ArH); 13C NMR (75 MHz; CDCl3) 19.8, 25.5-31.7 (signals superimposed), 38.0, 62.3, 67.7, 99.4, 123.0, 132.1, 134.3, 168.4 (CdO); m/z (ES+) 466 (MNa+, 25%), 377 (100). To 2-(14-tetrahydropyran-2-yloxy-tetradecyl)-isoindole-1,3dione (64 mg, 0.15 mmol) in methanol (2 mL) was added dilute hydrochloric acid (6 N; 0.5 mL). The reaction was heated at reflux for 2 h, ether was added (20 mL) and the solution washed with sodium carbonate solution (2 × 20 mL), saturated sodium chloride solution (1 × 20 mL) and dried (MgSO4). After evaporation in vacuo, the product was purified by flash silica chromatography (hexane/EtOAc, 1:1) to give 2-(14-hydroxytetradecyl)-isoindole-1,3-dione (37 mg, 67%) (17). 1H NMR (300 MHz; CDCl3) 1.29 (m, 18H), 1.60 (m, 6H), 3.60 (m, 4H), 7.68 (m, 2H, ArH), 7.78 (m, 2H, ArH); 13C NMR (75 MHz; CDCl3) 25.7-29.6 (signals superimposed), 32.8, 38.1, 63.1, 123.2, 132.2, 133.8, 168.5 (CdO); m/z (ES+) 360 (MH+, 10%). To 2-(14-hydroxytetradecyl)-isoindole-1,3-dione (37 mg, 0.10 mmol) in acetone (1 mL) was added Jones reagent (0.085 mL; for 1 mL of Jones reagent- 0.23 mL H2SO4, 270 mg CrO3, 0.77 mL H2O). The reaction was stirred at rt for 1 h, 2-propanol (5 mL) was added and the mixture diluted with water (5 mL), then the acetone removed in vacuo. The solution was extracted with CH2Cl2 (2 × 20 mL), dried (MgSO4) and evaporated, then purified by flash silica chromatography (hexane/EtOAc, 3:2) to give 14-(1,3-dioxo-1,3-dihydroisoindol-2-yl)tetradecanoic acid (20 mg, 50%). 1H NMR (300 MHz; CDCl3) 1.22 (m, 18H), 1.57 (m, 4H), 2.26 (m, 2H), 3.63 (m, 2H, CH2N), 7.68 (m, 2H, ArH), 7.78 (m, 2H, ArH); 13C NMR (75 MHz; CDCl3) 25.729.6 (signals superimposed), 32.4, 34.1, 38.1, 123.2, 132.2, 133.8, 168.5 (CdO), 171.3 (CdO acid). Hydrazine hydrate (0.25 mL, 0.50 mmol) was added to 14(1,3-dioxo-1,3-dihydroisoindol-2-yl)-tetradecanoic acid (20 mg, 0.05 mmol) in ethanol (1 mL). The reaction was heated at reflux for 4 h, and the solvents were evaporated in vacuo. The residue was dissolved in CH2Cl2, and the precipitated phthalimide was removed by filtration. The CH2Cl2 was removed in vacuo to

Bioconjugate Chem., Vol. 18, No. 1, 2007 201

give 14-aminotetradecanoic acid (7) (10 mg, 80%) (16) as a colorless solid. mp 176-178 °C (CH2Cl2); 1H NMR (300 MHz; CDCl3) 1.10 (m, 18H), 1.36 (m, 4H), 2.05 (t, J 7.5 Hz, 2H, CH2CO2H), 2.57 (m, 2H, CH2N); 13C NMR (75 MHz; CDCl3) 25.7-29.6 (signals superimposed), 32.9, 34.1, 40.1, 171.2 (Cd O acid); m/z (ES+) 261 (M+ + H2O, 100%), 258 (M+ + NH3, 21), 244 (MH+, 5). 16-Aminohexadecanoic Acid Hydrochloride (8). A solution of methyl 16-phthalimide hexadecanoate (19) (200 mg, 0.48 mmol) and hydrazine hydrate (97 mg, 1.94 mmol) in ethanol (5 mL) was heated at reflux for 3 h. The solvents were evaporated in vacuo, the residue was dissolved in ethanol (5 mL) and dilute hydrochloric acid (2 N; 0.5 mL), and the mixture was heated at reflux for a further 3 h. Ethanol (2 mL) was removed in vacuo and the precipitated phthalimide removed by filtration. Dilute sodium hydroxide solution (6 N; 0.5 mL) was added to the filtrate and the mixture was stirred for 4 h, then neutralized with 2 N hydrochloric acid solution. The remaining ethanol was removed in vacuo, and the suspension was filtered, then washed with water (20 mL) and CH2Cl2 (3 × 10 mL) to give 8 (16) as a colorless powder (125 mg, 84%), which was used directly in the next step (see synthesis of 10 below). 14-(Ferrocenoylamide-3-oxapentanyloxycarbonylamino)tetradecanoic Acid (9). The reaction was carried out under anhydrous conditions. Compound 3 was activated as the N-hydroxysuccinimide ester as previously described (13). The NHS intermediate (38 mg, 0.08 mmol) was dissolved in THF (1 mL) and triethylamine (14 µL, 0.09 mmol) was added. To this solution was added 7 (10 mg, 0.04 mmol), in a mixture of water/DMF (1 mL/1.3 mL). The reaction was stirred at rt for 18 h, and the solvents were evaporated in vacuo. The resulting oil was purified by flash silica chromatography (5% methanol in CH2Cl2) to afford 9 (2 mg, 62%) as an orange oil. 1H NMR (300 MHz; CDCl3) 1.13 (m, 18H), 1.46 (m, 4H), 2.15 (t, 2H, J 7.5 Hz, CH2CO2H), 3.08 (m, 2H, CH2NH), 3.54 (m, 2H, CH2NH), 3.65 (m, 4H, CH2O), 4.21 (s, 5H, C5H5), 4.23 (m, 2H, CH2OCO), 4.32 (m, 2H, C5H2), 4.72 (m, 2H, C5H2), 4.90 (br m, 1H, NH), 6.59 (br m, 1H, NH); m/z (ES+) 609 (MNa+, 10%), 340 (100). 16-(Ferrocenoylamide-3-oxapentanyloxycarbonylamino)hexadecanoic Acid (10). The reaction was carried out under anhydrous conditions. Compound 3 was activated as the N-hydroxysuccinimide ester as previously described (13). The NHS intermediate (50 mg, 0.11 mmol) was dissolved in THF (5 mL), and triethylamine (34 µL, 0.24 mmol) and 8 (37 mg, 0.12 mmol) in a mixture of water/THF (3 mL/2 mL) were added. The reaction was stirred at rt for 18 h, and the solvents were evaporated in vacuo. The resulting oil was solubilized in EtOAc (20 mL), washed with water (2 × 20 mL), dried (Na2SO4), evaporated, and purified by flash silica chromatography (100% EtOAc, then EtOAc/methanol 90:10) to afford 10 (20 mg, 30%) as an orange oil. 1H NMR (300 MHz; CDCl3) 1.26 (m, 20H), 1.45 (m, 2H), 1.65 (m, 2H), 2.38 (t, 2H, J 7.5 Hz, CH2CO2H), 3.15 (m, 2H, CH2NH), 3.59 (m, 2H, CH2NH), 3.64 (m, 2H, CH2O), 3.70 (m, CH2O), 4.21 (s, 5H, C5H5), 4.26 (m, 2H, CH2OCO), 4.34 (m, 2H, C5H2), 4.72 (m, 3H, C5H2 + NH), 6.25 (br m, 1H, NH); m/z HRMS calculated for C32H50N2O6FeNa (MNa)+ 637.29105, found (ES+) 637.28895. Electrochemistry. Materials and Cells. The basic reagents were phosphate buffered saline (PBS: 0.01 M phosphate buffer, 0.137 M NaCl, 0.0027 M KCl, pH 7.4), sodium oleate, human serum albumin (all Sigma) and Nafion perfluorinated ionexchange resin 10 wt % dispersion in water (Aldrich). All solutions were filtered using a 0.2 µm cutoff filter. All measurements were performed with electrodes screen-printed onto alumina tiles using polymer-bonded, heat-cured inks (Gwent Electronic Materials Ltd), with the cell area defined by


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over-printing an insulator with a circular hole to expose the electrodes: carbon working (area ) 3.0 × 10-2 cm2) and counter electrodes and a silver/silver chloride reference electrode. In repeat measurements of cyclic voltammetry of the ferrocene probe molecules in PBS, cells mass-produced in this way reproduced peak currents to better than ( 15% and E1/2 to better than ( 5 mV (2 standard deviations). Nafion coating of the electrodes was achieved by pipetting 3 µL of a 1% v/v dilution in water of the as-supplied Nafion solution onto the cell so as to cover the entire exposed disc with the working, counter and reference electrodes. The resulting assembly was left to dry at room temperature in the dark. The volume and dilution required were determined empirically. Given the cell area (0.12 cm2) this implied a fully dense membrane thickness of approximately 0.1 µm. When the sample was fully swollen in water, the thickness would be significantly greater. The electrode coating method was not optimized and introduced a degree of variability into the results (variation in peak current ( 20%). All electrochemical measurements were performed with a general purpose electrochemical system (Autolab: Eco Chemie B.V Netherlands). For measurement, 20 µL of sample was applied to the sample/ working area of the electrode ensuring complete coverage of the working, counter, and reference electrodes. All samples were premixed before being applied to the electrode, and a new electrode tile was used for each measurement. All ferrocenelabeled fatty acid probe molecule stock solutions were dissolved in methanol due to poor solubility at high concentrations in PBS. Subsequent dilutions were made with PBS, and the final concentration of methanol was no more than 5% v/v. Methanol binds weakly to albumin through the methylene group (20) and will probably compete on the drug binding sites. This competition will introduce an error into the determination of binding constants, which is assessed in the Discussion. Cyclic voltammetry measurements were made immediately on uncoated electrodes and after a 2 min period on Nafion-coated electrodes. All potentials are quoted with respect to the Ag/AgCl reference electrode in the measurement solution. The potential ramp was a digitally generated staircase. Cyclic voltammetry scan parameters were as follows, hold at 0 V for 5 s, start potential 0 V, vertex potential 0.7 V, staircase step potential 0.002 V, and scan rate 0.05 V s-1. Electrochemical System Design. The binding of the probe to a protein, the competition with other molecules in the solution, and measurement through electrochemistry is overall a complicated system. The electrochemical process has a second-order reaction preceding the electrochemical step and probably another following. Also, as we showed in our previous paper (13), the ferricenium product can decompose to electrochemically inactive species. kd

E + A [\ ] EA k a

+

E / E + ekd,1

E+ + A [\ ] E+A k

RESULTS

a,1

8 products E+ 9 k

so that E and E+ (eq 1) can be considered at equilibrium at the electrode surface, with concentrations determined by the Nernst equation, then the ratio of electrochemical to chemical rate constants is V ) VF/(RT{(ka,1 + ka,2)cE,T + kd,1 + kd,2 + k2}) where cE,T ) cE + cE+ + cEA + cE+A is the total concentration of the species E. With decrease of V below unity, reactions in the boundary layer on the time scale of the scan have a profound effect on the wave shape and peak height in cyclic voltammetry. Literature data for fatty acid binding (21) show one should expect ka,1cE,T + kd,1 ∼ 1 s-1, and hence for potential scan rates around 50 mV s-1, indeed V ∼ 1. Although the full system is complex, the behavior can be calculated easily using finite difference solution of the diffusion-reaction equations. We used a spreadsheet to do this. Figure 2 illustrates the wave shift that could be caused, calculated using representative parameters deduced in Results and from the literature. Figure 2 also shows that, in these circumstances, the concentration of “unbound” probe in the presence of protein cannot be obtained by using a simple calibration in the absence of the protein. Therefore, for quantitation of the equilibrium solution concentrations of the probe, E, the complicating effects of the preceding and following chemical reactions need to be avoided. While in principle a sufficiently high scan rate would suffice, quantitation is most conveniently achieved by use of a membrane that defines the boundary layer and excludes the protein from it. Figure 2 also illustrates the theoretical effect of a membrane on the voltammetry of a second-order CEC process. If the membrane is sufficiently thick, then the concentration changes of E and E+ near the surface, that determine the electrochemical response, are confined within the membrane and are largely uninfluenced by the protein. The peak current, though obviously dependent on the membrane thickness, the diffusion coefficient of E and E+ within the membrane and the decomposition rate of E+ within the membrane, becomes the same for the same bulk solution concentration of free label whether or not protein is present. The theory shows that even a very thin membrane could allow deduction of the bulk solution concentration of free label from a calibration made in the absence of protein, without too much error. A compromise is clearly achievable wherein the voltammetry can be simplified and a reasonable calibration obtained without too much diminution of the peak current. A suitably chosen membrane also has the advantage of mitigating the effects of protein and probe adsorption on the electrode surface. Proteins adsorb strongly on carbon and can unfold across the hydrophobic surface. There is also the possibility that the long-chain fatty acid analogues used as electrochemical probes could adsorb on a hydrophobic carbon surface. These considerations all indicate the use of a hydrophilic, protein-excluding membrane over the electrode. The major interferences in electrochemistry in blood are due to the anionic species ascorbate and uric acid. A cationic membrane should exclude these species. We indeed found that a Nafion membrane essentially eliminated the interference, and in view of the other considerations outlined above, used such coated electrodes for the measurements reported here.

(1)

2

The behavior is determined by the value of the characteristic time constants for the chemical processes relative to the characteristic time constant for the electrochemical experiment, which for cyclic voltammetry is VF/RT where V denotes the potential scan rate, F is Faraday’s constant, R is the gas constant and T is the Kelvin temperature. If the electrochemistry is fast,

Electrochemical Characterization. In the previous paper (13) the optimal linkers A and B and the ferrocene solubilizing group were identified in order to obtain molecules with the desired electrochemistry, solubility and stability. The selected structure 2 consisted of a monosubstituted ferrocene with an amide bond linking the cyclopentadienyl ring to a short (two unit) PEG spacer group, linked to the fatty acid through a carbamate bond (Figure 1). Compounds 4-6 (m ) 2, 5, 7) showed half-wave potentials at approximately 350 mV Ag/AgCl (Table 1). A further increase



Table 1. Electrochemical Data for the Probe Moleculesa compd

m

E1/2/V Ag,AgCl

Ea,p - Ea,p/2/V

Ea,p - Ec,/Vp

Ic,p/ Ia,p

4 5 6 2 9 10

2 5 7 10 13 15

0.349 0.356 0.351 0.363 0.379 0.507

0.063 0.065 0.061 0.059 0.071 0.105

0.074 0.072 0.071 0.072 0.095 0.125

0.88 0.91 0.91 0.65 0.43 0.06

a 50 mM in PBS with MeOH content for 10 ) 5%, others approximately 1-2%, on uncoated screen-printed carbon electrodes, recorded at 50 mV s-1 scan rate. E1/2 was taken as the mean of the peak potentials for anodic and cathodic peaks. The peak current ratio was determined by the extrapolation method illustrated in ref 22.

Figure 3. Correlation of peak current ratio Ic,p/Ia,p with half-wave potential E1/2 for the ferrocene probe molecules. The dotted line illustrates the correlation for the series of different compounds given in ref 13.

Figure 2. Effects of the CEC process described by eqs 1 on linear sweep voltammetry, illustrating the effect of a membrane. (a) Simulation of linear sweep voltammetry. 1: E only, no membrane; 2: E, protein, and membrane 1 (current density on right-hand axis); 3: E and protein, no membrane. (b) Simulated voltammetric peak current density, ip, dependence on the concentration of electrochemically active species, E, that is not bound to protein, cunbound, in the absence of a membrane, showing that a calibration deduced in the absence of protein would result in a significant error in cunbound; (c) as for (b), but with a membrane covering the electrode, illustrating that, with a sufficiently thick membrane a calibration deduced in the absence of protein would give a reasonably reliable measure of cunbound. Parameters assumed: cE,T ) 5 × 10-5 mol dm-3, cA,T ) cA + cEA + cE+A ) 5 × 10-4 mol dm-3, kd ) kd,1 ) 1 s-1, binding constant, K ) kd/ka ) kd,1/ka,1 ) 1 × 10-4 mol dm-3, solution diffusion coefficients of E and E+ 1 × 10-6 cm2 s-1, scan rate 50 mV s-1, start potential F(E - E0)/RT ) -5. Assumed membrane characteristics are that protein, A, is totally excluded and membrane 1, thickness 5 × 10-4 cm, diffusion coefficient of E and E+ 1 × 10-7 cm2 s-1; membrane 2: thickness 1 × 10-3cm, diffusion coefficient of E and E+ 5 × 10-8 cm2 s-1. The diffusion coefficients of the protein species A, EA, and E+A are assumed much less than those of the electrochemically active species E and E+. The decomposition of E+ is neglected.

in m caused E1/2 to move anodic. For a given concentration, the peak current generally increased with increasing m. When the length of the carbon chain in the fatty acid was longer than C11 (e.g., compound 10) the oxidation potential shifted significantly anodic and the ferricenium reduction wave virtually disappeared. As noted in a previous paper (13), there was a systematic trend of half-wave potential, E1/2 and anodic/cathodic peak current ratio with the length of the substituent chain (Figure 3). These effects are consistent with the trend in electrochemical behavior that was identified in previous work (13). The difference between the potential at the anodic peak and that at half the peak current, Ea,p - Ea,p/2, was ∼60 mV for m ) 10 or less, and somewhat higher for greater m, (Table 1). Figure 4 shows the effect of the Nafion membrane on the voltammetry of the ferrocene compounds. The half-wave potential was shifted significantly cathodic, to around 0.28 V Ag/ AgCl and the variation in E1/2 with substituent chain length was eliminated. The cathodic peak height, for reduction of ferricenium, was significantly increased. Binding to Human Serum Albumin. The ferrocene labeled fatty acid probe molecules investigated were ferrocene methanol (FcCH2OH), 4 (m ) 2), 5 (m ) 5), 6 (m ) 7), 2 (m ) 10), and 10 (m ) 15). Experiments with no membrane coating the electrodes showed the expected effects of the CEC process on the wave shape in cyclic voltammetry, different for different chain lengths, m (Figure 5). The effects on the peak position and wave shape were eliminated by the use of the Nafion membrane (Figure 5). Therefore, in view of the theory given in the System Design section, the membrane coating used was assumed sufficiently thick to allow reasonably reliable determination of the concentration of the probe free in solution, based on a calibration made in the absence of protein. The binding of the ferrocene labeled poly(oxoethylene)-fatty acid probe molecules as a function of the number of carbons in


Tranchant et al.

Figure 4. Cyclic voltammetry of ferrocenyl compounds illustrating the effect of a Nafion membrane. The curves are labeled with the compound number. (a) On uncoated electrodes; (b) on Nafion-coated electrodes. Compounds 2 and 10 at 50 µM in PBS with MeOH content 10 ) 5%, 2 ) 2%, recorded at 50 mV s-1 scan rate.

the fatty acid chain is further illustrated in Figure 6. The degree of binding of the probes to HSA can be changed by varying the fatty acid carbon chain length, as expected. The data show a gradual decrease in the degree of binding from 15 to 5 carbons. There was little detectable binding for two and five carbons. However, FcCH2OH (no poly(oxoethylene) chain) did bind to HSA. Electrochemical Probe of Site Occupancy on HSA. We explored the possibility of an electrochemical measurement of site occupancy on albumin, using 2 (m ) 10) and 10 (m ) 15), as examples of a medium chain length and a long-chain length probe, respectively, competing with decanoate and oleate. Figures 7 and 8 show the amount of free 2 and 10, respectively, taken from Ip in cyclic voltammetry on membrane-coated electrodes, in a solution containing different concentrations of HSA. Comparison has been made with the calculated amounts that would be bound, based on the binding constants of other fatty acids given by Spector et al. (11) (multiple binding sites and different binding constants to each site). Compound 2 did not bind as strongly to HSA as a fatty acid of similar chain length such as decanoate, but more like one with eight methylene groups. The binding of 2 was also well-represented by a threesite model, each site having dissociation constant, Kd ) 1 × 10-4 mol dm-3. However, the measurements at higher albumin concentration showed unbound probe concentration, determined electrochemically in the manner described, higher than expected. Figure 7 also illustrates that the fluorescent probe 11-(dansylamino)undecanoic acid (DAUDA) bound much more strongly to HSA than either the electrochemical probe, or fatty acids of similar chain length. Compound 10, with 15 methylene groups in the hydrocarbon chain, bound with similar strength to a fatty acid with 12-14 methylene groups. Curry et al. (7) described seven binding sites for fatty acids on albumin. A simple model in which 10 binds equally to seven sites gave reasonable

Figure 5. Effect of HSA on cyclic voltammetry of 2 and 5 at uncoated carbon electrodes and of 2 at a Nafion-coated electrode. The dotted lines are added to show the change in peak positions. PBS; compound concentration 50 µM, 50 mV s-1.

agreement to the data with Kd ) 5 × 10-6 mol dm-3. Again, the measurement at highest albumin concentration showed a higher than expected unbound probe concentration, determined electrochemically in the manner described. The competition of compounds 2 and 10 with fatty acids for binding sites on HSA is shown in Figures 9-11. Comparison is made with theoretical predictions assuming binding to different classes of site on HSA. The binding constants determined using the stepwise equilibrium model for oleate (12) and decanoate (11) have been used. In competition with decanoate, there was a steady increase in the concentration of 2 in the solution with an increasing molar ratio of decanoate to albumin. However, even when decanoate was at 10 times excess of HSA, there was still significant 2 bound to HSA. Competition of 2 with oleate showed a different pattern of behavior. For molar ratios oleate/HSA up to approximately 5, 2 was not significantly displaced by oleate. At higher ratios, 2 was completely displaced by oleate. The behavior of compound 10 was different, as expected. At low molar ratio



Figure 6. Binding of ferrocene labeled fatty acid probes (50 µM) to HSA, determined by the % decrease in anodic peak height in cyclic voltammetry on membrane-covered electrodes, as a function of the number of carbon atoms in the fatty acid chain. The points for m ) 1 are for FcCH2OH (ferrocene methanol).

Figure 8. Measurement of bulk solution unbound concentration of compound 10, cfree, (symbols) by cyclic voltammetry at a Nafion-coated carbon electrode, as a function of concentration of HSA, [HSA]. Total concentration of 10 in the solution 55 µM. Solid lines show cfree calculated at the same total concentration for different fatty acids, using literature values for the binding constants (11, 12).

Figure 7. Measurement of bulk solution unbound concentration of compound 2, cfree, (symbols) by cyclic voltammetry at a Nafion-coated carbon electrode, as a function of concentration of HSA, [HSA]. Total concentration of 2 in the solution 40 µM. Solid lines show cfree calculated at the same total concentration for different fatty acids, for 11-(dansylamino)undecanoic acid (DAUDA), using literature values for the binding constants (9, 11), and for a simple three-site binding model with each site having Kd ) 1 × 10-4 mol dm-3.

Figure 9. Competition of 2 with decanoate for sites on HSA. [2]total ) 25 µM, and [HSA]total ) 50 µM. Solid lines correspond to calculated values of the concentration of 2 free in the solution, cfree, assuming that decanoate binds to HSA at five sites according to Spector et al. (11), numbered in order of decreasing affinity, and that 2 binds to three of them, specified in the legend, with Kd ) 1 × 10-4 mol dm-3. Site 6 in the calculation is an additional site for which it is assumed that 2 binds but decanoate does not; cfree determined from Ip in cyclic voltammetry at Nafion-coated carbon electrodes.

oleate/HSA, the solution concentration of 10 increased, indicating its displacement from the higher affinity sites. Compound 10 was essentially completely displaced into solution at molar ratio oleate/HSA > 8.

DISCUSSION Electrochemistry of the Ferrocenes. In our previous paper (13), we deduced that the electrochemical behavior of variously substituted ferrocenes was strongly determined by the effect of the substituent on the stability of the ferricenium cation. The ability of the substituent to disperse the positive charge on ferricenium, and steric effects that caused the two cyclopentadiene rings to move out of coplanarity were also identified as important. The peak shape, as measured by the difference between peak and half-peak potential, indicated that the electrochemical reaction was fast on the time scale of the scan in cyclic voltammetry. The ratio of cathodic to anodic peak current implied decomposition of the ferricenium product in the boundary layer near the electrode. The behavior reported here (Figure 3, Table 1) is consistent with this interpretation. The peak potential difference implied that the electrochemical

process was fast in comparison with the time scale of the scan, perhaps decreasing in rate with increasing chain length, m. The variation of half-wave potential and peak current ratio indicated decreasing stability of the ferricenium cation with increasing substituent chain length. The observed increase of anodic peak current with increasing substituent chain length is similarly consistent with the interpretation: increasing the size of the molecule should decrease the diffusion coefficient and so decrease the peak current; however, if the electrochemistry is fast and the product decomposes then the peak current would be increased. Effect of the Nafion Membrane on the Electrochemistry of the Ferrocenes. The increase of the cathodic peak current, the cathodic shift of the wave, and the removal of the effect of the chain length on the half-wave potential and peak current ratio (Figure 4) can be interpreted simply as a consequence of stabilization of the ferricenium cation in the membrane. The cathodic peak current is significantly increased. We showed, by holding the potential at the anodic limit of the scan for a time, that ferricenium could be accumulated in the cation exchange membrane. The decrease in current with increasing m is attributable to interaction of the hydrocarbon chain with


Figure 10. Competition of 2 with oleate for sites on HSA. [2]total ) 50 µM, and [HSA]total ) 50 µM. Solid lines correspond to calculated values of concentration of 2 free in the solution, cfree, assuming that oleate binds to HSA at seven sites numbered in order of decreasing affinity, and that 2 binds to three of them, specified in the legend, with Kd ) 1 × 10-4 mol dm-3. Sites 1-6 in the calculation are those identified by Richieri et al. (12). Site 7 is an assumed additional weak binding site with Kd ) 1 × 10-5 mol dm-3; cfree determined from Ip in cyclic voltammetry at Nafion-coated carbon electrodes.

Figure 11. Competition of 10 with oleate for sites on HSA [10]total ) 50 µM, and [HSA]total ) 50 µM. Symbols give the concentration of 10 free in the solution, cfree, determined from Ip in cyclic voltammetry at Nafion-coated carbon electrodes. Solid lines correspond to calculated values cfree assuming that oleate binds to HSA at seven sites numbered in order of decreasing affinity. Sites 1-6 in the calculation are those identified by Richieri et al. (11). Site 7 is an additional weak binding site with assumed Kd ) 1 × 10-5 mol dm-3. Compound 10 is assumed to bind to all seven sites with equal binding constant, Kd, specified in the legend.

the polymer membrane decreasing the diffusion coefficient through the membrane. Electrochemistry in the Presence of Albumin. The effects on cyclic voltammetry at an uncoated electrode (Figure 5) were consistent with a dissociation rate of any complex increasing significantly, and a binding affinity decreasing significantly with decreasing chain length. The decrease in the presence of albumin of cathodic peak current relative to anodic peak current indicated that the ferricenium cation was also complexed by albumin. For compound 2, taken with the estimates of binding constant deduced below, the effect on the voltammetry was consistent with a dissociation rate constant ∼1 s-1, which is consistent with values reported in the literature (21). With a Nafion membrane, the protein had no effect on either the peak position or the relative peak height. On the basis of the theory given under Electrochemical System Design, it was therefore reasonable to assume that calibration in the absence of protein would

Tranchant et al.

give a reliable measure of the unbound concentration of the probe. However, the theory (Figure 2) also showed that this assumption could fail as the unbound concentration increased. The assumption could also fail at higher protein concentration. The effect would be to indicate a free probe concentration higher than that actually present, because the effect of the complexation with protein would be to increase the concentration gradients in the boundary layer immediately outside the membrane and hence increase the observed current. Binding of the Ferrocene Probes to Albumin. There appeared to be three effects operative (Figure 6): binding of the ferrocene group, binding of the hydrocarbon chain, and the effect of the poly(oxoethylene) part of the chain in decreasing the binding to albumin. With m ) 7, 10, 15 (compounds 6, 2, and 10), the carboxylic acid chain was long enough to interact with the fatty acid binding sites of HSA, and the observed good binding of these molecules to HSA is likely to be due to the fatty acid part. The result with ferrocene methanol showed that ferrocene itself could bind reasonably strongly to HSA. Albumin has sites that bind drug molecules (2), for example, paracetamol and aspirin, which have a conjugated ring as does ferrocene, and one of these sites may be involved in the ferrocene binding. The results show that the addition to ferrocene of a PEG chain, if this was terminated by a sufficiently short carboxylic acid chain (m ) 2, 5: compounds 4 and 5), prevented the ferrocene from binding to HSA. A likely explanation is improved solvation by water conferred by the PEG. However, effects caused by the chain and the carbamate linking group such as steric hindrance of binding, or restriction in the orientation of the ferrocene with respect to the protein, cannot be discounted. Figures 7 and 8 show that the system can reasonably measure the binding of the probes to albumin, although with a deviation from the expected behavior at high protein concentration. The deviation is expected in view of the theory above, and the solution to the problem would be to increase the membrane thickness. The binding of the ferrocene probes was similar to that of fatty acids with a slightly shorter chain length. We can attribute the reduction in binding affinity to the steric effects of the carbamate linker and the affinity for water conferred by the short PEG chain. The result indicates the possibility of finetuning the probe by altering the balance of hydrophilicity and hydrophobicity in the molecule. The behavior contrasts to that of DAUDA. Wilton (9) has interpreted data on binding to HSA of DAUDA, a fluorescently labeled fatty acid with 10 CH2 units in the carbon chain, to deduce three binding sites with Kd < 10-7 M at the high-affinity site, and Kd ) 8 × 10-7 M at the other two sites (8). The calculated binding of DAUDA to HSA, using the Kd given by Wilton, plotted in Figure 7, demonstrates the much stronger binding of that compound than either fatty acids or the ferrocene probe of similar chain length. As noted below, the bilirubin binding site was deduced as one major binding site for DAUDA implying that, for this compound, binding was dominated by the effect of the large aromatic fluorescent label. The competition studies (Figures 9-11) showed that the ferrocene probes did indeed bind to the fatty acid binding sites of albumin, with behavior modified by the presence of PEG and carbamate linker. Thus, Figure 9 shows that 2 bound to the sites accessed by decanoate, since it could be displaced from albumin by decanoate. However, compound 2 was not completely displaced, implying the presence of another site. The ferrocene probe appeared to bind weakly to a site not accessed by fatty acids. Given the binding observed for ferrocene methanol, it is reasonable to presume that this was a site that bound the cyclopentadienyl ring, perhaps one of the drug binding sites. Figure 9 shows that it is possible to make quantitative comparison with a site binding model and deduce the binding constants. Reliable deduction of the binding constants would


require further development of the electrochemical method and better design of the membrane coating of the electrode. Figure 10 shows that the high-affinity sites accessed by oleate were not accessed by 2. Compound 2 could only access sites with low affinity for oleate, from which it was displaced at a sufficiently high oleate/HSA ratio. The results are consistent with previous measurements carried out with DAUDA (10 CH2 fatty acid chain). Wilton (9) deduced that DAUDA does not bind to the high-affinity sites for long-chain fatty acids, since it was displaced only at higher fatty acid/HSA ratios. Similarly, DAUDA was not completely displaced by medium-chain fatty acids. However, bilirubin displaced DAUDA significantly indicating that DAUDA shared with this molecule its major binding site, termed drug site I (2). As noted above, it is probable that compound 2 also bound weakly at this site, perhaps through the ferrocene group. Extending the fatty acid chain of the ferrocene allowed these probes to access the high-affinity binding sites for long-chain fatty acids. Figure 11 shows that compound 10 was displaced from HSA at low molar ratios of oleate to HSA. Hence, this compound accessed the highest affinity sites for oleate. As indicated in Figure 11, the data could be interpreted with a simple competitive binding model assuming seven binding sites accessible to both speciessthe six sites identified by Richieri et al. (12) together with an additional weak binding site to account for the data when oleate/HSA > 6. Interaction with Methanol. The possible interaction with methanol used to introduce the probes and consequently present at concentrations up to 5% v/v is a complication that does not, however, affect the major conclusions of the work. Alcohols have a large effect on albumin in acid solution, promoting R-helix formation. However, the effects at neutral pH are small (23). Lubas et al. (20) showed a weak interaction of methanol with albumin, through the methylene group. By using the ethanol binding constant reported by Avdulov et al. (24) and comparing the results for methanol and ethanol in ref 20, an approximate binding constant for methanol can be deduced: 0.17 mol dm-3. Ha et al. (25) showed that ethanol could displace drugs such as warfarin from HSA. However, Rosenberg et al. (23) showed no effect of ethanol on the binding of methyl orange to HSA. The effect of methanol will be less than that of ethanol (20). If the added methanol competed equally with the probes for all the binding sites, with a binding constant of 0.17 mol dm-3, then the binding constants for the probes, deduced above, could be a factor of 10 times too high. If, however, the methanol competed only for the drug binding site where we have deduced that the ferrocene group also bound, then only the estimate of binding constant for this site would be in error, although it could be substantially so.

CONCLUSION We have shown how to design both an instrumentally simple electrochemical system and a set of probe molecules that can be used to determine the occupancy of different classes of fatty acid binding sites on human serum albumin. We have shown how variation in the fatty acid chain length on the probe results in interaction with different classes of sites on HSA and deduced that the interaction can be changed from primarily with a drug binding site, when the probe is ferrocene methanol, to one interacting primarily with medium-chain fatty acid binding sites, by adding a poly(oxoethylene)-fatty acid substituent, to one interacting primarily with high-affinity long-chain fatty acid binding sites as the fatty acid chain length in ferrocene-poly(oxoethylene)-fatty acid molecules is increased. The results strongly imply that the binding could be further tailored by relatively simple modifications to the probe, for example, to change the balance of hydrophobicity and hydrophilicity. The


approach is a general one, in that other small molecules might be electrochemically labeled using similar methods and these probe molecules used to access different classes of binding sites.

ACKNOWLEDGMENT We thank Unipath for funding (IT) and for permission to publish this article.

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