Design and Synthesis Of Ferrocene Probe Molecules for Detection by

Bioconjugate Chem. 2006, 17, 1256−1264

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Design and Synthesis Of Ferrocene Probe Molecules for Detection by Electrochemical Methods Isabelle Tranchant,‡ Anne-Cećile Herve´,†,§ Stephen Carlisle,† Phillip Lowe,†,| Christopher J. Slevin,‡,| Camilla Forssten,† John Dilleen,†,| David E. Williams,†,⊥ Alethea B. Tabor, 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 July 17, 2006

A series of ferrocenyl conjugates to fatty acids have been designed and synthesized to establish the key properties required for use in biomolecular binding studies. Amperometric detection of the ferrocene conjugates was sought in the region of 0.3 V (vs Ag/AgCl) for use in protein/blood solutions. Different linkers and solubilizing moieties were incorporated to produce a conjugate with optimal electrochemical properties. In electrochemical studies, the linker directly attached to the ferrocene was found to affect significantly the E1/2 value and the stability of the ferrocenium cation. Ester-linked ferrocene conjugates had E1/2 ranging from +400 to +410 mV, while amidelinked compounds ranged from +350 to +370 mV and the amines +260 to +270 mV. Folding of long-chain substituents around the ferrocene, also significantly affected by the choice of linker, was inferred as a secondary effect that increased E1/2. The stability of the ferrocenium cation decreased systematically as E1/2 increased. Disubstituted ferrocene ester and amide conjugates, with oxidation potentials of +640 and +570 mV, respectively, showed only a barely discernible reduction wave in cyclic voltammetry at 50 mV/s. Electrochemical measurements identified two lead compounds with the common structural characteristics of an amide and carbamate linker (compounds 17 and 21) with a C11 fatty acid chain attached. It is envisaged that such molecules can be used to mimic and study the biomolecular binding interaction between fatty acids and molecules such as human serum albumin.

INTRODUCTION Molecular interactions in biological systems have been studied using many different techniques such as surface plasmon resonance and fluorescence-based methodologies. There are, however, few reports in the literature utilizing electrochemical labels to study such interactions, with the exception of antibodyantigen interactions, where historically ferrocene-labeled molecules have been used. Metalloimmunoassays were first described in the 1970s when ferrocene was used to label steroid antigens with detection using flameless atomic absorption spectroscopy (1). Subsequently, an immunoassay incorporating a ferrocene electrochemical label was reported using a ferrocene-morphine conjugate whose electrochemistry was perturbed on binding to an antibody, resulting in a homogeneous competitive assay not requiring the separation of a free and antibody-bound label (2). Since then, a range of carbonylmetalloimmunoassays (CMIA) have been developed using metalcarbonyl-labeled molecules, in particular, incorporating ferrocene, which has been used as an electrochemical tracer and signaling molecule (3). The range of ferrocene derivatives available, ease of conjugation to a range of functionalities, reversible electrochemistry, and ability to synthetically tailor redox properties for a particular application makes it ideally * Corresponding author. E-mail: [email protected]. Fax: +44 (0)20 7679 7463. † Unipath Ltd. ‡ Current address: Diatos SA, Therapeutic Chemistry Department, 3-5 Impasse Reille, 75014 Paris, France. § Current address: Diatos NV, Brusselsestraat 69, B-3000 Leuven, Belgium. | 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.

placed for use in electrochemical sensing systems. More recent applications include the utilization of ferrocene as an oligonucleotide label in DNA probes (4-6) and as an electrochemical sensor for the detection of proteins (7). Ferrocene immunoassays with reduced glucose oxidase incorporating enzyme amplification (8), an amperometric immunoassay for lidocaine (9), and a homogeneous ferrocene-theophylline conjugate electrochemical assay (10) have also been reported. Our aim was to demonstrate the utility of ferrocene for the study of biomolecular interactions and its potential in such applications. Interactions between fatty acids and their naturally occurring ligands have been intensively studied and thus provide a well-characterized system in which to study rationally designed synthetic electrochemical probe molecules; to our knowledge, no such probes have been reported in the literature. Fatty acidhuman serum albumin (HSA) binding interactions are particularly important due to the effect on the transport of therapeutic drugs, for example, the reduced capacity of HSA to bind warfarin in the presence of bound fatty acids (11). Therefore, the synthesis of a series of ferrocene-labeled fatty acids would be a suitable model system for study but could also provide a greater understanding of the interactions between fatty acids and HSA. Conventionally such fatty acid ligand interactions have been studied by a number of techniques. Bhattacharya et al. have studied complexes of medium- and long-chain fatty acids and HSA using X-ray crystallography, identifying medium- and long-chain fatty acid binding sites (12). X-ray crystallography has also been used to study the complex of intestinal fatty acid binding protein and palmitate (13). Acrylodanmodified HSA has been used to study, using fluorescent techniques, the effect of oleic acid binding to the microenvironment around residue cysteine-34 in HSA (14). The binding characteristics of the fluorescently labeled fatty acid probe 11(dansylamino)undecanoic acid with HSA (15) and a Fascilo

10.1021/bc060038m CCC: $33.50 © 2006 American Chemical Society Published on Web 08/30/2006

Design/Synthesis of Ferrocene Probe Molecules

Figure 1. Ferrocene multifunctional conjugates.

hepatica fatty acid binding protein have also been studied (16). In addition, acrylodan-modified intestinal FABP (ADIFAB) has been used to investigate the binding of a range of fatty acids, allowing measurement of Kd values by fluorescent measurement (17). However, such measurements can only normally be performed in protein solution or plasma, while electrochemical measurements can potentially be performed in whole blood. In the present study, we have designed and synthesized a range of electrochemically labeled molecules to establish the key properties required for use in binding studies in the presence of serum, such as E1/2, reversibility, and aqueous compound solubility. The final component in the molecular design was the introduction of a moiety for recognition or binding purposes. In our studies, we conjugated a fatty acid as the specific biorecognition for potential applications in fatty acid binding studies. However, alternative functionalities can be introduced depending on the molecular interaction being investigated. The molecular conjugates were designed as shown in Figure 1, incorporating ferrocene as the electrochemical detection method and a solubilizing spacer group together with the binding moiety, in these studies a fatty acid. Poly(ethylene glycol) (PEG)-modified ferrocenes have previously been reported incorporating long polymeric PEG units and have exhibited enhanced aqueous solubilities for use in homogeneous redox membrane biosensors (18, 19). Commercially available PEG spacers were therefore incorporated as solubilizing moieties. However, short, defined PEG units were used to ensure that the spacer group did not interfere with any binding or recognition properties. Also, two linkers, A and B, were introduced to enable the rapid synthesis of a range of analogues that could be modified to alter molecular properties and stabilities. The ferrocene conjugate selected (1) is shown in Figure 1 with linkers A and B, PEG repeat unit n, the option of a second functionality on ferrocene (X), and fatty acid pendant group containing a methylene chain of length m.

EXPERIMENTAL PROCEDURES Materials. Unless otherwise noted, solvents and reagents were reagent grade from commercial suppliers and used without further purification. 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 (for ureas), 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. 1-tButyloxycarbonyl-1,8-diamino-3,6-dioxaoctane 7 (20), methyl 16-hydroxyhexadecanoate (21), and methyl 11-aminoundecanoate (22) were prepared as previously reported. Ferrocenoyl-8-hydroxy-3,6-dioxaoctanoate (3). The reaction was carried out under anhydrous conditions. To a solution of tri(ethylene glycol) (3.26 g, 21.7 mmol) in CH2Cl2 (50 mL) was added a solution of ferrocenecarboxylic acid 2 (0.500 g, 2.17 mmol) in CH2Cl2 (10 mL). After 15 min, DCC (0.525 g,

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2.30 mmol) and DMAP (0.03 g, 0.02 mmol) were added. The solution was stirred at room temperature for 18 h, then the solution was concentrated and purified by flash silica chromatography (EtOAc/hexane, 1:1) to afford 3 (0.55 g, 70%) as an orange oil. 1H NMR (300 MHz; CDCl3) 3.40 (br s, 1H, OH), 3.55 (t, J ) 4.5 Hz, 2H), 3.58 (m, 6H), 3.72 (t, J ) 4.8 Hz, 2H), 4.14 (s, 5H, C5H5), 4.28 (m, 2H, C5H2), 4.32 (m, 2H, CO2CH2), 4.75 (m, 2H, C5H2); 13C NMR (75 MHz; CDCl3) 61.8, 63.1, 69.5, 69.8, 70.2, 70.4, 70.7, 70.8, 71.4, 72.6, 171.7 (CdO); m/z HRMS calculated for C17H22O5FeNa (MNa)+ 385.07089, found (ES+) 385.07110. Ferrocenoyl-(8-ethanoate)-3,6-dioxaoctanoate (4). The reaction was carried out under anhydrous conditions. To compound 2 (100 mg, 0.27 mmol) in CH2Cl2 (5 mL) was added pyridine (0.25 mL) and DMAP (3 mg, 0.03 mmol). The solution was cooled to 0 °C and acetic anhydride (0.032 mL, 0.66 mmol) added dropwise. After 30 min, the solution was warmed to room temperature (rt) and stirred for 2 h. The solvents were evaporated and the resulting oil purified by flash silica chromatography (EtOAc/hexane, 7:3) to afford compound 4 (88 mg, 80%) as an orange oil. 1H NMR (300 MHz; CDCl3) 2.03 (s, 3H, COCH3), 3.69 (m, 6H), 3.76 (t, J ) 4.8 Hz, 2H), 4.17 (s, 5H, C5H5), 4.18 (m, 2H), 4.34 (m, 2H), 4.36 (m, 2H, C5H2), 4.79 (m, 2H, C5H2); 13C NMR (75 MHz; CDCl3) 20.8 (COCH3), 63.1, 63.5, 69.1, 69.4, 69.7, 70.1, 70.5, 70.9, 71.2, 170.8 (Cd O), 171.5 (CdO); m/z HRMS calculated for C19H25O6Fe (MH)+ 404.09168, found (CI+) 404.09263. N-(5-Hydroxy-3-oxapentanyl)ferrocenoylamide (5). The reaction was carried out under anhydrous conditions. Ferrocene carboxylic acid 2 (3.00 g, 13.0 mmol) and HOBt (1.94 g, 14.3 mmol) were dissolved in CH2Cl2 (200 mL), and the reaction was stirred at rt for 10 min. EDCI (2.85 g, 13.0 mmol) and triethylamine (3.60 mL, 26.0 mmol) were then added, and after 30 min, 2-(2-aminoethoxy)ethanol (1.37 g, 13.0 mmoles) was added and the solution stirred at rt for 4 h. The solution was concentrated and purified by flash silica chromatography (gradient: 100% EtOAc to 15% methanol in EtOAc) to afford the title compound (3.47 g, 84%) as an orange oil. 1H NMR (300 MHz; CDCl3) 2.90 (br, 1H, OH), 3.44 (td, J ) 5.2 and 5.2 Hz, 2H), 3.51 (m, 4H), 3.64 (t, J ) 4.5 Hz, 2H), 4.07 (s, 5H, C5H5), 4.17 (m, 2H, C5H2), 4.63 (m, 2H, C5H2), 6.58 (br s, 1H, NH); 13C NMR (75 MHz; CDCl3) 39.2 (CH2N), 61.3, 68.0, 69.4, 69.9-70.0 (signals superimposed), 72.2, 170.6 (CdO); m/z HRMS calculated for C15H19NO3FeNa (MNa)+ 340.06066, found (ES+) 340.06126. N-(8-Amino-3,6-dioxaoctanyl)ferrocenoylamide (8). The reaction was carried out under anhydrous conditions. DCC (88 mg, 0.43 mmol) was added to compound 7 (95 mg, 0.39 mmol) and ferrocene carboxylic acid (80 mg, 0.35 mmol) in CH2Cl2 (20 mL), and the reaction was stirred for 18 h at rt. The solvent was evaporated in vacuo and the resulting oil purified by flash silica chromatography (100% EtOAc) to afford the Bocprotected intermediate (82 mg, 46%) as an orange oil. 1H NMR (300 MHz; CDCl3) 1.36 (s, 9H, 3 × CH3), 3.24 (td, J ) 5.2 and 5.1 Hz, 2H), 3.47 (m, 4H), 3.51 (m, 6H), 4.11 (s, 5H, C5H5), 4.24 (m, 2H, C5H2), 4.62 (m, 2H, C5H2), 4.98 (br s, 1H, NH), 6.24 (br s, 1H, NH); 13C NMR (75 MHz; CDCl3) 28.4 (BocCH3), 39.3 (CH2N), 40.3 (CH2N), 68.2, 69.8, 70.2-70.4 (signals superimposed), 79.3 (Boc-C), 156.0 (CdO Boc), 170.4 (CdO amide). The protected amine (82 mg, 0.18 mmol) was dissolved in a mixture of CH2Cl2/TFA (4 mL/4 mL), and the solution was stirred at rt for 2 h. The solvents were evaporated and the resulting oil purified by flash silica chromatography (CH2Cl2/ MeOH/NEt3; 85/10/5) to afford 8 (45 mg, 70%) as an orange oil. 1H NMR (300 MHz; CDCl3/CD3OD) 3.09 (m, 2H), 3.28 (td, J ) 5.3 and 5.3 Hz, 2H), 3.43 (m, 8H), 3.95 (s, 5H, C5H5),

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4.14 (m, 2H, C5H2), 4.56 (m, 2H, C5H2), 6.70 (br s, 1H, NH); 13C NMR (75 MHz; CDCl ) 39.5 (CH N), 39.8 (CH N), 68.4, 3 2 2 69.9, 70.1-70.9 (signals superimposed), 75.5, 172.5 (CdO); m/z HRMS calculated for C17H25N2O3Fe (MH)+ 361.12091, found (ES+) 361.12111; m/z (ES+) 383 (MNa+, 41%), 361 (MH+, 100). Ferrocenylmethylamine N-(5-hydroxy-3-oxapentanyl) (10). The reaction was carried out under anhydrous conditions. Ferrocene carboxaldehyde 9 (150 mg, 0.70 mmol) and 2-(2aminoethoxy)ethanol (147 mg, 1.40 mmol) in toluene (30 mL) were heated at reflux for 18 h. After cooling to rt, sodium borohydride (79.8 mg, 2.10 mmol) was added, and the reaction was stirred at rt for 6 h. Water (5 mL) was added, the solvent removed in vacuo, and the residue suspended in CH2Cl2. The organic phase was washed with water (10 mL), dried (sodium sulfate), and evaporated. The residue was purified by flash silica chromatography (gradient: 100% EtOAc to 20% methanol in EtOAc) to give 10 (74 mg, 35%) as a dark orange oil. 1H NMR (300 MHz; CDCl3) 2.83 (t, J ) 5.3 Hz, 2H, CH2CH2N), 3.53 (s, 2H, CH2-ferrocenyl), 3.57-3.64 (m, 4H), 3.70 (t, J ) 4.4 Hz, 2H), 4.10 (m, 2H, C5H2), 4.12 (s, 5H, C5H5), 4.18 (m, 2H, C5H2); 13C NMR (75 MHz; CDCl3) 48.9 (C-1), 61.9 (C-5), 67.9, 68.4, 68.5, 70.1, 72.4, 86.3; m/z (ES+) 326 (MNa+, 91%), 304 (MH+, 100). Ferrocenylmethylamine N-methyl-N-(5-hydroxy-3-oxapentanyl) (11). The reaction was carried out under anhydrous conditions. To a solution of 10 (70 mg, 0.23 mmol) in CH3CN (30 mL) was added formaldehyde (37%, 38 µL, 0.46 mmol). After stirring for 10 min, sodium borohydride (26 mg, 0.69 mmol) was added in portions, and the mixture was stirred for 5 h. Water (5 mL) was added, the solvent removed in vacuo, and the residue was suspended in CH2Cl2. The organic layer was washed with water (10 mL), dried (sodium sulfate), and evaporated, and the residue was purified by flash silica chromatography (gradient: 100% EtOAc to 20% methanol in EtOAc) to give 11 (26 mg, 35%) as an orange oil. 1H NMR (300 MHz; CDCl3) 2.21 (s, 3H, Me), 2.55 (t, J ) 5.6 Hz, 2H, CH2CH2N), 3.47 (s, 2H, CH2-ferrocenyl), 3.58 (m, 4H), 3.68 (t, J ) 4.5 Hz, 2H), 4.11 (m, 7H, C5H5, C5H2), 4.18 (m, 2H, C5H2); 13C NMR (75 MHz; CDCl3) 41.2 (NMe), 55.6, 57.2, 62.0, 67.8, 68.3, 68.5, 70.4, 72.6; m/z (ES+) 340 (MNa+, 100%), 318 (MH+, 18). 1,1′-Ferrocenoyl-(8-ethoxy-3,6-dioxaoctanoate) (13). The reaction was carried out under anhydrous conditions. DCC (1.19 mmol, 245 mg) and DMAP (5 mg), were added to a solution of 1,1′-ferrocenedicarboxylic acid 12 (0.54 mmol, 150 mg) and tri(ethylene glycol) monoethyl ether (2.16 mmol, 385 mg) in CH2Cl2 (30 mL). The reaction was stirred at rt for 18 h. After filtration, the filtrate was concentrated in vacuo, dissolved in Et2O, and urea was removed by filtration. The solvent was removed under reduced pressure and the residue purified by flash silica chromatography (EtOAc/hexane: 60/40) to give 13 (96 mg, 30%) as an orange oil. 1H NMR (300 MHz; CDCl3) 1.22 (t, J ) 6.9 Hz, 6H, 2 × CH3), 3.54 (q, J ) 6.9 Hz, 4H, 2 × CH2CH3), 3.55-3.70 (m, 16H), 3.79 (m, 4H), 4.38 (m, 4H, CH2OCO), 4.42 (m, 4H, 2 × C5H2), 4.86 (m, 4H, 2 × C5H2); 13C NMR (75 MHz; CDCl ) 15.2 (CH ), 63.5, 66.7, 69.4, 69.9, 3 3 70.7, 70.8, 71.7, 72.6, 73.1, 170.4, 212.1 (CdO); m/z (ES+) 617 (MNa+, 100%), 433 (28). N-(5-Hydroxy-3-oxapentanyl)-1,1′-ferrocenoylamide (14). The reaction was carried out under anhydrous conditions. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.20 mmol, 420 mg) (which had been previously neutralized with 2.20 mmol Et3N) and HOBt (2.20 mmol, 295 mg) were added to a solution of 1,1′-ferrocenedicarboxylic acid 12 (0.54 mmol, 150 mg) in CH2Cl2 (20 mL), and the mixture was stirred for 30 min. A solution of aminoethoxyethanol (2.16 mmol, 227 mg) in CH2-

Tranchant et al.

Cl2 (5 mL) was then added and the reaction stirred for 18 h at rt. The organic phase was washed with water (5 mL), dried (sodium sulfate), and the solvent removed under reduced pressure. The residue was purified by flash silica chromatography (gradient: 100% hexane to 100% EtOAc, then CH2Cl2/ MeOH 95/5) to give 14 (107 mg, 50%) as an orange oil. 1H NMR (300 MHz; CDCl3) 3.62-3.75 (m, 16H), 4.35 (m, 4H, 2 × C5H2), 4.57 (m, 4H, 2 × C5H2), 7.32 (br, 2H, NH); 13C NMR (75 MHz; CDCl3) 39.8, 61.6, 69.9, 70.9, 71.3, 72.5, 78.0, 170.9, 212.0 (CdO); m/z (ES+) 471 (MNa+, 100%), 449 (MH+, 25). 11-(Ferrocenoyl-3,6-dioxaoctanyloxycarbonylamino)undecanoic acid (15). The reaction was carried out under anhydrous conditions. To compound 3 (680 mg, 1.87 mmol) in acetonitrile (20 mL) at 0 °C was added N,N-disuccinimidyl carbonate (574 mg, 2.24 mmol). The solution was stirred at 0 °C for 30 min and at rt for 18 h. The solvent was then evaporated and the resulting oil purified by flash silica chromatography (hexane/EtOAc, 1:4) to afford the succinimidyl carbonate activated 3 (254 mg, 27%) as an orange oil. 1H NMR (300 MHz; CDCl3) 2.83 (s, 4H, CH2 succinimidyl), 3.69 (m, 4H), 3.78 (m, 4H), 4.22 (s, 5H, C5H5), 4.40 (m, 4H), 4.45 (m, 2H), 4.82 (s, 2H, C5H2); 13C NMR (75 MHz; CDCl3) 25.4 (CH2-succinimidyl), 63.2, 68.4, 69.5, 69.8, 70.1, 70.6-70.9 (signals superimposed), 71.3, 152.3, 162.4, 168.9 (CdO ester); m/z (ES+) 526 (MNa+, 46%), 429 (100). To 11-aminoundecanoic acid (12 mg, 0.06 mmol) in THF/ H2O (0.5 mL/1 mL) was added the succinimidyl carbonate activated 3 (15 mg, 0.03 mmol) in THF (0.5 mL). The reaction was stirred at rt for 18 h, then the solvents were evaporated under reduced pressure. The remaining oil was purified by flash silica chromatography (MeOH/CH2Cl2, 5/95) in to afford 15 (13 mg, 74%) as an orange oil. 1H NMR (300 MHz; CDCl3) 1.27 (m, 12H), 1.46 (quint, J ) 6.8 Hz, 2H), 1.62 (quint, 2H, J ) 6.8 Hz), 2.33 (t, J ) 7.2 Hz, 2H, CH2CO2H), 3.13 (dt, J ) 6.6 and 6.6 Hz, 2H, CH2N), 3.68 (m, 6H), 3.79 (t, J ) 4.8 Hz, 2H), 4.19 (m, 2H), 4.20 (s, 5H, C5H5), 4.38 (m, 2H, C5H2), 4.39 (m, 2H), 4.79 (m, 2H, C5H2), 4.81 (br, 1H, NH); 13C NMR (75 MHz; CDCl3/CD3OD) 24.8, 26.6, 28.9, 29.1-29.7 (signals superimposed), 34.2, 40.8, 63.7, 68.1, 69.3-70.6 (signals superimposed), 75.4, 156.8 (CdO carbamate), 171.7, 177.9; m/z HRMS calculated for C29H43NO8FeNa (MNa)+ 612.22303, found (ES+) 612.22331. 16-(Ferrocenoyl-3,6-dioxaoctanoyl)hexadecanoic acid (16). The reaction was carried out under anhydrous conditions. DCC (89 mg, 0.435 mmol) was added to a stirred solution of hexadecanedioic acid (230 mg, 0.829 mmol) in CH2Cl2 (20 mL), and the reaction was stirred at rt for 2 h. To this was added 3 (150 mg, 0.414 mmol), and DMAP (50 mg, 0.041 mol), and the reaction was stirred at rt overnight. Insoluble urea was filtered from the reaction and washed with CH2Cl2 (10 mL), and the combined organic extracts were removed under reduced pressure, leaving a brown viscous oil. The remaining oil was purified by flash silica chromatography (100% EtOAc) to afford the title compound (20 mg, 4%) as an orange oil. 1H NMR (300 MHz; CDCl3) 1.25 (m, 20H), 1.59 (m, 4H), 2.27 (m, 4H), 3.68 (m, 6H), 3.77 (t, J ) 4.8 Hz, 2H), 4.22 (m, 7H), 4.38 (m, 4H), 4.81 (m, 2H, C5H2); 13C NMR (75 MHz; CDCl3) 24.7, 29.2, 29.3, 29.5, 29.6, 29.8, 30.0-30.1 (signals superimposed), 34.2, 34.6, 63.2, 63.3, 69.3, 69.5, 69.8, 70.2, 70.5-70.6 (signals superimposed), 71.4, 170.5, 172.2, 174.2; m/z HRMS calculated for C33H50O8FeNa (MNa)+ 653.27473, found (ES+) 653.27595. 11-(Ferrocenoylamide-3-oxapentanyloxycarbonylamino)undecanoic acid (17). The reaction was carried out under anhydrous conditions. Compound 5 (50 mg, 0.16 mmol) was dissolved in acetonitrile (10 mL), and triethylamine (45 µL, 0.32 mmol) was added. The solution was cooled to 0 °C, and N,Ndisuccinimidyl carbonate (49 mg, 0.19 mmol) was added. The


solution was stirred at 0 °C for 30 min and at rt for 18 h. The solvent was then evaporated in vacuo and the resulting oil purified by flash silica chromatography (20% hexane in EtOAc) to afford the activated intermediate (64 mg, 87%) as an oil. 1H NMR (300 MHz; CDCl3) 2.76 (s, 4H, succinimide), 3.49 (m, 2H, CH2NH), 3.62-3.74 (m, 4H), 4.11 (s, 5H, C5H5), 4.16 (m, 2H, C5H2), 4.45 (m, 2H, CH2OCO), 4.68 (m, 2H, C5H2), 6.35 (br s, 1H, NH); 13C NMR (CDCl3; 75 MHz) 25.5, 39.4 (CH2N), 68.1, 68.2, 69.7, 69.8, 69.9, 70.2, 70.5, 151.6 (CdO), 168.8 (CdO), 172.5 (CdO amide); m/z (ES+) 481 (MNa+, 100%), 439 (48). The intermediate (50 mg, 0.11mmol) was dissolved in DMF (0.7 mL), and triethylamine (18 µL, 0.13 mmol) was added. To this was added 11-aminoundecanoic acid (22 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 then evaporated in vacuo. The resulting oil was purified by flash silica chromatography (5% methanol in CH2Cl2) to afford 17 (54 mg, 90%) as an orange oil. 1H NMR (300 MHz; CDCl3) 1.23 (m, 12H), 1.41 (m, 2H), 1.59 (m, 2H), 2.32 (t, J ) 7.5 Hz, 2H, CH2CO2H), 3.10 (dt, J ) 4.8 and 4.6 Hz, 2H, (CH2)9CH2NH), 3.56-3.66 (m, 6H), 4.18 (s, 5H, C5H5), 4.21 (m, 2H, CH2O), 4.31 (m, 2H, C5H2), 4.72 (m, 2H, C5H2), 4.90 (br, 1H, NH carbamate), 6.47 (br, 1H, NH amide); 13C NMR (75 MHz; CDCl3) 24.7, 26.6, 28.9, 29.0 (2 signals), 29.1, 29.2, 29.6, 34.0, 39.4, 41.0, 63.6, 68.2, 69.6, 69.7, 69.9, 70.5, 75.5, 156.4 (Cd O carbamate), 171.2 (CdO amide), 177.9 (CdO acid); m/z HRMS calculated for C27H40N2O6Fe M+ 544.22356, found (FAB+) 544.22408. 2-(8-Hydroxy-3,6-dioxaoctanyl)isoindole-1,3-dione (19). The reaction was carried out under anhydrous conditions. 1-Chloro3,6-dioxaoctan-1-ol 18 (5.00 g, 29.7 mmol) and potassium phthalimide (7.14 g, 28.6 mmol) were dissolved in DMF (20 mL) and heated at 40 °C for 4 h. The mixture was extracted with water and CH2Cl2. The organic phase was dried (magnesium sulfate) and evaporated to afford 19 (5.76 g, 70%) as a yellow oil, which was used without further purification. 1H NMR (300 MHz; CDCl3) 3.57 (m, 4H), 3.62 (m, 6H), 3.71 (m, 2H), 7.77 (m, 4H, H-phthalimide); 13C NMR (CDCl3; 75 MHz) 37.1, 62.3 (C-8), 68.9, 71.2, 71.5, 73.7, 124.2, 133.0, 135.4, 169.7 (CdO); m/z HRMS calculated for C14H17NO5Na (MNa)+ 302.09989, found (ES+) 302.09998. 11-(8-[1,3-Dioxo-1,3-dihydroisoindol-2-yl]-3,6-dioxaoctanyloxycarbonylamino)undecanoic acid methyl ester (20). The reaction was carried out under anhydrous conditions. Compound 19 (1.00 g, 3.57 mmol) in acetonitrile (35 mL) was cooled to 0 °C, and N,N-disuccinimidyl carbonate (1.48 g, 4.28 mmol) was added. The solution was stirred at 0 °C for 30 min and at rt for 18 h. The solvent was evaporated in vacuo and the resulting oil purified by flash silica chromatography (20% hexane in EtOAc) to afford NHS activated 19 (910 mg, 61%) as a white solid. 1H NMR (300 MHz; CDCl3) 2.80 (s, 4H, H succinimidyl), 3.59 (m, 4H), 3.67 (m, 4H), 3.85 (t, J ) 5.6 Hz, 2H), 4.32 (m, 2H), 7.67 (m, 2H, H-phthalimide), 7.80 (m, 2H, H-phthalimide); 13C NMR (CDCl ; 75 MHz) 25.3 (CH succinimidyl), 37.0 3 2 (CH2N), 67.6, 68.0, 69.8, 70.4, 71.1, 122.9, 132.5, 133.6 (C aromatic), 151.4 (CdO carbonate), 167.9 (CdO succinimidyl), 168.8 (CdO phthalimide); m/z (ES+) 443 (MNa+, 18%). The subsequent reaction was carried out under anhydrous conditions. 11-Aminoundecanoic acid methyl ester (285 mg, 1.32 mmol) was added to the NHS activated intermediate (556 mg, 1.32 mmol) in CH2Cl2 (30 mL), and the reaction was stirred at rt for 18 h. The solvent was evaporated and the resulting oil purified by flash silica chromatography (6:4 hexane/EtOAc) to afford 20 (228 mg, 34%). 1H NMR (300 MHz; CDCl3) 1.24 (m, 12H), 1.45 (m, 2H), 1.58 (m, 2H), 2.27 (t, J ) 7.5 Hz, 2H, CH2CO2Me), 3.12 (q, J ) 6.7 Hz, 2H, CH2N), 3.59 (m, 6H),


3.63 (s, 3H, O-Me), 3.71 (t, J ) 5.3 Hz, 2H), 3.87 (t, J ) 6.0 Hz, 2H), 4.08 (m, 2H), 7.69 (m, 2H, H-phthalimide), 7.70 (m, 2H, H-phthalimide); 13C NMR (CDCl3; 75 MHz) 24.8, 25.4, 26.5, 26.6, 29.0-29.6 (signals superimposed), 33.8, 36.7, 37.1, 39.4, 40.8, 51.2 (O-Me), 63.4, 67.7, 69.4, 69.8, 70.4, 123.0 (CHAr), 131.8, 133.7 (CH-Ar), 156.2 (CdO carbamate), 168.0 (Cd O phthalimide), 174.0 (CdO ester); m/z (ES+) 543 (MNa+, 40%). 11-(Ferrocenoylamide-3,6-dioxaoctanyloxycarbonylamino)undecanoic acid (21). A solution of 20 (228 mg, 0.44 mmol) and hydrazine hydrate (110 mg, 2.2 mmol) in ethanol (3 mL) was heated at reflux for 2 h. The solvent was evaporated and the resulting product dissolved in CH2Cl2. The precipitate was removed by filtration and the solvent evaporated to afford the amine intermediate as an oil (167 mg, 93%), which was used without further purification. 1H NMR (300 MHz; CDCl3) 1.26 (m, 12H), 1.38 (m, 2H), 1.54 (m, 2H), 2.27 (t, J ) 7.5 Hz, 2H, CH2CO2Me), 3.12 (q, J ) 5.5 Hz, 2H, CH2N), 3.53 (m, 6H), 3.61 (s, 3H, OMe), 3.66 (t, J ) 5.6 Hz, 2H), 3.83 (t, J ) 5.6 Hz, 2H), 4.05 (m, 2H); 13C NMR (CDCl3; 75 MHz) 25.8, 26.9, 29.1-29.9 (signals superimposed), 31.2, 34.1, 38.1, 41.0, 51.5 (OMe), 63.7, 67.9, 69.6, 69.9, 70.5, 156.4 (CdO carbamate), 174.2 (CdO ester); m/z HRMS calculated for C19H39N2O6 (MH)+ 391.28026, found (ES+) 391.28031. The subsequent reaction was carried out under anhydrous conditions. To the amine intermediate (167 mg, 0.41 mmol) and ferrocenecarboxylic acid 2 (190 mg, 0.82 mmol) in CH2Cl2 (20 mL) was added EDCI (85 mg, 0.43 mmol), and the reaction was stirred for 18 h at rt. The solvent was evaporated and the resulting oil purified by flash silica chromatography (20% hexane in EtOAc) to afford the ferrocene-coupled methyl ester (35 mg, 15%) as an orange oil. 1H NMR (300 MHz; CDCl3) 1.25 (m, 12H), 1.39 (m, 2H), 1.49 (m, 2H), 2.28 (t, J ) 7.5 Hz, 2H, CH2CO2Me), 3.14 (q, J ) 5.5 Hz, 2H, CH2N), 3.61 (m, 6H), 3.63 (s, 3H, O-Me), 3.68 (m, 2H), 3.75 (m, 2H), 4.06 (s, 5H, C5H5), 4.14 (m, 2H), 4.22 (m, 2H, C5H2), 4.61 (m, 2H, C5H2), 4.80 (br s, 1H, NH), 6.44 (br s, 1H, NH); 13C NMR (CDCl3; 75 MHz) 24.8, 26.9, 29.2-29.9 (signals superimposed), 30.2, 34.1, 38.1, 40.9, 51.53 (OMe), 63.7, 68.1, 69.7, 67.9, 69.6-70.5, 156.4 (CdO carbamate), 171.4 (CdO amide), 174.2 (CdO ester). The ester (35 mg, 0.06 mmol) was dissolved in dioxane (3 mL), and to the solution was added lithium hydroxide (5 mg, 0.12 mmol) in water (2 mL). The reaction was stirred at rt for 3 h, and the dioxane removed in vacuo. The crude product was dissolved in CH2Cl2 (20 mL), the aqueous layer neutralized, and the organic layer was separated, dried (MgSO4), and concentrated. The resulting oil was purified by flash silica chromatography (100% ethyl acetate to 5% MeOH in EtOAc) to afford 21 (20 mg, 60%) as an orange oil. 1H NMR (300 MHz; CDCl3) 1.29 (m, 12H), 1.41 (m, 2H), 1.54 (m, 2H), 2.22 (t, J ) 7.5 Hz, 2H, CH2CO2H), 3.15 (q, J ) 5.5 Hz, 2H, CH2N), 3.64 (m, 6H), 3.69 (m, 2H), 3.77 (m, 2H), 4.09 (s, 5H, C5H5), 4.16 (m, 2H), 4.28 (m, 2H, C5H2), 4.64 (m, 2H, C5H2), 4.85 (br s, 1H, NH), 6.45 (br s, 1H, NH); 13C NMR (CDCl3; 75 MHz) 24.8, 26.9, 29.1-29.9 (signals superimposed), 30.3, 34.1, 39.1, 41.9, 64.4, 68.1, 69.8-70.2 (signals superimposed), 70.6, 156.9 (CdO carbamate), 171.9 (CdO amide), 174.2 (CdO acid); m/z HRMS calculated for C29H44N2O7Fe M+ 588.24977, found (CI+) 588.25104. 16-(2,5-Dioxopyrrolidin-1-yloxycarbonyloxy)hexadecanoic acid methyl ester (23). The reaction was carried out under anhydrous conditions. To methyl 16-hydroxyhexadecanoate (450 mg, 1.60 mmol) and triethylamine (0.240 mL) and acetonitrile (30 mL) at 0 °C was added N,N-disuccinimidyl carbonate (487 mg, 1.88 mmol). The reaction was stirred at 0 °C for 30 min and at rt for 18 h. The solvent was evaporated and the resulting


oil purified by flash silica chromatography (20% hexane in EtOAc) to afford 23 (590 mg, 85%) as an oil. 1H NMR (300 MHz; CDCl3) 1.19 (m, 22H), 1.71 (m, 2H), 1.68 (m, 2H), 2.23 (t, J ) 7.5 Hz, 2H, CH2CO2Me), 2.77 (s, 4H, CH2 succinimidyl), 3.59 (s, 3H, OMe), 4.25 (t, J ) 6.7 Hz, 2H, CH2O); 13C NMR (75 MHz; CDCl3) 25.4, 25.7, 26.2, 29.5, 29.6-30.1 (signals superimposed), 30.3, 34.0, 34.5, 51.5 (OMe), 68.4, 151.7, 168.4, 175.3 (CdO ester). 16-[8-(2-tert-Butoxycarbonylamino)-3,6-dioxaoctanylaminocarbamoyloxy]hexadecanoic acid methyl ester (24). The reaction was carried out under anhydrous conditions. Compound 7 (415 mg, 1.68 mmol) was added to 23 (590 mg, 1.4 mmol) in CH2Cl2 (30 mL), and the reaction was stirred at rt for 18 h. The solvent was evaporated and the resulting oil purified by flash silica chromatography (6:4, hexane/EtOAc) to afford 24 (311 mg, 42%). 1H NMR (300 MHz; CDCl3) 1.25 (m, 22H), 1.35 (m, 9H, CH3-Boc), 1.58 (m, 4H), 2.26 (t, J ) 7.5 Hz, 2H, CH2CO2Me), 3.28 (m, 2H), 3.33 (m, 2H), 3.57 (m, 8H), 3.62 (s, 3H, O-Me), 4.00 (m, 2H); 13C NMR (CDCl3, 75 MHz) 24.5, 25.5, 28.0, 28.7-29.2 (signals superimposed) 33.6, 40.0, 40.4, 51.0 (OMe), 64.4, 70.1 (signals superimposed), 78.6, 155.6 (Cd O carbamate), 156.4 (CdO carbamate), 173.7 (CdO ester); m/z (ES+) 460 ([MH - Boc]+, 100%). 16-(Ferrocenylamide-3,6-dioxactanylaminocarbamoyloxy)hexadecanoic acid (25). The protected amine 24 (310 mg, 0.58 mmol) in a mixture of CH2Cl2/TFA (3 mL/3 mL) was stirred at rt for 2 h. The solvents were removed in vacuo, and the resulting oil was evaporated several times with methanol and CH2Cl2 to give the amine (250 mg, 99%) as a yellow oil. 1H NMR (300 MHz; CDCl3) 1.27 (m, 22H), 1.59 (m, 4H), 2.35 (t, J ) 7.5 Hz, 2H, CH2CO2Me), 3.21-3.39 (m, 4H), 3.59 (m, 2H), 3.64 (m, 7H), 3.67 (m, 2H), 4.03 (m, 2H); 13C NMR (CDCl3, 75 MHz) 25.1, 25.8, 28.4 , 28.9-29.5 (signals superimposed), 31.7, 34.2, 34.5, 40.1, 51.9 (OMe), 65.3, 66.1, 69.7-69.7 (signals superimposed), 157.8 (CdO carbamate), 174.5 (CdO ester); m/z (ES+) 461 (MH+, 100%), 449 (95). The reaction was carried out under anhydrous conditions. To the amine intermediate (250 mg, 0.54 mmol) and triethylamine (0.113 mL) in CH2Cl2 (10 mL) was added ferrocenecarboxylic acid 2 (186 mg, 0.81 mmol) and DCC (165 mg, 0.81 mmol), and the reaction was stirred for 18 h. After removal of the solvents in vacuo, the resulting oil was purified by flash silica chromatography (100% EtOAc) to afford the ferrocenyl methyl ester intermediate (167 mg, 46%). 1H NMR (300 MHz; CDCl3) 1.20 (m, 22H), 1.53 (m, 4H), 2.32 (t, J ) 7.5 Hz, 2H), 3.33 (m, 2H), 3.63 (m, 10H), 3.65 (s, 3H, OMe), 3.99 (m, 2H), 4.13 (s, 5H, C5H5), 4.26 (m, 2H, C5H2), 4.65 (m, 2H, C5H2), 5.24 (br s, 1H, NH), 6.13 (br s, 1H, NH); 13C NMR (CDCl3, 75 MHz) 24.9, 25.8, 29.0-29.6 (signals superimposed), 34.1, 39.3, 40.7, 51.9 (OMe), 65.1, 68.2, 69.7-70.3 (signals superimposed), 76.1, 156.9 (CdO carbamate), 170.3 (CdO amide), 174.3 (CdO ester). The ferrocene conjugate (167 mg, 0.25 mmol) was dissolved in dioxane (3 mL), and sodium hydroxide (15 mg, 0.38 mmol) in water (2 mL) was added. The reaction was stirred at rt for 3 h, the dioxane was removed in vacuo, and then CH2Cl2 (10 mL) was added. The aqueous layer was neutralized and the organic layer separated, dried (MgSO4), and concentrated. The resulting oil was purified by flash silica chromatography (100% EtOAc to 5% MeOH in EtOAc) to afford 25 (50 mg, 32%) as an orange oil. 1H NMR (300 MHz; CDCl3) 1.23 (m, 22H), 1.55 (m, 4H), 2.22 (t, J ) 7.5 Hz, 2H, CH2CO2H), 3.37 (m, 2H), 3.51-3.65 (m, 10H), 4.01 (m, 2H), 4.17 (s, 5H, C5H5), 4.29 (m, 2H, C5H2), 4.67 (m, 2H, C5H2), 5.31 (br s, 1H, NH), 6.23 (br s, 1H, NH); 13C NMR (CDCl3, 75 MHz) 24.9, 25.9, 29.129.6 (signals superimposed), 34.1, 39.3, 40.8, 65.1, 68.2, 69.770.3 (signals superimposed), 71.5, 156.9 (CdO carbamate),

Tranchant et al.

Figure 2. Cyclic voltammetry of substituted ferrocenes (∼100 µM in PBS). Curves 1,2, background, illustrating the variability between different screen-printed carbon electrodes; 3, compound 17; 4, compound 13.

170.4 (CdO amide), 177.3 (CdO acid); m/z HRMS calculated for C34H54N2O7FeNa (MNa)+ 681.31727, found (ES+) 681.31763. Electrochemical Methods. All measurements were performed with screen-printed electrodes consisting of carbon working (area 0.03 cm2) and counter electrodes and a silver/ silver chloride reference electrode. All electrochemical measurements were performed with an Autolab General Purpose Electrochemical System (Eco Chemie B.V. Netherlands). All signal molecule stock solutions (approximately 100 µM) were dissolved in methanol due to poor solubility at high concentrations in phosphate-buffered saline (PBS: 0.137 M NaCl, 0.0027 M KCl, 0.01 M phosphate buffer pH 7.4). Subsequent dilutions were made with PBS, and the final concentration of methanol was never more than 5% v/v, which was found to have no effect on experiments. All solutions were filtered using a 0.2 µm cutoff filter. 20 µL of solution 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 was used for each measurement. Cyclic voltammetry measurements were made immediately. The potential ramp was digitally generated and therefore in the form of a staircase, with a small step height. Cyclic voltammogram scan parameters were as follows: pretreatment of 0 V potential for 5 s, start potential 0 V, second vertex potential 0.7 V, step potential 0.00198 V, and scan rate 0.05 V/s. All potentials are quoted with respect to Ag/AgCl in PBS. Carbon electrodes prepared by screen printing have a small inherent variability associated with some porosity of the electrodes, that gives rise to a variable background and has some effect upon peak shape in cyclic voltammetry. Typical variability of background caused by electrode variability is illustrated in Figure 2.

RESULTS The nature of linker A was initially explored with a range of spacers (together with either no group or a small group at position B), since this was likely to significantly affect the electrochemical properties of the ferrocene due to their close proximity and potential shielding properties. Three types of linker were synthesized: ester, amide, and amine. The synthesis of ferrocenyl esters has been reported using DCC as a coupling reagent, which was found to be effective unless the alcohol had poor solubility when rearrangement of the O-acyl urea occurred (23, 24). This was not the case with tri(ethylene glycol), which readily reacted with ferrocenecarboxylic acid 2, DCC, and


Design/Synthesis of Ferrocene Probe Molecules Scheme 1. Synthesis of Ferrocene Conjugates with Different Linkers at Aa

a (i) Tri(ethylene glycol), DCC, DMAP, CH2Cl2, 70%; (ii) (CH3CO)2O, pyridine, DMAP, 80%; (iii) 2-(2-aminoethoxy)ethanol, EDCI, HOBt, CH2Cl2, Et3N, 84%; (iv) Boc2O, CH2Cl2, 30%; (v) 2, EDCI, HOBt, CH2Cl2, Et3N, 46%; (vi) TFA, CH2Cl2, 70%; (vii) 2-(2-aminoethoxy)ethanol, NaBH4, 35%; (viii) CH2O, NaBH4, 35%; (ix) tri(ethylene glycol), DCC, DMAP, CH2Cl2, 30%; (x) 2-(2-aminoethoxy)ethanol, EDCI, HOBt, CH2Cl2, Et3N, 50%.

DMAP to give the ester 3 in 70% yield (Scheme 1). As a control, the ferrocene alcohol 3 was converted into the corresponding acetate 4, to assess electrochemical properties compared to other analogues. For the synthesis of the ferrocene amide, commercially available 2-(2-aminoethoxy)ethanol was reacted with ferrocenecarboxylic acid 2 and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI), that has been used in ferrocenecarboxamide synthesis (24), together with HOBt to give 5 in 84% isolated yield. The synthesis of a second amide linker with an alternative spacer possessing a terminal amine group was also investigated. The monoprotection of N,N′-bis(2-hydroxylethyl)ethylenediamine 6 was achieved using Boc anhydride to give 7 (20), which was coupled to ferrocenecarboxylic acid 2 again using EDCI (Scheme 1) in 46% yield. Removal of the Boc group using 50% TFA in CH2Cl2 generated 8 in 70% yield. To synthesize amine-linked conjugates, ferrocenecarboxaldehyde 9 was used. This was coupled to 2-(2-aminoethoxy)ethanol to afford the corresponding imine, which was then reduced in situ using sodium borohydride to the amine 10. The amine 10 was also methylated to give the tertiary amine 11, since this derivative could potentially be used in further coupling reactions to avoid a competing reaction at the nitrogen center. 1,1′-Ferrocenedicarboxylic acid 12 (X ) CO2H, Figure 1) was used to prepare disubstituted ferrocenes to assess the effect of difunctionalization on the electrochemical properties: the diester 13 from tri(ethylene glycol), and DCC and DMAP, and diamide 14 from 2-(2-aminoethoxy)ethanol, EDCI, and HOBt (Scheme 1). From the initial electrochemical data (see below), the esterlinked compound 3, amides 5 and 8, and amine 11 were the most suitable ferrocenyl scaffolds. However, as the amine was generated in generally low yields, only the ester and amides were carried through for further structural elaboration. The nature of the solubilizing spacer together with the linker B were then explored to assess the influence of the spacer and second linker on electrochemical properties. Depending on the types of spacer and linker incorporated, three different functionalized

acids were attached, either 11-aminoundecanoic acid, hexadecanedioic acid, or 16-hydroxyhexadecanoic acid. Initially, a carbamate linkage was synthesized using the ferrocenyl ester with a PEG-3 spacer. Accordingly, alcohol 3 was activated using N,N′-disuccinimidyl carbonate (DSC) to give the corresponding N-hydroxysuccinimidyl ester which was converted into 15 via coupling with 11-aminoundecanoic acid in THF/H2O in 74% isolated yield (Scheme 2). The diester-linked analogue 16 was also prepared from 3 and hexadecanedioic acid using DCC and DMAP in low yield, due to isolation problems and side-product formation; however, sufficient material was prepared for electrochemical analysis. The synthesis of an amide analogue of 15 to establish any benefits of a longer PEG spacer was carried out using an identical method. Compound 5 was activated as the Nhydroxysuccinimidyl ester, which was isolated and coupled with 11-aminoundecanoic acid to afford 17 in 78% yield over the two steps. To evaluate the potential benefits of a longer PEG spacer, compound 21 (an analogue of 17) was also prepared. Synthesis of the spacer with a terminal amine and alcohol was required, and accordingly, 2-[2-(2-chloroethoxy)ethoxy]ethanol 18 was reacted with potassium phthalimide to give 19 in 70% yield. The hydroxyl group was then activated as before using DSC, and the isolated intermediate was coupled with methyl 11aminoundecanoate to generate 20. Removal of the phthalimide group was achieved by treatment with hydrazine in 93% yield, and ferrocenecarboxylic acid 2 was attached to the resulting free amino group using EDCI. Finally, hydrolysis of the methyl ester afforded compound 21. Finally, an analogue of 21 with the carbamate reversed was synthesized. To do this, initially methyl 16-hydroxyhexadecanoate 22 was activated and isolated as the N-hydroxysuccinimidyl ester 23 in 85% yield. The activated alcohol was coupled with the mono-Boc-protected diamine, 7, to give compound 24. The Boc group was removed in quantitative yield using TFA and the resulting amine coupled to ferrocenecarboxylic acid 2, using DCC and triethylamine. Finally, hydrolysis of the methyl ester revealed the reverse carbamate analogue 25 (Scheme 2).


Tranchant et al.

Scheme 2. Synthesis of Ferrocene Conjugates with a Range of Spacers and Linkers at Ba

a (i) DSC, CH3CN, 27%; (ii) 11-aminoundecanoic acid, THF/H2O, 74%; (iii) hexadecanedioic acid, DCC, DMAP, CH2Cl2, 4%; (iv) DSC, CH3CN, 87%; (v) 11-aminoundecanoic acid, DMF/H2O, 90%; (vi) potassium phthalimide, DMF, 70%; (vii) DSC, CH3CN, 61%; (viii) methyl 11-aminoundecanoate, THF/H2O, 34%; (ix) N2H4, EtOH, reflux, 2 h, 93%; (x) 2, EDCI, CH2Cl2, 15%; (xi) LiOH, dioxane/H2O, 60%; (xii) 22, DSC, CH3CN, 85%; (xiii) 7, CH2Cl2, 42%; (xiv) TFA, CH2Cl2, 50:50, 99%; (xv) 2, Et3N, DCC, CH2Cl2, 46%; (xvi) NaOH, dioxane/H2O, 32%.

Table 1. Electrochemical Properties Measured for Ferrocene Compoundsa

compound

A linker

B linker

n

3 4 15 16 5 8 17 21 25 10 11 13 14

ester ester ester ester amide amide amide amide amide sec-amine tert-amine ester amide

H COMe H ester O(CO) H (CH2)2NH2 carbamate O(CO)NH carbamate O(CO)NH carbamate O(CO)NH H H Et Et

3 3 2 3 2 2 2 3 2 2 2 3 2

X

E1/2/ mV Ag,AgCl (PBS)

Ip,c/Ip,a

H H H H H H H H H H H CO2(CH2CH2O)3Et CONH(CH2CH2O)2H

402 402 408 560 349 365 357 370 449 266 258 638 568

0.68 0.71 0.55 nd 0.94 0.80 0.76 0.67b 0.53 0.89 0.80 0 0.28

m

10 14 10 10 15

Ep,a - Ep,c/ mV

Ep,a - Ep,a/2/ mV

81 81 77 nd 82 80 76 98 68 80 100

59 61 58 nd 63 65 58 64 66 66 86 77 66

96

a

Electrochemical data were obtained in PBS on printed carbon electrodes with scan rate 50 mV/s. E1/2 was taken as the mean of the peak potentials for anodic and cathodic peaks or as the half-wave potential where there was no cathodic peak (compound 13). The peak current ratio was determined by the extrapolation method illustrated in ref 29. nd: not determined. b Shoulder at E1/2 ) 250 mV.

The properties of the ferrocene conjugates were then studied using cyclic voltammetry. In blood samples, electrochemical measurements are complicated due to the presence of several electrochemically active compounds including uric acid, glycine, ascorbic acid, and urea (25). These compounds have an oxidation potential of approximately +350 to +550 mV (vs Ag/AgCl reference). The various components of the blood including proteins such as HSA produce a significant electrochemical background signal at approximately +550 mV. For use in binding studies, compounds with reversible voltammograms and an E1/2 of approximately +350 mV are preferred to limit the interference from such an electrochemical background. The range of electrochemical behavior observed is given in Table 1 and illustrated in Figure 2. For some compounds (the

esters and the disubstituted compounds), a second process appearing as a shoulder at lower Ea,p was occasionally observed. The probable explanation, since the appearance of the effect correlated with the time between synthesis and electrochemical measurement (up to several weeks in some cases) is that it was due to ferrocene carboxylic acid formed by hydrolysis of the compound. For comparison, the cyclic voltammetry curve for ferrocenecarboxylic acid (FcCOOH) has a half-wave potential E1/2 of approximately +300 mV (vs Ag/AgCl) (26). A oneelectron reversible electrochemical process would have peak to half-peak and anodic peak to cathodic peak potential differences (Ep,a - Ep,a/2 and Ep,a - Ep,c) of approximately 60 mV, in the absence of effects due to ohmic drop between working and reference electrodes, and would have the peak current ratio Ip,a/Ip,c ) 1. The compounds all essentially satisfied


Figure 3. Variation with half-wave potential, E1/2, of ratio of cathodic to anodic peak current in cyclic voltammetry, Ic,p/Ia,p The points are labeled with the compound number.


ester, then E1/2 increases, with the amide being more effective at stabilizing Fc+ than the ester. The disubstituted ferrocenes are most likely distorted: this was supported by the 13C NMR spectroscopic data with the two carbonyl signals separated by more than 30 ppm. The second effect relates to the way in which long-chain substituents can potentially fold around the ferrocene (5, 6). The effect seems to be determined by the length of the chain and functional group at the linker. Thus, we have identified that, to enable electrochemical measurements to be made in protein solutions, plasma, and blood, an amide at linker A, carbamate at linker B, short PEG unit, and fatty acid moiety are required for use in biomolecular binding studies, to mimic fatty acid binding. Applications of the model compound 17 and analogues as an electrochemical probe of biochemical interaction will be described in a future paper (30).

the criteria for the peak potential differences, but not for the peak current ratio. A satisfactory interpretation is that the oxidized product of the anodic reaction (ferrocenium) decomposes to an electroinactive species at a rate dependent upon the substituents. There was a systematic trend with the nature of linker A and the length of the substituent chain, shown in Figure 3: as E1/2 increased, indicating relative destabilization of the ferrocenium cation, the decomposition rate of ferrocenium, measured by Ip,c/Ip,a, also increased.

ACKNOWLEDGMENT

DISCUSSION

(1) Cais, M., Slovin, E., and Snarsky, L. (1978) Metalloimmunoassay II. Iron-metallohaptens from estrogen steroids. J. Organomet. Chem. 160, 223-230. (2) Weber, S. G., and Purdy, W. C. (1979) Homogeneous voltammetric immunoassay: a preliminary study. Anal. Lett. 12, 1-19. (3) Tiefenauer, L. X., Kossek, S., Padeste, C., Thiebaud, P. (1997) Towards Amperometric Immunosensor Devices. Biosens. Bioelectron. 12, 213-223. (4) Yu, C. J., Wang, H., Wan, Y., Yowanto, H., Lie, J., Tao, C., James, M. D., Tan, C. L., Blackburn, G. F., and Meade, T. J. (2001) 2-Ribose-ferrocene oligonucleotides for electronic detection of nucleic acids. J. Am. Chem. Soc. 123, 11155-11161. (5) Hillier, S. C., Flower, S. E., Frost, C. G., Jenkins, A. T. A., Keay, R., Braven, H., Clarkson, J. (2004) An electrochemical gene detection assay utilizing T7 exonuclease activity on complementary probetarget oligonucleotide sequences. Electrochem. Commun. 6, 12271232. (6) Hillier, S. C., Frost, C. G., Jenkins, A. T. A., Braven, H., Keay, R. W., Flower, S. E., Clarkson, J. M. (2004) An electrochemical study of enzymatic oligonucleotide digestion. Bioelectrochemistry 63, 307-310. (7) Plumb, K., and Kraatz, H.-B. (2003) Interaction of a ferrocenylmodified peptide with papain: toward protein-sensitive electrochemical probes. Bioconjugate Chem. 14, 601-606. (8) Forrow, N. J., Sanghera, G. S., and Walters, S. J. (2002) The influence of structure in the reaction of electrochemically generated ferrocenium derivatives with reduced glucose oxidase. J. Chem. Soc., Dalton Trans. 3187-3194. (9) Di Gleria, K., Hill, H. A. O., McNeil, C. J., and Green, M. J. (1988) Homogeneous ferrocene-mediated amperometric immunoassay. Anal. Chem. 58, 1203-1205. (10) Forrow, N. J., Foulds, N. C., Frew, J. E., and Law, J. T. (2004) Synthesis, characterization, and evaluation of ferrocene-theophylline conjugates for use in electrochemical enzyme immunoassay. Bioconjugate Chem. 15, 137-144. (11) Zatoń, A. M. L., Ferrer, J. M., Ruizde Gorda, J. C., and Marquıń, M. A. (1995) Binding of coumarins to site I of human serum albumin. Effects of fatty acids. Chem.-Biol. Interact. 97, 169-174. (12) Bhattacharya, A. A., Gru¨ne, T., and Curry, S. (2000) Crystallographic analysis reveals common modes of binding medium and long chain fatty acids to human serum albumin. J. Mol. Biol. 303, 721-732. (13) Sacchettini, J. C., Gordon, J. I., and Banaszak, L. J. (1989) Crystal structure of rat intestinal fatty acid binding protein. Refinment and analysis of the Escherichia coli-derived protein with bound palmitate. J. Mol. Biol. 208, 327-339.

Since the carboxylate anion can stabilize ferrocenium more than an amide group, conversion of ferrocene carboxylic acid to an amide or ester should cause a shift to a higher redox potential, as observed. The shift in E1/2 caused by substitution onto the ferrocene correlates with the expected relative ability of the substituent groups to disperse the charge on the ferrocenium cation: amine > amide > ester > diamide > diester. The amide 8, possessing a slightly longer PEG unit than the amide conjugate 5 and a terminal amine group, had a similar E1/2 value, indicating that further substitution should not affect the electrochemical properties significantly. Indeed, previous studies on ferrocenoyl peptides have indicated that the electrochemical behavior was relatively insensitive to the amino acid R group (27). However, there was a discernible effect in each group of compounds of the length of the alkyl chainsthe longer the chain, the lower the ratio Ic,p/Ia,p and, to a degree perhaps dependent on the nature of the second linker, the greater E1/2. These results indicated that when the ferrocenyl esters were linked through either a carbamate or an ester to a fatty moiety, E1/2 increased such that compounds 15 and 16 were unsuitable for application in an electrochemistry in protein solutions, plasma, and blood. In contrast, ferrocenyl amides linked to a fatty moiety through a carbamate unit, compounds 17 and 21, showed E1/2 values close to that observed without the linked fatty acid chain. Interestingly, however, the reverse carbamate 25 had significantly increased E1/2, possibly due to hydrogenbonding effects and folding of the molecule. The best properties were displayed by compounds 17 and 21, but 17 was more synthetically accessible. This was therefore selected as our model compound for further synthetic and electrochemical studies. In summary, two structural characteristics can be identified that determine the reversibility and half-wave potential for oxidation. The first is stabilization of ferrocenium (Fc+) by electron donation from ring substituents. Thus, the lowest E1/2 is found for ferrocene carboxylic acid (stabilization of Fc+ by carboxylate attached directly to the ring) and for amines 10 and 11 where the nitrogen is separated from the ring by a methylene group: ferrocene methanol has a similar E1/2 value (28). If the carboxylic acid is derivatized as the corresponding amide or

We thank Unipath for funding (IT) and for permission to publish this article. Supporting Information Available: NMR spectra of synthesized compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

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Tranchant et al. (24) Schetter, B., and Speiser, B. (2004) Reaction of ferrocenecarboxylic acid with N,N-disubstituted carbodiimides: synthesis, spectroscopic and X-ray crystallographic analysis of N,N′-disubstituted N-ferrocenoylureas and identification of a one-pot coupling reagent for the formation of ferrocenecarboxamides in a non-aqueous solvent. J. Organomet. Chem. 689, 1472-1480. (25) Pravda, M., Junjar, C. M., Iwuoha, E. I., Smyth, M. R., Vytras, K., and Ivaska, A. (1995) Evaluation of amperometeric glucose biosensors based on co-immobilisation of glucose oxidase with an osmium redox polymer in electrochemically generated polyphenol films. Anal. Chim. Acta 304, 127-138. (26) Rossier, J. S., Roberts, M. A., Ferringo, R., and Girault, H. H. (1999) Electrochemical detection in polymer microchannels. Anal. Chem. 71, 4294-4299. (27) Sheehy, M. J., Gallagher, J. F., Yamashita, M., Ida, Y., WhiteColangelo, J., Johnson, J., Orlando, R., and Kenny, P. T. M. (2004) Synthesis and structural characterization of redox-responsive Nferrocenoyl amino acid esters; the X-ray crystal structure of N-ferrocenoyl alanine methyl ester; electrochemical anion recognition and 1H NMR complexation studies. J. Organomet. Chem. 689, 1511-1520. (28) Yoon, H. C., Yang, H., and Byun, S. Y. (2004) Ferritin immunosensing on microfabricated electrodes based on the integration of immunoprecipitation and electrochemical signaling reactions. Anal. Sci. 20, 1249-1253. (29) Southampton Electrochemistry Group (1990) Instrumental methods in electrochemistry, p 182, Ellis Horwood, Chichester, U.K. (30) Tranchant, I., Herve´, A.-C., Carlisle, S., Lowe, P., Slevin, C. J., Forssten, C., Dilleen, J., Tabor, A. B., Williams, D. E., and Hailes, H. C., manuscript in preparation. BC060038M

Design and Synthesis Of Ferrocene Probe Molecules for Detection by

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