1256 Bioconjugate Chem.

2006, 17, 1256−1264

Design and Synthesis Of Ferrocene Probe Molecules for Detection by

Electrochemical Methods
Isabelle Tranchant,‡ Anne-Cécile Hervé,†,§ 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 amide-
linked 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 placed for use in electrochemical sensing systems. More recent

applications include the utilization of ferrocene as an oligo-
Molecular interactions in biological systems have been studied
using many different techniques such as surface plasmon nucleotide label in DNA probes (4-6) and as an electrochemical
resonance and fluorescence-based methodologies. There are, sensor for the detection of proteins (7). Ferrocene immunoassays
however, few reports in the literature utilizing electrochemical with reduced glucose oxidase incorporating enzyme amplifica-
labels to study such interactions, with the exception of antibody- tion (8), an amperometric immunoassay for lidocaine (9), and
antigen interactions, where historically ferrocene-labeled mol- a homogeneous ferrocene-theophylline conjugate electrochemi-
ecules have been used. Metalloimmunoassays were first de- cal assay (10) have also been reported.
scribed in the 1970s when ferrocene was used to label steroid Our aim was to demonstrate the utility of ferrocene for the
antigens with detection using flameless atomic absorption study of biomolecular interactions and its potential in such
spectroscopy (1). Subsequently, an immunoassay incorporating applications. Interactions between fatty acids and their naturally
a ferrocene electrochemical label was reported using a fer- occurring ligands have been intensively studied and thus provide
rocene-morphine conjugate whose electrochemistry was per- a well-characterized system in which to study rationally designed
turbed on binding to an antibody, resulting in a homogeneous synthetic electrochemical probe molecules; to our knowledge,
competitive assay not requiring the separation of a free and no such probes have been reported in the literature. Fatty acid-
antibody-bound label (2). Since then, a range of carbonylmet- human serum albumin (HSA) binding interactions are particu-
alloimmunoassays (CMIA) have been developed using metal- larly important due to the effect on the transport of therapeutic
carbonyl-labeled molecules, in particular, incorporating fer- drugs, for example, the reduced capacity of HSA to bind
rocene, which has been used as an electrochemical tracer and warfarin in the presence of bound fatty acids (11). Therefore,
signaling molecule (3). The range of ferrocene derivatives the synthesis of a series of ferrocene-labeled fatty acids would
available, ease of conjugation to a range of functionalities, be a suitable model system for study but could also provide a
reversible electrochemistry, and ability to synthetically tailor greater understanding of the interactions between fatty acids
redox properties for a particular application makes it ideally and HSA. Conventionally such fatty acid ligand interactions
have been studied by a number of techniques. Bhattacharya et
* Corresponding author. E-mail: h.c.hailes@ucl.ac.uk. Fax: +44 al. have studied complexes of medium- and long-chain fatty
(0)20 7679 7463. acids and HSA using X-ray crystallography, identifying medi-
†
Unipath Ltd. um- and long-chain fatty acid binding sites (12). X-ray crystal-
‡
Current address: Diatos SA, Therapeutic Chemistry Department, lography has also been used to study the complex of intestinal
3-5 Impasse Reille, 75014 Paris, France. fatty acid binding protein and palmitate (13). Acrylodan-
§
Current address: Diatos NV, Brusselsestraat 69, B-3000 Leuven,
Belgium.
modified HSA has been used to study, using fluorescent
|
Current address: Sterling Medical Innovations, Unit 10, Scion techniques, the effect of oleic acid binding to the microenvi-
House, Stirling University Innovation Park, Stirling, FK9 4NF. ronment around residue cysteine-34 in HSA (14). The binding
⊥ characteristics of the fluorescently labeled fatty acid probe 11-
Current address: Department of Chemistry, University of Auck-
land, Private Bag, Auckland, New Zealand. (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 Bioconjugate Chem., Vol. 17, No. 5, 2006 1257

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 chroma-
tography (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,
Figure 1. Ferrocene multifunctional conjugates. 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
hepatica fatty acid binding protein have also been studied (16). (CdO); m/z HRMS calculated for C17H22O5FeNa (MNa)+
In addition, acrylodan-modified intestinal FABP (ADIFAB) has 385.07089, found (ES+) 385.07110.
been used to investigate the binding of a range of fatty acids, Ferrocenoyl-(8-ethanoate)-3,6-dioxaoctanoate (4). The re-
allowing measurement of Kd values by fluorescent measurement action was carried out under anhydrous conditions. To com-
(17). However, such measurements can only normally be pound 2 (100 mg, 0.27 mmol) in CH2Cl2 (5 mL) was added
performed in protein solution or plasma, while electrochemical pyridine (0.25 mL) and DMAP (3 mg, 0.03 mmol). The solution
measurements can potentially be performed in whole blood. was cooled to 0 °C and acetic anhydride (0.032 mL, 0.66 mmol)
In the present study, we have designed and synthesized a added dropwise. After 30 min, the solution was warmed to room
range of electrochemically labeled molecules to establish the temperature (rt) and stirred for 2 h. The solvents were evaporated
key properties required for use in binding studies in the presence and the resulting oil purified by flash silica chromatography
of serum, such as E1/2, reversibility, and aqueous compound (EtOAc/hexane, 7:3) to afford compound 4 (88 mg, 80%) as
solubility. The final component in the molecular design was an orange oil. 1H NMR (300 MHz; CDCl3) 2.03 (s, 3H,
the introduction of a moiety for recognition or binding purposes. COCH3), 3.69 (m, 6H), 3.76 (t, J ) 4.8 Hz, 2H), 4.17 (s, 5H,
In our studies, we conjugated a fatty acid as the specific C5H5), 4.18 (m, 2H), 4.34 (m, 2H), 4.36 (m, 2H, C5H2), 4.79
biorecognition for potential applications in fatty acid binding (m, 2H, C5H2); 13C NMR (75 MHz; CDCl3) 20.8 (COCH3),
studies. However, alternative functionalities can be introduced 63.1, 63.5, 69.1, 69.4, 69.7, 70.1, 70.5, 70.9, 71.2, 170.8 (Cd
depending on the molecular interaction being investigated. O), 171.5 (CdO); m/z HRMS calculated for C19H25O6Fe (MH)+
The molecular conjugates were designed as shown in Figure 404.09168, found (CI+) 404.09263.
1, incorporating ferrocene as the electrochemical detection N-(5-Hydroxy-3-oxapentanyl)ferrocenoylamide (5). The
method and a solubilizing spacer group together with the binding reaction was carried out under anhydrous conditions. Ferrocene
moiety, in these studies a fatty acid. Poly(ethylene glycol) carboxylic acid 2 (3.00 g, 13.0 mmol) and HOBt (1.94 g, 14.3
(PEG)-modified ferrocenes have previously been reported mmol) were dissolved in CH2Cl2 (200 mL), and the reaction
incorporating long polymeric PEG units and have exhibited was stirred at rt for 10 min. EDCI (2.85 g, 13.0 mmol) and
enhanced aqueous solubilities for use in homogeneous redox triethylamine (3.60 mL, 26.0 mmol) were then added, and after
membrane biosensors (18, 19). Commercially available PEG 30 min, 2-(2-aminoethoxy)ethanol (1.37 g, 13.0 mmoles) was
spacers were therefore incorporated as solubilizing moieties. added and the solution stirred at rt for 4 h. The solution was
However, short, defined PEG units were used to ensure that concentrated and purified by flash silica chromatography
the spacer group did not interfere with any binding or recogni- (gradient: 100% EtOAc to 15% methanol in EtOAc) to afford
tion properties. Also, two linkers, A and B, were introduced to the title compound (3.47 g, 84%) as an orange oil. 1H NMR
enable the rapid synthesis of a range of analogues that could (300 MHz; CDCl3) 2.90 (br, 1H, OH), 3.44 (td, J ) 5.2 and
be modified to alter molecular properties and stabilities. The 5.2 Hz, 2H), 3.51 (m, 4H), 3.64 (t, J ) 4.5 Hz, 2H), 4.07 (s,
ferrocene conjugate selected (1) is shown in Figure 1 with 5H, C5H5), 4.17 (m, 2H, C5H2), 4.63 (m, 2H, C5H2), 6.58 (br s,
linkers A and B, PEG repeat unit n, the option of a second 1H, NH); 13C NMR (75 MHz; CDCl3) 39.2 (CH2N), 61.3, 68.0,
functionality on ferrocene (X), and fatty acid pendant group 69.4, 69.9-70.0 (signals superimposed), 72.2, 170.6 (CdO);
containing a methylene chain of length m. m/z HRMS calculated for C15H19NO3FeNa (MNa)+ 340.06066,
found (ES+) 340.06126.
EXPERIMENTAL PROCEDURES N-(8-Amino-3,6-dioxaoctanyl)ferrocenoylamide (8). The
Materials. Unless otherwise noted, solvents and reagents reaction was carried out under anhydrous conditions. DCC (88
were reagent grade from commercial suppliers and used without mg, 0.43 mmol) was added to compound 7 (95 mg, 0.39 mmol)
further purification. THF was dried by distillation from a and ferrocene carboxylic acid (80 mg, 0.35 mmol) in CH2Cl2
sodium/benzophenone suspension under a dry N2 atmosphere. (20 mL), and the reaction was stirred for 18 h at rt. The solvent
CH2Cl2 was dried by distillation from CaH2 under a dry N2 was evaporated in vacuo and the resulting oil purified by flash
atmosphere. All moisture-sensitive reactions were performed silica chromatography (100% EtOAc) to afford the Boc-
under a nitrogen atmosphere using oven-dried glassware. protected intermediate (82 mg, 46%) as an orange oil. 1H NMR
Reactions were monitored by TLC on Kieselgel 60 F254 plates (300 MHz; CDCl3) 1.36 (s, 9H, 3 × CH3), 3.24 (td, J ) 5.2
with detection by UV or permanganate, ninhydrin (for ureas), and 5.1 Hz, 2H), 3.47 (m, 4H), 3.51 (m, 6H), 4.11 (s, 5H, C5H5),
and phosphomolybdic acid stains. Flash column chromatography 4.24 (m, 2H, C5H2), 4.62 (m, 2H, C5H2), 4.98 (br s, 1H, NH),
was carried out using silica gel (particle size 40-63 µm). 6.24 (br s, 1H, NH); 13C NMR (75 MHz; CDCl3) 28.4 (Boc-
Melting points are uncorrected. 1H NMR and 13C NMR spectra CH3), 39.3 (CH2N), 40.3 (CH2N), 68.2, 69.8, 70.2-70.4 (signals
were recorded in CDCl3 at the field indicated. 1-tButyloxycar- superimposed), 79.3 (Boc-C), 156.0 (CdO Boc), 170.4 (CdO
bonyl-1,8-diamino-3,6-dioxaoctane 7 (20), methyl 16-hydroxy- amide).
hexadecanoate (21), and methyl 11-aminoundecanoate (22) were The protected amine (82 mg, 0.18 mmol) was dissolved in a
prepared as previously reported. mixture of CH2Cl2/TFA (4 mL/4 mL), and the solution was
Ferrocenoyl-8-hydroxy-3,6-dioxaoctanoate (3). The reac- stirred at rt for 2 h. The solvents were evaporated and the
tion was carried out under anhydrous conditions. To a solution resulting oil purified by flash silica chromatography (CH2Cl2/
of tri(ethylene glycol) (3.26 g, 21.7 mmol) in CH2Cl2 (50 mL) MeOH/NEt3; 85/10/5) to afford 8 (45 mg, 70%) as an orange
was added a solution of ferrocenecarboxylic acid 2 (0.500 g, oil. 1H NMR (300 MHz; CDCl3/CD3OD) 3.09 (m, 2H), 3.28
2.17 mmol) in CH2Cl2 (10 mL). After 15 min, DCC (0.525 g, (td, J ) 5.3 and 5.3 Hz, 2H), 3.43 (m, 8H), 3.95 (s, 5H, C5H5),
1258 Bioconjugate Chem., Vol. 17, No. 5, 2006 Tranchant et al.

4.14 (m, 2H, C5H2), 4.56 (m, 2H, C5H2), 6.70 (br s, 1H, NH); Cl2 (5 mL) was then added and the reaction stirred for 18 h at
13C NMR (75 MHz; CDCl ) 39.5 (CH N), 39.8 (CH N), 68.4, rt. The organic phase was washed with water (5 mL), dried
3 2 2
69.9, 70.1-70.9 (signals superimposed), 75.5, 172.5 (CdO); (sodium sulfate), and the solvent removed under reduced
m/z HRMS calculated for C17H25N2O3Fe (MH)+ 361.12091, pressure. The residue was purified by flash silica chromatog-
found (ES+) 361.12111; m/z (ES+) 383 (MNa+, 41%), 361 raphy (gradient: 100% hexane to 100% EtOAc, then CH2Cl2/
(MH+, 100). MeOH 95/5) to give 14 (107 mg, 50%) as an orange oil. 1H
Ferrocenylmethylamine N-(5-hydroxy-3-oxapentanyl) (10). NMR (300 MHz; CDCl3) 3.62-3.75 (m, 16H), 4.35 (m, 4H, 2
The reaction was carried out under anhydrous conditions. × C5H2), 4.57 (m, 4H, 2 × C5H2), 7.32 (br, 2H, NH); 13C NMR
Ferrocene carboxaldehyde 9 (150 mg, 0.70 mmol) and 2-(2- (75 MHz; CDCl3) 39.8, 61.6, 69.9, 70.9, 71.3, 72.5, 78.0, 170.9,
aminoethoxy)ethanol (147 mg, 1.40 mmol) in toluene (30 mL) 212.0 (CdO); m/z (ES+) 471 (MNa+, 100%), 449 (MH+, 25).
were heated at reflux for 18 h. After cooling to rt, sodium 11-(Ferrocenoyl-3,6-dioxaoctanyloxycarbonylamino)-
borohydride (79.8 mg, 2.10 mmol) was added, and the reaction undecanoic acid (15). The reaction was carried out under
was stirred at rt for 6 h. Water (5 mL) was added, the solvent anhydrous conditions. To compound 3 (680 mg, 1.87 mmol) in
removed in vacuo, and the residue suspended in CH2Cl2. The acetonitrile (20 mL) at 0 °C was added N,N-disuccinimidyl
organic phase was washed with water (10 mL), dried (sodium carbonate (574 mg, 2.24 mmol). The solution was stirred at 0
sulfate), and evaporated. The residue was purified by flash silica °C for 30 min and at rt for 18 h. The solvent was then evaporated
chromatography (gradient: 100% EtOAc to 20% methanol in and the resulting oil purified by flash silica chromatography
EtOAc) to give 10 (74 mg, 35%) as a dark orange oil. 1H NMR (hexane/EtOAc, 1:4) to afford the succinimidyl carbonate
(300 MHz; CDCl3) 2.83 (t, J ) 5.3 Hz, 2H, CH2CH2N), 3.53 activated 3 (254 mg, 27%) as an orange oil. 1H NMR (300 MHz;
(s, 2H, CH2-ferrocenyl), 3.57-3.64 (m, 4H), 3.70 (t, J ) 4.4 CDCl3) 2.83 (s, 4H, CH2 succinimidyl), 3.69 (m, 4H), 3.78 (m,
Hz, 2H), 4.10 (m, 2H, C5H2), 4.12 (s, 5H, C5H5), 4.18 (m, 2H, 4H), 4.22 (s, 5H, C5H5), 4.40 (m, 4H), 4.45 (m, 2H), 4.82 (s,
C5H2); 13C NMR (75 MHz; CDCl3) 48.9 (C-1), 61.9 (C-5), 67.9, 2H, C5H2); 13C NMR (75 MHz; CDCl3) 25.4 (CH2-succinim-
68.4, 68.5, 70.1, 72.4, 86.3; m/z (ES+) 326 (MNa+, 91%), 304 idyl), 63.2, 68.4, 69.5, 69.8, 70.1, 70.6-70.9 (signals superim-
(MH+, 100). posed), 71.3, 152.3, 162.4, 168.9 (CdO ester); m/z (ES+) 526
Ferrocenylmethylamine N-methyl-N-(5-hydroxy-3-oxap- (MNa+, 46%), 429 (100).
entanyl) (11). The reaction was carried out under anhydrous To 11-aminoundecanoic acid (12 mg, 0.06 mmol) in THF/
conditions. To a solution of 10 (70 mg, 0.23 mmol) in CH3CN H2O (0.5 mL/1 mL) was added the succinimidyl carbonate
(30 mL) was added formaldehyde (37%, 38 µL, 0.46 mmol). activated 3 (15 mg, 0.03 mmol) in THF (0.5 mL). The reaction
After stirring for 10 min, sodium borohydride (26 mg, 0.69 was stirred at rt for 18 h, then the solvents were evaporated
mmol) was added in portions, and the mixture was stirred for under reduced pressure. The remaining oil was purified by flash
5 h. Water (5 mL) was added, the solvent removed in vacuo, silica chromatography (MeOH/CH2Cl2, 5/95) in to afford 15
and the residue was suspended in CH2Cl2. The organic layer (13 mg, 74%) as an orange oil. 1H NMR (300 MHz; CDCl3)
was washed with water (10 mL), dried (sodium sulfate), and 1.27 (m, 12H), 1.46 (quint, J ) 6.8 Hz, 2H), 1.62 (quint, 2H,
evaporated, and the residue was purified by flash silica J ) 6.8 Hz), 2.33 (t, J ) 7.2 Hz, 2H, CH2CO2H), 3.13 (dt, J )
chromatography (gradient: 100% EtOAc to 20% methanol in 6.6 and 6.6 Hz, 2H, CH2N), 3.68 (m, 6H), 3.79 (t, J ) 4.8 Hz,
EtOAc) to give 11 (26 mg, 35%) as an orange oil. 1H NMR 2H), 4.19 (m, 2H), 4.20 (s, 5H, C5H5), 4.38 (m, 2H, C5H2),
(300 MHz; CDCl3) 2.21 (s, 3H, Me), 2.55 (t, J ) 5.6 Hz, 2H, 4.39 (m, 2H), 4.79 (m, 2H, C5H2), 4.81 (br, 1H, NH); 13C NMR
CH2CH2N), 3.47 (s, 2H, CH2-ferrocenyl), 3.58 (m, 4H), 3.68 (75 MHz; CDCl3/CD3OD) 24.8, 26.6, 28.9, 29.1-29.7 (signals
(t, J ) 4.5 Hz, 2H), 4.11 (m, 7H, C5H5, C5H2), 4.18 (m, 2H, superimposed), 34.2, 40.8, 63.7, 68.1, 69.3-70.6 (signals
C5H2); 13C NMR (75 MHz; CDCl3) 41.2 (NMe), 55.6, 57.2, superimposed), 75.4, 156.8 (CdO carbamate), 171.7, 177.9; m/z
62.0, 67.8, 68.3, 68.5, 70.4, 72.6; m/z (ES+) 340 (MNa+, 100%), HRMS calculated for C29H43NO8FeNa (MNa)+ 612.22303,
318 (MH+, 18). found (ES+) 612.22331.
1,1′-Ferrocenoyl-(8-ethoxy-3,6-dioxaoctanoate) (13). The 16-(Ferrocenoyl-3,6-dioxaoctanoyl)hexadecanoic acid (16).
reaction was carried out under anhydrous conditions. DCC (1.19 The reaction was carried out under anhydrous conditions. DCC
mmol, 245 mg) and DMAP (5 mg), were added to a solution (89 mg, 0.435 mmol) was added to a stirred solution of
of 1,1′-ferrocenedicarboxylic acid 12 (0.54 mmol, 150 mg) and hexadecanedioic acid (230 mg, 0.829 mmol) in CH2Cl2 (20 mL),
tri(ethylene glycol) monoethyl ether (2.16 mmol, 385 mg) in and the reaction was stirred at rt for 2 h. To this was added 3
CH2Cl2 (30 mL). The reaction was stirred at rt for 18 h. After (150 mg, 0.414 mmol), and DMAP (50 mg, 0.041 mol), and
filtration, the filtrate was concentrated in vacuo, dissolved in the reaction was stirred at rt overnight. Insoluble urea was
Et2O, and urea was removed by filtration. The solvent was filtered from the reaction and washed with CH2Cl2 (10 mL),
removed under reduced pressure and the residue purified by and the combined organic extracts were removed under reduced
flash silica chromatography (EtOAc/hexane: 60/40) to give 13 pressure, leaving a brown viscous oil. The remaining oil was
(96 mg, 30%) as an orange oil. 1H NMR (300 MHz; CDCl3) purified by flash silica chromatography (100% EtOAc) to afford
1.22 (t, J ) 6.9 Hz, 6H, 2 × CH3), 3.54 (q, J ) 6.9 Hz, 4H, 2 the title compound (20 mg, 4%) as an orange oil. 1H NMR (300
× CH2CH3), 3.55-3.70 (m, 16H), 3.79 (m, 4H), 4.38 (m, 4H, MHz; CDCl3) 1.25 (m, 20H), 1.59 (m, 4H), 2.27 (m, 4H), 3.68
CH2OCO), 4.42 (m, 4H, 2 × C5H2), 4.86 (m, 4H, 2 × C5H2); (m, 6H), 3.77 (t, J ) 4.8 Hz, 2H), 4.22 (m, 7H), 4.38 (m, 4H),
13C NMR (75 MHz; CDCl ) 15.2 (CH ), 63.5, 66.7, 69.4, 69.9, 4.81 (m, 2H, C5H2); 13C NMR (75 MHz; CDCl3) 24.7, 29.2,
3 3
70.7, 70.8, 71.7, 72.6, 73.1, 170.4, 212.1 (CdO); m/z (ES+) 29.3, 29.5, 29.6, 29.8, 30.0-30.1 (signals superimposed), 34.2,
617 (MNa+, 100%), 433 (28). 34.6, 63.2, 63.3, 69.3, 69.5, 69.8, 70.2, 70.5-70.6 (signals
N-(5-Hydroxy-3-oxapentanyl)-1,1′-ferrocenoylamide (14). superimposed), 71.4, 170.5, 172.2, 174.2; m/z HRMS calculated
The reaction was carried out under anhydrous conditions. for C33H50O8FeNa (MNa)+ 653.27473, found (ES+) 653.27595.
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.20 mmol, 11-(Ferrocenoylamide-3-oxapentanyloxycarbonylamino)-
420 mg) (which had been previously neutralized with 2.20 mmol undecanoic acid (17). The reaction was carried out under
Et3N) and HOBt (2.20 mmol, 295 mg) were added to a solution anhydrous conditions. Compound 5 (50 mg, 0.16 mmol) was
of 1,1′-ferrocenedicarboxylic acid 12 (0.54 mmol, 150 mg) in dissolved in acetonitrile (10 mL), and triethylamine (45 µL, 0.32
CH2Cl2 (20 mL), and the mixture was stirred for 30 min. A mmol) was added. The solution was cooled to 0 °C, and N,N-
solution of aminoethoxyethanol (2.16 mmol, 227 mg) in CH2- disuccinimidyl carbonate (49 mg, 0.19 mmol) was added. The
Design/Synthesis of Ferrocene Probe Molecules Bioconjugate Chem., Vol. 17, No. 5, 2006 1259

solution was stirred at 0 °C for 30 min and at rt for 18 h. The 3.63 (s, 3H, O-Me), 3.71 (t, J ) 5.3 Hz, 2H), 3.87 (t, J ) 6.0
solvent was then evaporated in vacuo and the resulting oil Hz, 2H), 4.08 (m, 2H), 7.69 (m, 2H, H-phthalimide), 7.70 (m,
purified by flash silica chromatography (20% hexane in EtOAc) 2H, H-phthalimide); 13C NMR (CDCl3; 75 MHz) 24.8, 25.4,
to afford the activated intermediate (64 mg, 87%) as an oil. 1H 26.5, 26.6, 29.0-29.6 (signals superimposed), 33.8, 36.7, 37.1,
NMR (300 MHz; CDCl3) 2.76 (s, 4H, succinimide), 3.49 (m, 39.4, 40.8, 51.2 (O-Me), 63.4, 67.7, 69.4, 69.8, 70.4, 123.0 (CH-
2H, CH2NH), 3.62-3.74 (m, 4H), 4.11 (s, 5H, C5H5), 4.16 (m, Ar), 131.8, 133.7 (CH-Ar), 156.2 (CdO carbamate), 168.0 (Cd
2H, C5H2), 4.45 (m, 2H, CH2OCO), 4.68 (m, 2H, C5H2), 6.35 O phthalimide), 174.0 (CdO ester); m/z (ES+) 543 (MNa+,
(br s, 1H, NH); 13C NMR (CDCl3; 75 MHz) 25.5, 39.4 (CH2N), 40%).
68.1, 68.2, 69.7, 69.8, 69.9, 70.2, 70.5, 151.6 (CdO), 168.8 11-(Ferrocenoylamide-3,6-dioxaoctanyloxycarbonylamino)-
(CdO), 172.5 (CdO amide); m/z (ES+) 481 (MNa+, 100%), undecanoic acid (21). A solution of 20 (228 mg, 0.44 mmol)
439 (48). and hydrazine hydrate (110 mg, 2.2 mmol) in ethanol (3 mL)
The intermediate (50 mg, 0.11mmol) was dissolved in DMF was heated at reflux for 2 h. The solvent was evaporated and
(0.7 mL), and triethylamine (18 µL, 0.13 mmol) was added. the resulting product dissolved in CH2Cl2. The precipitate was
To this was added 11-aminoundecanoic acid (22 mg, 0.11 mmol) removed by filtration and the solvent evaporated to afford the
dissolved in a mixture of water/DMF (1.1 mL/0.4 mL). The amine intermediate as an oil (167 mg, 93%), which was used
reaction was stirred at rt for 18 h, and the solvents then without further purification. 1H NMR (300 MHz; CDCl3) 1.26
evaporated in vacuo. The resulting oil was purified by flash (m, 12H), 1.38 (m, 2H), 1.54 (m, 2H), 2.27 (t, J ) 7.5 Hz, 2H,
silica chromatography (5% methanol in CH2Cl2) to afford 17 CH2CO2Me), 3.12 (q, J ) 5.5 Hz, 2H, CH2N), 3.53 (m, 6H),
(54 mg, 90%) as an orange oil. 1H NMR (300 MHz; CDCl3) 3.61 (s, 3H, OMe), 3.66 (t, J ) 5.6 Hz, 2H), 3.83 (t, J ) 5.6
1.23 (m, 12H), 1.41 (m, 2H), 1.59 (m, 2H), 2.32 (t, J ) 7.5 Hz, Hz, 2H), 4.05 (m, 2H); 13C NMR (CDCl3; 75 MHz) 25.8, 26.9,
2H, CH2CO2H), 3.10 (dt, J ) 4.8 and 4.6 Hz, 2H, (CH2)9CH2- 29.1-29.9 (signals superimposed), 31.2, 34.1, 38.1, 41.0, 51.5
NH), 3.56-3.66 (m, 6H), 4.18 (s, 5H, C5H5), 4.21 (m, 2H, (OMe), 63.7, 67.9, 69.6, 69.9, 70.5, 156.4 (CdO carbamate),
CH2O), 4.31 (m, 2H, C5H2), 4.72 (m, 2H, C5H2), 4.90 (br, 1H, 174.2 (CdO ester); m/z HRMS calculated for C19H39N2O6
NH carbamate), 6.47 (br, 1H, NH amide); 13C NMR (75 MHz; (MH)+ 391.28026, found (ES+) 391.28031.
CDCl3) 24.7, 26.6, 28.9, 29.0 (2 signals), 29.1, 29.2, 29.6, 34.0, The subsequent reaction was carried out under anhydrous
39.4, 41.0, 63.6, 68.2, 69.6, 69.7, 69.9, 70.5, 75.5, 156.4 (Cd conditions. To the amine intermediate (167 mg, 0.41 mmol)
O carbamate), 171.2 (CdO amide), 177.9 (CdO acid); m/z and ferrocenecarboxylic acid 2 (190 mg, 0.82 mmol) in CH2-
HRMS calculated for C27H40N2O6Fe M+ 544.22356, found Cl2 (20 mL) was added EDCI (85 mg, 0.43 mmol), and the
(FAB+) 544.22408. reaction was stirred for 18 h at rt. The solvent was evaporated
2-(8-Hydroxy-3,6-dioxaoctanyl)isoindole-1,3-dione (19). The and the resulting oil purified by flash silica chromatography
reaction was carried out under anhydrous conditions. 1-Chloro- (20% hexane in EtOAc) to afford the ferrocene-coupled methyl
3,6-dioxaoctan-1-ol 18 (5.00 g, 29.7 mmol) and potassium ester (35 mg, 15%) as an orange oil. 1H NMR (300 MHz;
phthalimide (7.14 g, 28.6 mmol) were dissolved in DMF (20 CDCl3) 1.25 (m, 12H), 1.39 (m, 2H), 1.49 (m, 2H), 2.28 (t, J
mL) and heated at 40 °C for 4 h. The mixture was extracted ) 7.5 Hz, 2H, CH2CO2Me), 3.14 (q, J ) 5.5 Hz, 2H, CH2N),
with water and CH2Cl2. The organic phase was dried (magne- 3.61 (m, 6H), 3.63 (s, 3H, O-Me), 3.68 (m, 2H), 3.75 (m, 2H),
sium sulfate) and evaporated to afford 19 (5.76 g, 70%) as a 4.06 (s, 5H, C5H5), 4.14 (m, 2H), 4.22 (m, 2H, C5H2), 4.61 (m,
yellow oil, which was used without further purification. 1H NMR 2H, C5H2), 4.80 (br s, 1H, NH), 6.44 (br s, 1H, NH); 13C NMR
(300 MHz; CDCl3) 3.57 (m, 4H), 3.62 (m, 6H), 3.71 (m, 2H), (CDCl3; 75 MHz) 24.8, 26.9, 29.2-29.9 (signals superimposed),
7.77 (m, 4H, H-phthalimide); 13C NMR (CDCl3; 75 MHz) 37.1, 30.2, 34.1, 38.1, 40.9, 51.53 (OMe), 63.7, 68.1, 69.7, 67.9,
62.3 (C-8), 68.9, 71.2, 71.5, 73.7, 124.2, 133.0, 135.4, 169.7 69.6-70.5, 156.4 (CdO carbamate), 171.4 (CdO amide), 174.2
(CdO); m/z HRMS calculated for C14H17NO5Na (MNa)+ (CdO ester).
302.09989, found (ES+) 302.09998. The ester (35 mg, 0.06 mmol) was dissolved in dioxane (3
11-(8-[1,3-Dioxo-1,3-dihydroisoindol-2-yl]-3,6-dioxaocta- mL), and to the solution was added lithium hydroxide (5 mg,
nyloxycarbonylamino)undecanoic acid methyl ester (20). The 0.12 mmol) in water (2 mL). The reaction was stirred at rt for
reaction was carried out under anhydrous conditions. Compound 3 h, and the dioxane removed in vacuo. The crude product was
19 (1.00 g, 3.57 mmol) in acetonitrile (35 mL) was cooled to 0 dissolved in CH2Cl2 (20 mL), the aqueous layer neutralized,
°C, and N,N-disuccinimidyl carbonate (1.48 g, 4.28 mmol) was and the organic layer was separated, dried (MgSO4), and
added. The solution was stirred at 0 °C for 30 min and at rt for concentrated. The resulting oil was purified by flash silica
18 h. The solvent was evaporated in vacuo and the resulting chromatography (100% ethyl acetate to 5% MeOH in EtOAc)
oil purified by flash silica chromatography (20% hexane in to afford 21 (20 mg, 60%) as an orange oil. 1H NMR (300 MHz;
EtOAc) to afford NHS activated 19 (910 mg, 61%) as a white CDCl3) 1.29 (m, 12H), 1.41 (m, 2H), 1.54 (m, 2H), 2.22 (t, J
solid. 1H NMR (300 MHz; CDCl3) 2.80 (s, 4H, H succinimidyl), ) 7.5 Hz, 2H, CH2CO2H), 3.15 (q, J ) 5.5 Hz, 2H, CH2N),
3.59 (m, 4H), 3.67 (m, 4H), 3.85 (t, J ) 5.6 Hz, 2H), 4.32 (m, 3.64 (m, 6H), 3.69 (m, 2H), 3.77 (m, 2H), 4.09 (s, 5H, C5H5),
2H), 7.67 (m, 2H, H-phthalimide), 7.80 (m, 2H, H-phthalimide); 4.16 (m, 2H), 4.28 (m, 2H, C5H2), 4.64 (m, 2H, C5H2), 4.85
13C NMR (CDCl ; 75 MHz) 25.3 (CH succinimidyl), 37.0 (br s, 1H, NH), 6.45 (br s, 1H, NH); 13C NMR (CDCl3; 75
3 2
(CH2N), 67.6, 68.0, 69.8, 70.4, 71.1, 122.9, 132.5, 133.6 (C MHz) 24.8, 26.9, 29.1-29.9 (signals superimposed), 30.3, 34.1,
aromatic), 151.4 (CdO carbonate), 167.9 (CdO succinimidyl), 39.1, 41.9, 64.4, 68.1, 69.8-70.2 (signals superimposed), 70.6,
168.8 (CdO phthalimide); m/z (ES+) 443 (MNa+, 18%). 156.9 (CdO carbamate), 171.9 (CdO amide), 174.2 (CdO
The subsequent reaction was carried out under anhydrous acid); m/z HRMS calculated for C29H44N2O7Fe M+ 588.24977,
conditions. 11-Aminoundecanoic acid methyl ester (285 mg, found (CI+) 588.25104.
1.32 mmol) was added to the NHS activated intermediate (556 16-(2,5-Dioxopyrrolidin-1-yloxycarbonyloxy)hexadecano-
mg, 1.32 mmol) in CH2Cl2 (30 mL), and the reaction was stirred ic acid methyl ester (23). The reaction was carried out under
at rt for 18 h. The solvent was evaporated and the resulting oil anhydrous conditions. To methyl 16-hydroxyhexadecanoate (450
purified by flash silica chromatography (6:4 hexane/EtOAc) to mg, 1.60 mmol) and triethylamine (0.240 mL) and acetonitrile
afford 20 (228 mg, 34%). 1H NMR (300 MHz; CDCl3) 1.24 (30 mL) at 0 °C was added N,N-disuccinimidyl carbonate (487
(m, 12H), 1.45 (m, 2H), 1.58 (m, 2H), 2.27 (t, J ) 7.5 Hz, 2H, mg, 1.88 mmol). The reaction was stirred at 0 °C for 30 min
CH2CO2Me), 3.12 (q, J ) 6.7 Hz, 2H, CH2N), 3.59 (m, 6H), and at rt for 18 h. The solvent was evaporated and the resulting
1260 Bioconjugate Chem., Vol. 17, No. 5, 2006 Tranchant et al.

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-dioxaoctanylami-
nocarbamoyloxy]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, Figure 2. Cyclic voltammetry of substituted ferrocenes (∼100 µM in
CH2CO2Me), 3.28 (m, 2H), 3.33 (m, 2H), 3.57 (m, 8H), 3.62 PBS). Curves 1,2, background, illustrating the variability between
(s, 3H, O-Me), 4.00 (m, 2H); 13C NMR (CDCl3, 75 MHz) 24.5, different screen-printed carbon electrodes; 3, compound 17; 4, com-
pound 13.
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 170.4 (CdO amide), 177.3 (CdO acid); m/z HRMS calcu-
O carbamate), 156.4 (CdO carbamate), 173.7 (CdO ester); m/z lated for C34H54N2O7FeNa (MNa)+ 681.31727, found (ES+)
(ES+) 460 ([MH - Boc]+, 100%). 681.31763.
16-(Ferrocenylamide-3,6-dioxactanylaminocarbamoyloxy- Electrochemical Methods. All measurements were per-
)hexadecanoic acid (25). The protected amine 24 (310 mg, 0.58 formed with screen-printed electrodes consisting of carbon
mmol) in a mixture of CH2Cl2/TFA (3 mL/3 mL) was stirred working (area 0.03 cm2) and counter electrodes and a silver/
at rt for 2 h. The solvents were removed in vacuo, and the silver chloride reference electrode. All electrochemical measure-
resulting oil was evaporated several times with methanol and ments were performed with an Autolab General Purpose
CH2Cl2 to give the amine (250 mg, 99%) as a yellow oil. 1H Electrochemical System (Eco Chemie B.V. Netherlands).
NMR (300 MHz; CDCl3) 1.27 (m, 22H), 1.59 (m, 4H), 2.35 (t, All signal molecule stock solutions (approximately 100 µM)
J ) 7.5 Hz, 2H, CH2CO2Me), 3.21-3.39 (m, 4H), 3.59 (m, were dissolved in methanol due to poor solubility at high
2H), 3.64 (m, 7H), 3.67 (m, 2H), 4.03 (m, 2H); 13C NMR concentrations in phosphate-buffered saline (PBS: 0.137 M
(CDCl3, 75 MHz) 25.1, 25.8, 28.4 , 28.9-29.5 (signals NaCl, 0.0027 M KCl, 0.01 M phosphate buffer pH 7.4).
superimposed), 31.7, 34.2, 34.5, 40.1, 51.9 (OMe), 65.3, 66.1, Subsequent dilutions were made with PBS, and the final
69.7-69.7 (signals superimposed), 157.8 (CdO carbamate), concentration of methanol was never more than 5% v/v, which
174.5 (CdO ester); m/z (ES+) 461 (MH+, 100%), 449 (95). was found to have no effect on experiments. All solutions were
The reaction was carried out under anhydrous conditions. To filtered using a 0.2 µm cutoff filter.
the amine intermediate (250 mg, 0.54 mmol) and triethylamine 20 µL of solution was applied to the sample/working area of
(0.113 mL) in CH2Cl2 (10 mL) was added ferrocenecarboxylic the electrode ensuring complete coverage of the working,
acid 2 (186 mg, 0.81 mmol) and DCC (165 mg, 0.81 mmol), counter, and reference electrodes. All samples were premixed
and the reaction was stirred for 18 h. After removal of the before being applied to the electrode, and a new electrode was
solvents in vacuo, the resulting oil was purified by flash silica used for each measurement. Cyclic voltammetry measurements
chromatography (100% EtOAc) to afford the ferrocenyl methyl were made immediately. The potential ramp was digitally
ester intermediate (167 mg, 46%). 1H NMR (300 MHz; CDCl3) generated and therefore in the form of a staircase, with a small
1.20 (m, 22H), 1.53 (m, 4H), 2.32 (t, J ) 7.5 Hz, 2H), 3.33 (m, step height. Cyclic voltammogram scan parameters were as
2H), 3.63 (m, 10H), 3.65 (s, 3H, OMe), 3.99 (m, 2H), 4.13 (s, follows: pretreatment of 0 V potential for 5 s, start potential 0
5H, C5H5), 4.26 (m, 2H, C5H2), 4.65 (m, 2H, C5H2), 5.24 (br s, V, second vertex potential 0.7 V, step potential 0.00198 V, and
1H, NH), 6.13 (br s, 1H, NH); 13C NMR (CDCl3, 75 MHz) scan rate 0.05 V/s. All potentials are quoted with respect to
24.9, 25.8, 29.0-29.6 (signals superimposed), 34.1, 39.3, 40.7, Ag/AgCl in PBS. Carbon electrodes prepared by screen printing
51.9 (OMe), 65.1, 68.2, 69.7-70.3 (signals superimposed), 76.1, have a small inherent variability associated with some porosity
156.9 (CdO carbamate), 170.3 (CdO amide), 174.3 (CdO of the electrodes, that gives rise to a variable background and
ester). has some effect upon peak shape in cyclic voltammetry. Typical
The ferrocene conjugate (167 mg, 0.25 mmol) was dissolved variability of background caused by electrode variability is
in dioxane (3 mL), and sodium hydroxide (15 mg, 0.38 mmol) illustrated in Figure 2.
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 RESULTS
mL) was added. The aqueous layer was neutralized and the The nature of linker A was initially explored with a range of
organic layer separated, dried (MgSO4), and concentrated. The spacers (together with either no group or a small group at
resulting oil was purified by flash silica chromatography (100% position B), since this was likely to significantly affect the
EtOAc to 5% MeOH in EtOAc) to afford 25 (50 mg, 32%) as electrochemical properties of the ferrocene due to their close
an orange oil. 1H NMR (300 MHz; CDCl3) 1.23 (m, 22H), 1.55 proximity and potential shielding properties. Three types of
(m, 4H), 2.22 (t, J ) 7.5 Hz, 2H, CH2CO2H), 3.37 (m, 2H), linker were synthesized: ester, amide, and amine. The synthesis
3.51-3.65 (m, 10H), 4.01 (m, 2H), 4.17 (s, 5H, C5H5), 4.29 of ferrocenyl esters has been reported using DCC as a coupling
(m, 2H, C5H2), 4.67 (m, 2H, C5H2), 5.31 (br s, 1H, NH), 6.23 reagent, which was found to be effective unless the alcohol had
(br s, 1H, NH); 13C NMR (CDCl3, 75 MHz) 24.9, 25.9, 29.1- poor solubility when rearrangement of the O-acyl urea occurred
29.6 (signals superimposed), 34.1, 39.3, 40.8, 65.1, 68.2, 69.7- (23, 24). This was not the case with tri(ethylene glycol), which
70.3 (signals superimposed), 71.5, 156.9 (CdO carbamate), readily reacted with ferrocenecarboxylic acid 2, DCC, and
Design/Synthesis of Ferrocene Probe Molecules Bioconjugate Chem., Vol. 17, No. 5, 2006 1261

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, acids were attached, either 11-aminoundecanoic acid, hexade-
the ferrocene alcohol 3 was converted into the corresponding canedioic acid, or 16-hydroxyhexadecanoic acid.
acetate 4, to assess electrochemical properties compared to other Initially, a carbamate linkage was synthesized using the
analogues. ferrocenyl ester with a PEG-3 spacer. Accordingly, alcohol 3
For the synthesis of the ferrocene amide, commercially was activated using N,N′-disuccinimidyl carbonate (DSC) to give
available 2-(2-aminoethoxy)ethanol was reacted with ferrocen- the corresponding N-hydroxysuccinimidyl ester which was
ecarboxylic acid 2 and N-(3-dimethylaminopropyl)-N′-ethyl- converted into 15 via coupling with 11-aminoundecanoic acid
carbodiimide hydrochloride (EDCI), that has been used in in THF/H2O in 74% isolated yield (Scheme 2).
ferrocenecarboxamide synthesis (24), together with HOBt to The diester-linked analogue 16 was also prepared from 3 and
give 5 in 84% isolated yield. The synthesis of a second amide hexadecanedioic acid using DCC and DMAP in low yield, due
linker with an alternative spacer possessing a terminal amine to isolation problems and side-product formation; however,
group was also investigated. The monoprotection of N,N′-bis- sufficient material was prepared for electrochemical analysis.
(2-hydroxylethyl)ethylenediamine 6 was achieved using Boc The synthesis of an amide analogue of 15 to establish any
anhydride to give 7 (20), which was coupled to ferrocenecar- benefits of a longer PEG spacer was carried out using an
boxylic acid 2 again using EDCI (Scheme 1) in 46% yield. identical method. Compound 5 was activated as the N-
Removal of the Boc group using 50% TFA in CH2Cl2 generated hydroxysuccinimidyl ester, which was isolated and coupled with
8 in 70% yield. 11-aminoundecanoic acid to afford 17 in 78% yield over the
To synthesize amine-linked conjugates, ferrocenecarboxal- two steps.
dehyde 9 was used. This was coupled to 2-(2-aminoethoxy)- To evaluate the potential benefits of a longer PEG spacer,
ethanol to afford the corresponding imine, which was then compound 21 (an analogue of 17) was also prepared. Synthesis
reduced in situ using sodium borohydride to the amine 10. The of the spacer with a terminal amine and alcohol was required,
amine 10 was also methylated to give the tertiary amine 11, and accordingly, 2-[2-(2-chloroethoxy)ethoxy]ethanol 18 was
since this derivative could potentially be used in further coupling reacted with potassium phthalimide to give 19 in 70% yield.
reactions to avoid a competing reaction at the nitrogen center. The hydroxyl group was then activated as before using DSC,
1,1′-Ferrocenedicarboxylic acid 12 (X ) CO2H, Figure 1) was and the isolated intermediate was coupled with methyl 11-
used to prepare disubstituted ferrocenes to assess the effect of aminoundecanoate to generate 20. Removal of the phthalimide
difunctionalization on the electrochemical properties: the diester group was achieved by treatment with hydrazine in 93% yield,
13 from tri(ethylene glycol), and DCC and DMAP, and diamide and ferrocenecarboxylic acid 2 was attached to the resulting
14 from 2-(2-aminoethoxy)ethanol, EDCI, and HOBt (Scheme free amino group using EDCI. Finally, hydrolysis of the methyl
1). ester afforded compound 21.
From the initial electrochemical data (see below), the ester- Finally, an analogue of 21 with the carbamate reversed was
linked compound 3, amides 5 and 8, and amine 11 were the synthesized. To do this, initially methyl 16-hydroxyhexade-
most suitable ferrocenyl scaffolds. However, as the amine was canoate 22 was activated and isolated as the N-hydroxysuccin-
generated in generally low yields, only the ester and amides imidyl ester 23 in 85% yield. The activated alcohol was coupled
were carried through for further structural elaboration. The with the mono-Boc-protected diamine, 7, to give compound 24.
nature of the solubilizing spacer together with the linker B were The Boc group was removed in quantitative yield using TFA
then explored to assess the influence of the spacer and second and the resulting amine coupled to ferrocenecarboxylic acid 2,
linker on electrochemical properties. Depending on the types using DCC and triethylamine. Finally, hydrolysis of the methyl
of spacer and linker incorporated, three different functionalized ester revealed the reverse carbamate analogue 25 (Scheme 2).
1262 Bioconjugate Chem., Vol. 17, No. 5, 2006 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

E1/2/
mV Ag,AgCl Ep,a - Ep,c/ Ep,a - Ep,a/2/
compound A linker B linker n m X (PBS) Ip,c/Ip,a mV mV
3 ester H 3 H 402 0.68 81 59
4 ester COMe 3 H 402 0.71 81 61
15 ester H 2 10 H 408 0.55 77 58
16 ester ester O(CO) 3 14 H 560 nd nd nd
5 amide H 2 H 349 0.94 82 63
8 amide (CH2)2NH2 2 H 365 0.80 80 65
17 amide carbamate O(CO)NH 2 10 H 357 0.76 76 58
21 amide carbamate O(CO)NH 3 10 H 370 0.67b 98 64
25 amide carbamate O(CO)NH 2 15 H 449 0.53 68 66
10 sec-amine H 2 H 266 0.89 80 66
11 tert-amine H 2 H 258 0.80 100 86
13 ester Et 3 CO2(CH2CH2O)3Et 638 0 77
14 amide Et 2 CONH(CH2CH2O)2H 568 0.28 96 66
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 esters and the disubstituted compounds), a second process
using cyclic voltammetry. In blood samples, electrochemical appearing as a shoulder at lower Ea,p was occasionally observed.
measurements are complicated due to the presence of several The probable explanation, since the appearance of the effect
electrochemically active compounds including uric acid, gly- correlated with the time between synthesis and electrochemical
cine, ascorbic acid, and urea (25). These compounds have measurement (up to several weeks in some cases) is that it was
an oxidation potential of approximately +350 to +550 mV due to ferrocene carboxylic acid formed by hydrolysis of the
(vs Ag/AgCl reference). The various components of the blood compound. For comparison, the cyclic voltammetry curve for
including proteins such as HSA produce a significant electro- ferrocenecarboxylic acid (FcCOOH) has a half-wave potential
chemical background signal at approximately +550 mV. For E1/2 of approximately +300 mV (vs Ag/AgCl) (26). A one-
use in binding studies, compounds with reversible voltam- electron reversible electrochemical process would have peak
mograms and an E1/2 of approximately +350 mV are preferred to half-peak and anodic peak to cathodic peak potential
to limit the interference from such an electrochemical back- differences (Ep,a - Ep,a/2 and Ep,a - Ep,c) of approximately 60
ground. mV, in the absence of effects due to ohmic drop between
The range of electrochemical behavior observed is given in working and reference electrodes, and would have the peak
Table 1 and illustrated in Figure 2. For some compounds (the current ratio Ip,a/Ip,c ) 1. The compounds all essentially satisfied
Design/Synthesis of Ferrocene Probe Molecules Bioconjugate Chem., Vol. 17, No. 5, 2006 1263

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
Figure 3. Variation with half-wave potential, E1/2, of ratio of cathodic fatty acid binding. Applications of the model compound 17 and
to anodic peak current in cyclic voltammetry, Ic,p/Ia,p The points are analogues as an electrochemical probe of biochemical interaction
labeled with the compound number. will be described in a future paper (30).

the criteria for the peak potential differences, but not for the ACKNOWLEDGMENT
peak current ratio. A satisfactory interpretation is that the We thank Unipath for funding (IT) and for permission to
oxidized product of the anodic reaction (ferrocenium) decom- publish this article.
poses to an electroinactive species at a rate dependent upon the
substituents. There was a systematic trend with the nature of Supporting Information Available: NMR spectra of synthe-
linker A and the length of the substituent chain, shown in Figure sized compounds. This material is available free of charge via
3: as E1/2 increased, indicating relative destabilization of the the Internet at http://pubs.acs.org.
ferrocenium cation, the decomposition rate of ferrocenium,
measured by Ip,c/Ip,a, also increased. LITERATURE CITED
DISCUSSION (1) Cais, M., Slovin, E., and Snarsky, L. (1978) Metalloimmunoassay
II. Iron-metallohaptens from estrogen steroids. J. Organomet. Chem.
Since the carboxylate anion can stabilize ferrocenium more 160, 223-230.
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