Synthesis, Characterization, and Evaluation of Ferrocene

Bioconjugate Chem. 2004, 15, 137−144

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Synthesis, Characterization, and Evaluation of Ferrocene-Theophylline Conjugates for Use in Electrochemical Enzyme Immunoassay Nigel J. Forrow,* Nicola C. Foulds, Jane E. Frew, and John T. Law MediSense (UK) Ltd., Abbott Laboratories, 14/15 Eyston Way, Abingdon, Oxon OX14 1TR, UK. Received July 22, 2003; Revised Manuscript Received October 20, 2003

A series of 8-(ferrocenylalkyl)theophylline conjugates were synthesized for evaluation in a homogeneous, competitive electrochemical immunoassay for theophylline with amperometric detection of the ferrocene label at +320 mV. The electrical signal was amplified via redox cycling with the glucose oxidase/ glucose system. The resulting catalytic current was strongly inhibited upon binding of the conjugates to anti-theophylline antibodies such that a large excess of theophylline was required to achieve complete reversal leading to an assay with poor sensitivity in the clinical range. A study of the nonspecific interaction of the antibodies with various ferrocene derivatives indicated that this was reduced when a charged functional group was present on the metallocene ring. Consequently, a conjugate was synthesized with a quaternary ammonium group which when incorporated into the assay resulted in improved sensitivity.

INTRODUCTION

Many different types of immunoassays have been developed that avoid the use of radioisotopic labels with their associated problems of short shelf life, disposal, and safety. Fluorescent and chemiluminescent markers along with enzymes have received the most attention as alternative labels for nonisotopic assay systems (1). The use of metal complexes as probes was first reported by Cais et al. who described the principle of metalloimmunoassay (MIA) in which, for example, steroid antigens were labeled with ferrocene with detection by flameless atomic absorption spectroscopy (2). Recently, this type of immunoassay has been expanded to include the use of metal carbonyl labels which can be detected by Fourier transform infrared spectroscopy (FT-IR). The term carbonylmetalloimmunoassay (CMIA) has been suggested for this class of assay (3, 4). Metal complexes, in particular ferrocene, are also of interest for use as electrochemical tracers in immunoassays. Here, the wide variety of substituted derivatives of ferrocene makes it an attractive label for conjugation to antigens. The relative ease of synthesis of ferrocene-labeled molecules, coupled with reversible electrochemistry and the ability to alter redox potential if desired, has led to ferrocene being the first choice for use in electrochemical sensing systems. Thus, ferrocene has also found widespread use as an electrochemical label for oligonucleotides (5-9) in DNA probes and for host molecules (10, 11) in the detection of neutral and charged guest species. Also, ferrocene-peptide conjugates have been suggested to be useful in electrochemical sensors for the detection of proteins (12, 13). The use of electrochemical detection offers the possibility of cheap, miniature devices capable of performing rapid assays in cloudy test samples. For example, commercial biosensor devices such as the hand-held MediSense Precision QID glucose meter incorporate a fer* Corresponding author. Telephone: +44 (0)1235 542122. Fax: +44 (0)1235 467640. E-mail: [email protected].

rocene derivative in a mass-produced enzyme electrode for use by diabetics to determine their sugar levels in whole blood (14). The potential of ferrocene as an electrochemical label in immunoassays was first explored by Weber and Purdy (15) who prepared a ferrocene-morphine conjugate and demonstrated that its electrochemistry was perturbed on binding to the antibody so that a homogeneous competitive assay could, in principle, be configured in which separation of free and antibody-bound label was unnecessary. This procedure has been enhanced using enzyme amplification by exploiting the known (16, 17) ability of ferrocene derivatives to act as electron-transfer mediators to glucose oxidase (GOx). An amperometric immunoassay for lidocaine has been configured using this approach (18). Also, brief reports have appeared in a patent application (19) and a book (20) describing its use for an electrochemical theophylline assay. Here, a detailed report of the synthesis and characterization of some ferrocene-theophylline conjugates and the problems associated with configuring an immunoassay based on them are described. RESULTS AND DISCUSSION

Synthesis of 8-(Ferrocenylalkyl)theophylline Conjugates. 8-Phenyl and 8-benzyl derivatives of theophylline have been previously prepared by condensation of 5,6-diamino-1,3-dimethyluracil with the respective carboxylic acids in refluxing POCl3 followed by cyclization with NaOH (21). The preparation of 8-(ferrocenylmethyl)theophylline 2a from ferrocenylacetic acid 1a using an adaptation of this method has been briefly reported (19, 20). A high boiling solvent, N,N-dimethylaniline (DMA), is used in place of POCl3. The Experimental Procedures section of this article provides a detailed description of the reaction conditions for the synthesis of 2a and further applies them to the preparation of a series of ferrocenetheophylline conjugates 2b-f from the corresponding

10.1021/bc034131b CCC: $27.50 © 2004 American Chemical Society Published on Web 12/11/2003

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Scheme 1. Synthesis of Ferrocene-Theophylline Conjugatesa

a

(i) ∆, DMA; (ii) NaOH; (iii) 2a, Ac2O, AlCl3, CH2Cl2; (iv) 2a, MeI, Na2CO3, DMF.

ferrocene carboxylic acids 1b-f (Scheme 1). No identifiable conjugate was produced from the reaction of ferrocene carboxylic acid 1 (n ) 0, R1 ) R2 ) H) or ferrocene dicarboxylic acid with 5,6-diamino-1,3-dimethyluracil. The synthesis of substituted ferrocene-theophylline conjugates was of interest for a number of reasons. In the first instance, the introduction of electron-donating substituents such as methyl groups into the ferrocene cyclopentadienyl rings will alter the redox potential of the conjugate such that a lower operating potential is required in an electrochemical assay. This is advantageous for test media, such as blood or plasma, which may contain oxidizable species. Here, the dimethyl-substituted conjugate 2b can be synthesized from the readily available (1,1′-dimethylferrocenyl)acetic acid 1b. However, apart from 1b, substituted ferrocene carboxylic acids are not easily accessible for use as starting materials in the synthesis of ferrocene-theophylline conjugates. Direct substitution of a ferrocene ring in a conjugate was explored for the introduction of electron-donating substituents. Here, functional groups of this type were attractive for modifying the hydrophobic nature of the ferrocene-theophylline conjugates. For example, the conjugates 2a-f were almost completely insoluble in aqueous buffer solutions. As such, a carboxylate or amino substituent might be effective in enhancing the aqueous solubility of the conjugates as well as modulating the strength of their binding with anti-theophylline antibodies. The Friedel-Crafts acetylation of conjugate 2a proceeded remarkably cleanly to afford the acetyl derivative 3 in moderate yield (Scheme 1). Acetylation was observed to occur only in the unsubstituted ferrocene ring. It was hoped that the acetyl group could be further elaborated into a carboxylate group. However, known procedures for effecting this transformation in acetylated ferrocene derivatives using NaOH/Br2 (22) or I2/pyridine then NaOH (23) failed for the conjugate 3. The Mannich reaction was considered to be a possible direct route to a tertiary amino derivative of conjugate 2a which could then be converted simply to a hydrophilic quaternary ammonium compound by alkylation. It appeared feasible that alkylation could be confined to the tertiary amine group since reaction at N-7 of theophylline

Scheme 2. Aminomethylation of Ferrocene-Labeled Conjugate 2aa

a

(i) ∆, (Me2N)2CH2, H3PO4, AcOH; (ii) MeI, MeOH.

required the additional use of base to obtain the caffeine conjugate 4 from the corresponding theophylline compound 2a (Scheme 1). However, we were aware that aminomethylation of 2a could result in reaction at three different ferrocene ring positions [cf. methylferrocene (24)] to yield the isomers 2aa, 2ab, 2ac and also at the N-7 position of theophylline (25) to give 2ad (Scheme 2). Initially, we successfully repeated the known (25) Mannich reaction of theophylline itself using Et2NH/ HCHO to yield the unstable 7-(diethylaminomethyl)-

Ferrocene−Theophylline Conjugates

theophylline 5 which displayed a signal at δ 5.2 for the N-7 methylene group in the 1H NMR spectrum. No reac-

Bioconjugate Chem., Vol. 15, No. 1, 2004 139 Table 1. Electrochemical Data for Ferrocene-Theophylline Conjugates conjugatea

tion was observed when we applied the exact same reaction conditions to the ferrocene-theophylline conjugate 2a. This result was encouraging in that it seemed that the ferrocenylmethyl substituent in the 8-position of theophylline had the effect of reducing the acidity of the N-7 hydrogen of 2a such that it might be possible to restrict aminomethylation to the ferrocene rings of the conjugate. Next, we carried out an aminomethylation reaction on 2a with the reagents used for methylferrocene (24) and ferrocene (26), i.e., bis(dimethylamino)methane in the presence of phosphoric acid as a catalyst with glacial acetic acid as the solvent. A brown, water-soluble, oily product was obtained in low yield. The 1H NMR spectrum demonstrated that substitution had occurred at ferrocene to yield 2aa/2ab/2ac rather than at theophylline since the expected signal at approximately δ 5 for the N-7 aminomethylene group of 2ad was not observed. Further examination of the spectrum indicated that only two ferrocene ring isomers were present. These are presumed to be 2aa and 2ab by analogy with the isomer distribution seen for aminomethylated methylferrocene (24). Latterly, we did note that it was also possible to effect N-7 aminomethylation of theophylline using bis(dimethylamino)methane to afford 7-(dimethylaminomethyl)theophylline 6. Here, compound 6 displayed a methylene signal at δ 5.1 in the 1H NMR spectrum. Methylation of 2aa/2ab with MeI proceeds readily at NMe2 to afford the quaternary ammonium salt 2aa+/ 2ab+ (Scheme 2) as expected from standard ferrocene chemistry (24, 26). Further support for ferrocene ring substitution was indicated by the apparent stability of 2aa+/2ab+. Thus, attempts to form the quaternary ammonium salt of 7-(dimethylaminomethyl)theophylline 6 resulted only in the isolation of theophylline. No such decomposition to the starting conjugate was observed during the quaternization of the aminomethylated products of conjugate 2a. Also, it was noted earlier in this article that N-7 methylation of the theophylline unit with MeI to produce a caffeine derivative does not occur in the absence of base. Despite poor yields and limited characterization data, the water soluble salt 2aa+/2ab+ was nonetheless included in further studies as the only example of a hydrophilic ferrocene-theophylline conjugate. The structural assignments for 2aa+/2ab+ and 2aa/2ab were considered to be sound based on a combination of 1H NMR data and comparative reactions carried out on theophylline itself for the purpose of eliminating N-7 in favor of ferrocene ring substitution. Electrochemistry of Ferrocene-Theophylline Conjugates. The conjugates 2a-f are practically insoluble in aqueous buffer solution. Polyoxyethylene 9-lauryl ether (5%) was therefore employed to enhance the solubility of 2a-f such that their aqueous electrochemistry could be easily studied by cyclic voltammetry. In contrast, the aminomethylated methiodide conjugate

no.

E1/2, mVb

Fc-CH2-Theo

2a

Me2Fc-CH2-Theo Fc-(CH2)2-Theo Fc-(CH2)3-Theo Fc-(CH2)4-Theo Fc-(CH2)8-Theo Fc-CH2-Caff Theo-CH2-Fc-CH2NMe3+

2b 2c 2d 2e 2f 4 2aa+/2ab+

+235 +220c +135 +225 +205 +220 +235 +265 +220d

a Fc ) ferrocene, Me Fc ) 1,1′-dimethylferrocene, Theo ) 2 theophylline, Caff ) caffeine. b Unless otherwise stated, electrochemical data was obtained in 0.15 M phosphate/0.2 M NaCl buffer (pH 7.0), containing 5% polyoxyethylene 9-lauryl ether. c 0.1 M BES/0.15 M NaCl (pH 7.0), containing 1% THF. d 0.15 M phosphate/ 0.2 M NaCl buffer (pH 7.0), no polyoxyethylene 9-lauryl ether.

Figure 1. (a) Cyclic voltammogram (at 5 mV s-1) of 2a (0.25 mM) in a 0.6 mL of 0.15 M phosphate/0.2 M NaCl buffer (pH 7.0), containing 5% polyoxyethylene 9-lauryl ether, at a gold electrode (area 0.16 cm2); (b) as for a but with the addition of 100 µL of stock glucose oxidase solution at a final concentration of 2.1 µM.

2aa+/2ab+ is water soluble. Reversible redox waves due to the ferrocene/ferrocenium couple with midpoint potentials E1/2 in the range +135 to +265 mV were recorded for 2a-f and 2aa+/2ab+ (Table 1). In addition, conjugate 2aa+/2ab+ displayed a broad wave in the range +500 to +600 mV due to the oxidation of iodide. This provides further support for the assignment of 2aa+/2ab+ as a methiodide salt. A typical cyclic voltammogram for a ferrocene-theophylline conjugate is displayed in Figure 1a. As expected from previous work (17) on the aqueous electrochemistry of ferrocene derivatives, the dimethylsubstituted conjugate 2b was observed to have an E1/2 value 100 mV lower than the corresponding unsubstituted compound 2a. There was no clear trend in E1/2 value on increasing the number of methylene groups in the ferrocene-theophylline linker from one in 2a to eight in 2f. Here, E1/2 varied over a narrow range of +205 to +235 mV. The E1/2 value for the caffeine conjugate 4 is 30 mV higher than the corresponding to theophylline compound 2a. The use of 1% THF in place of 5%

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Figure 2. Variation of the catalytic current with glucose oxidase concentration as measured by chronoamperometry. Different amounts of enzyme were added to conjugate 2a (2 µM) in a cell containing glucose (0.1 M) in BES buffer.

polyoxyethylene 9-lauryl ether did not result in a large shift in the E1/2 value for the conjugate 2a. Redox mediation of Ferrocene-Theophylline Conjugates with Glucose Oxidase. An enhanced catalytic wave was observed during cyclic voltammetry of the conjugates 2a-f in the presence of glucose oxidase and a nonlimiting excess amount (0.1 M) of glucose substrate. Figure 1b displays a typical voltammogram for 2a recorded under these conditions. Here, the oxidized ferrocenium form of the conjugate mediated the transfer of electrons from reduced glucose oxidase, produced during oxidation of glucose by the enzyme, to the electrode. This is basis of the signal generation in the proposed immunosensor. Chronoamperometry is an appropriate electrochemical technique for the exploitation of this signal generation mechanism. A fixed potential (>E1/2) was applied to an electrode such that the ferrocenium form of the conjugate was initially generated and then recycled upon reduction by reduced glucose oxidase thereby amplifying the signal. A poise potential of +320 mV was found to be suitable in this respect for the conjugates 2a-f. Each of the compounds 2a-f yielded similar maximum catalytic currents in the region of 0.8 µA (5.0 µA cm-2) for 2 µM solutions in the presence of 0.1 M glucose and a minimum of 35 µM of glucose oxidase (Figure 2). Inhibition of Conjugate Catalytic Current in the Presence of Antiserum. The redox mediation reaction of conjugates 2a-f with glucose oxidase was inhibited by the anti-theophylline antiserum. For example, this is clearly demonstrated for 2a in Figure 3a, which was obtained by incubating a fixed amount of conjugate (2 µM) with increasing quantities of undiluted antiserum followed by performing chronoamperometry on the mixture in the presence of glucose oxidase and glucose to determine the catalytic current. Here, it is presumed that the conjugate binds to the antibody and is thereby prevented from interacting with the flavin cofactor inside the active site of glucose oxidase due to the large size of the conjugate-antibody complex. This effect has been reported previously for a ferrocenelidocaine conjugate in the presence of anti-lidocaine antiserum (18). Furthermore, a clear analogy can be drawn with cyclodextrin inclusion complexes of ferrocene derivatives, which are also known not to mediate with glucose oxidase (27). Partial reversal of the inhibition of the catalytic current was achieved upon addition of theophylline to the mixture of conjugate, antiserum, glucose, and glucose oxidase. This is illustrated in Figures 3b and 3c for 2a for different amounts of theophylline. Here, theophylline competed with the ferrocene-theophylline conjugates

Figure 3. (a) Inhibition of the catalytic current on addition of varying volumes of undiluted anti-theophylline antisera to conjugate 2a (2 µM) in the presence of glucose (0.1 M) and glucose oxidase (35 µM); (b) as for a but with the addition of theophylline at a final concentration of 25 µM; (c) as for a but with the addition of theophylline at a final concentration of 50 µM.

Figure 4. Theophylline calibration curve for electrochemical immunoassay mediated by conjugate 2a and using an antibody concentration of 125 µg mL-1.

2a-f for binding with the antibodies thereby releasing more electroactive label for redox mediation with glucose oxidase. The antibody interaction with the conjugates could not be reversed completely even in the presence of a large excess of theophylline. In this respect, 2a appeared to be the least antigenic and therefore further work was confined to this particular conjugate. Figure 4 displays a calibration curve for theophylline using 2a; this spans the therapeutic range for theophylline of 55100 µM. However, the assay has a low sensitivity in the clinical range due to the poor reversibility of the binding of 2a with the antibodies. The Influence of Antiserum on the Redox Mediation of Ferrocene Derivatives with Glucose Oxidase. It appeared unlikely that the ferrocene-theophylline conjugates were more antigenic toward the antiserum than theophylline itself. The nature of the interaction of ferrocene with proteins is unknown but other authors (13) have also commented on its strength. In the present system, a possible explanation might be that the hydrophobic nature of the ferrocene group in 2a leads to nonspecific interactions with the antibody. In this respect, we decided to investigate the influence of antitheophylline antibodies on the redox mediation of charged

Ferrocene−Theophylline Conjugates

Figure 5. Inhibition of the catalytic current on addition of varying volumes of undiluted anti-theophylline antisera to various ferrocene derivatives (2 µM) in the presence of glucose (0.1 M) and glucose oxidase (35 µM): (a/b) ferrocene carboxylic acid 9 (2) and dimethylaminomethyl-1,1′-dimethylferrocene methiodide 10 (O); (c) ferrocene 7 (×); (d) conjugate 2a (b) (for comparison); (e) 1,1′-dimethylferrocene 8 (0).

Bioconjugate Chem., Vol. 15, No. 1, 2004 141

Figure 6. Inhibition of the catalytic current on addition of varying volumes of undiluted anti-theophylline antisera to conjugate 2aa+/2ab+ (2 µM) in the presence of glucose (0.1 M) and glucose oxidase (35 µM).

and uncharged ferrocenes 7, 8, 9, and 10 with glucose oxidase in comparison with the conjugate 2a.

In a fashion similar to the conjugates 2a-f, the ability of the uncharged ferrocenes 7 and 8 to mediate with glucose oxidase was markedly inhibited in the presence of antiserum (Figures 5c and 5e). Even the addition of an excess (100 µM) of theophylline had no significant effect upon the observed decrease in catalytic current. In contrast, the charged ferrocene derivatives 9 and 10 displayed no such inhibition (Figure 5a/b). These results support the hypothesis that nonspecific hydrophobic interactions between the ferrocene moiety of conjugates 2a-f and the antibody contribute to the observed inhibition. Attempts to Reduce Nonspecific Interactions in the Assay System. In light of the above results, the charged conjugate 2aa+/2ab+ was synthesized since the ability of the ferrocene quaternary ammonium salt 10 to mediate with glucose oxidase was not inhibited by the presence of antiserum. Nonspecific interactions between the ferrocene unit and anti-theophylline antibodies should then, in theory, be reduced to a minimum. However, as for the uncharged 2a, the catalytic activity of the charged conjugate 2aa+/2ab+ with glucose oxidase was inhibited by antisera (Figure 6). Complete reversal of this inhibition could only be achieved through the addition of a 50fold excess of theophylline over 2aa+/2ab+. Therefore, although reducing the hydrophobic nature of the ferrocene moiety of the conjugate improves the reversibility of the inhibition, the sensitivity of the assay over the clinical range remains unsatisfactory (Figure 7).

Figure 7. Theophylline calibration curve for electrochemical immunoassay mediated by conjugate 2aa+/2ab+ and using an antibody concentration of 125 µg ml-1. CONCLUSION

8-(Ferrocenylalkyl)theophylline conjugates, with a range of linker arm chain lengths, were readily synthesized from the corresponding ferrocenylalkanoic acid and 5,6diamino-1,3-dimethyluracil in moderate yield. However, the conjugates were discovered to bind more strongly to anti-theophylline antibodies than theophylline itself. The resulting competitive, homogeneous immunoassay displayed poor sensitivity in the therapeutic range for theophylline. The nonspecific interaction of the ferrocene moiety with the antibodies was found to be reduced by introducing a charged functional group into the metallocene ring of one conjugate. The assay sensitivity using the modified conjugate was improved but remains unsatisfactory for development into a viable electrochemical immunoassay for theophylline. This work indicates that, unless nonspecific interactions with antibodies can be overcome, ferrocene may be an unsuitable electrochemical label for general use in immunoassays. EXPERIMENTAL PROCEDURES

Materials. All reactions were carried out under an atmosphere of nitrogen. Anhydrous solvents (Sure Seal grade; Aldrich) were used for reactions when required. All reagents and starting materials for reactions were purchased from Aldrich except for 1,1′-dimethylferrocene

142 Bioconjugate Chem., Vol. 15, No. 1, 2004

8 (Strem) and were used without further purification. Literature methods were used for the preparation of dimethylaminomethyl-1,1′-dimethylferrocene methiodide 10 (24), ferrocenylacetic acid 1a (28), 3-ferrocenylpropanoic acid 1c (29, 30), 4-ferrocenylbutanoic acid 1d (31), 5-ferrocenylpentanoic acid 1e (31), and 9-ferrocenylnonanoic acid 1f (32, 33). Dimethylaminomethyl-1,1′dimethylferrocene methiodide 10 was used as a mixture of the 2- and 3-isomers. Theophylline and polyoxyethylene 9-lauryl ether were obtained from Sigma. Rabbit antitheophylline antiserum Travenol H113, containing 2.5 mg/mL antibody, was purchased from Clinical Assays. Glucose oxidase (EC 1.1.3.4 from Aspergillus niger) was supplied by Rhodia. The commercial dilute enzyme was concentrated, using tangential flow filtration (30 kDa size exclusion membrane, Millipore), to a protein level of ca. 250 mg/mL (1.5 mM) in 0.2 M BES buffer pH 7.5. The concentration of glucose oxidase was expressed in terms of catalytically active FAD determined spectrophotometrically at 450 nm. A molar extinction coefficient of 1.31 × 104 M-1 cm-1 was used (34). The BES buffer solution of 0.1 M BES (with 0.15 M NaCl as supporting electrolyte), pH 7.0, and containing 0.1 M D-glucose was prepared using AnalaR reagents from VWR in deionized water. The solution was stored overnight at 4 °C prior to use to allow equilibration of the R- and β-anomers. The phosphate buffer (0.15 M, with 0.2 M NaCl), pH 7.0, and containing 0.1 M D-glucose was also prepared employing AnalaR reagents (VWR). Proton NMR spectra were recorded on Bru¨ker WH 300 (300 MHz) or AM 500 (500 MHz) (external NMR service, University of Oxford) and Hitachi R-24B (60 MHz) instruments. Spectra were referenced internally to either the solvent or SiMe4. Microanalyses were performed by C. H. N. Analysis Ltd., Leicester and Butterworth Laboratories, Teddington. Mass spectra (electron impact) were obtained via the external mass spectrometry service at the University of Oxford. 8-(Ferrocenylmethyl)theophylline (2a). Ferrocenylacetic acid 1a (20.0 g, 82.3 mmol) and 5,6-diamino-1,3dimethyluracil hydrate (16.7 g, 88.7 mmol) were dissolved in N,N-dimethylaniline and refluxed for 5 h using a Dean-Stark water separator. After cooling, NaOH solution (8% w/v, 400 mL) was added and the mixture was stirred for 18 h. The N,N-dimethylaniline was then removed by steam distillation. A heavy yellow precipitate was observed which partially dissolved upon acidification to pH 3-4 with 6 M HCl. The product was separated from the deep red solution by filtration, and then washed with acetonitrile followed by Et2O to afford 2a as an analytically pure yellow powder (11.9 g, 38%). Mp 270-272 °C (dec). Anal. Found: C, 56.93; H, 4.84; N, 14.78%; M+ 378. C18H18N4O2Fe requires C, 57.16; H, 4.80; N, 14.81%; M 378. IR (Nujol, cm-1): 1700, 1650 (υCO); 1H NMR (500 MHz; CDCl3): 3.43 (s, 3H, NMe), 3.62 (s, 3H, NMe), 3.94 (s, 2H, CH2), 4.19 (s, 5H, C5H5), 4.21 (m, 2H, C5H4), 4.25 (m, 2H, C5H4). 8-(Ferrocenylmethyl)caffeine (4). To a mixture of 8-(ferrocenylmethyl)theophylline 2a (1.25 g, 3.31 mmol) and Na2CO3 (0.53 g, 5.0 mmol) in DMF (100 mL) was added iodomethane (0.25 mL). After stirring for 3 d at room temperature, the reaction mixture was filtered and evaporated to dryness. The yellow residue was dissolved in CH2Cl2 and then filtered to remove inorganic salts. Removal of the solvent yielded 4 as a yellow solid (1.23 g, 95%). Mp 182-184 °C. 1H NMR (500 MHz; CDCl3): 3.42 (s, 3H, NMe), 3.62 (s, 3H, NMe), 3.90 (s, 2H, CH2), 3.92 (s, 3H, NMe), 4.18 (s + m, 9H, C5H5 + C5H4).

Forrow et al.

(1,1′-Dimethylferrocenyl)acetonitrile. Dimethylaminomethyl-1,1′-dimethylferrocene methiodide (10.0 g, 24 mmol) was suspended in water (100 mL), and KCN (7.87 g, 121 mmol) was added. The mixture was then refluxed for 2 h. A brown oil separated on cooling. The aqueous layer was decanted from the oil and washed with Et2O. The oil and the ether extracts were combined and dried (MgSO4). Removal of the solvent in vacuo gave the crude nitrile as an orange oil (6.10 g, 100%). IR (liquid film, cm-1): 2250 (υCN). (1,1′-Dimethylferrocenyl)acetic Acid (1b). Unpurified (1,1′-Dimethylferrocenyl)acetonitrile (6.10 g, 24 mmol) was dissolved in EtOH (50 mL), and a solution of KOH (14.0 g, 25 mmol) in water (200 mL) was added. The mixture was refluxed for 7 h. After cooling, the reaction mixture was washed twice with Et2O before being acidified with 6 M HCl to pH 1. A dark yellow oil was obtained. The oil was dissolved in CH2Cl2 (250 mL) and dried (MgSO4). The crude product 1b was obtained as a brown oil (4.76 g, 73%) after evaporation of the chlorinated solvent. IR (liquid film, cm-1): 1700 (υCO). 8-[(1,1′-Dimethylferrocenyl)methyl]theophylline (2b). Unpurified (1,1′-Dimethylferrocenyl)acetic acid 1b (4.76 g, 17.5 mmol) was dissolved in N,N-dimethylaniline (150 mL), and 1,3-dimethyl-5,6-diamino uracil hydrate (3.57 g, 19 mmol) was added. The reaction mixture was refluxed for 5 h using a Dean-Stark water separator. NaOH solution (8% w/v, 50 mL) was added, and the mixture was stirred for 18 h. N,N-Dimethylaniline was then removed by steam distillation. Upon cooling, a solid precipitated and this was collected by filtration. The filtrate was acidified with glacial acetic acid to pH 4-5 affording a yellow precipitate. The crude product was dissolved in CH2Cl2 and dried (MgSO4). An orange-yellow solid (3.10 g) was thus obtained which was subjected to column chromatography (Florisil). Elution with Et2O-Me2CO-MeOH (8:1:1) gave a yellow band from which the product 2b was obtained as a yellowgreen solid (1.46 g, 21%). Mp 220 °C (dec). Anal. Found: C, 59.02; H, 5.44; N, 13.80%; M+ 378. C20H22N4O2Fe requires C, 59.13; H, 5.46; N, 13.79%. 1H NMR (60 MHz; CDCl3): 1.90 (br m, 6H, C5H4Me + C5H3Me), 3.45 (br s, 3H, NMe), 3.55 (br s, 3H, NMe), 3.80 (9H, br m, CH2 + C5H4Me + C5H3Me). Preparation of Ferrocene-Theophylline Conjugates (2c-f). The above procedure for the synthesis of 8-[(1,1′-dimethylferrocenyl)methyl]theophylline 2b was followed substituting the appropriate ferrocenylalkanoic acid 1c-f for (1,1′-dimethylferrocenyl)acetic acid 1b. After column chromatography, the desired ferrocenetheophylline conjugates 2c-f were isolated as yellow solids in 20-30% yield. 8-(2-Ferrocenylethyl)theophylline (2c). Mp > 200 °C (dec). Anal. Found: C, 58.27; H, 4.55; N, 14.14%. C19H20N4O2Fe requires C, 58.18; H, 5.14; N, 14.28%. IR (Nujol, cm-1): 1720, 1630 (υCO); 1H NMR (300 MHz; CDCl3): 2.88 (t, J(HH) 6.9 Hz, 2H, CH2), 3.08 (t, J(HH) 6.9 Hz, 2H, CH2), 3.48 (s, 3H, NMe), 3.67 (s, 3H, NMe), 4.01 (m, 4H, C5H4), 4.11 (s, 5H, C5H5), 12.64 (s, 1H, NH). 8-(3-Ferrocenylpropyl)theophylline (2d). Mp 210212 °C (dec). Anal. Found: C, 58.90; H, 5.50; N, 13.70%. C20H22N4O2Fe requires C, 59.13; H, 5.46; N, 13.79%. IR (Nujol, cm-1): 1710, 1640 (υCO); 1H NMR (300 MHz; CDCl3): 2.07 (m, J(HH) 7.6 Hz, J(HH) 7.6 Hz, 2H, CH2), 2.44 (t, J(HH) 7.6 Hz, 2H, CH2), 2.90 (t, J(HH) 7.6 Hz, 2H, CH2), 3.44 (s, 3H, NMe), 3.65 (s, 3H, NMe), 4.05 (m, 4H, C5H4), 4.09 (s, 5H, C5H5), 12.61 (s, 1H, NH). 8-(4-Ferrocenylbutyl)theophylline (2e). Mp 192194 °C (dec). Anal. Found: C, 59.70; H, 6.22; N, 12.71%.

Ferrocene−Theophylline Conjugates

C21H24N4O2Fe requires C, 60.01; H, 5.76; N, 13.33%. IR (Nujol, cm-1): 1710, 1625 (υCO); 1H NMR (300 MHz; CDCl3): 1.59 (m, J(HH) 7.6 Hz, J(HH) 7.6 Hz, 2H, CH2), 1.88 (m, J(HH) 7.6 Hz, J(HH) 7.6 Hz, 2H, CH2), 2.38 (t, J(HH) 7.6 Hz, 2H, CH2), 2.90 (t, J(HH) 7.6 Hz, 2H, CH2), 3.47 (s, 3H, NMe), 3.64 (s, 3H, NMe), 4.02 (m, 4H, C5H4), 4.07 (s, 5H, C5H5), 12.74 (s, 1H, NH). 8-(8-Ferrocenyloctyl)theophylline (2f). Mp 157158 °C. Found: C, 63.63; H, 6.82; N, 11.82%. C25H32N4O2Fe requires C, 63.03; H, 6.77; N, 11.76%. IR (Nujol, cm-1): 1710, 1625 (υCO); 1H NMR (300 MHz; CDCl3): 1.30 (br m, 8H, CH2), 1.48 (br m, 2H, CH2), 1.84 (m, J(HH) 7.6 Hz, J(HH) 7.0 Hz, 2H, CH2), 2.30 (t, J(HH) 7.7 Hz, 2H, CH2), 2.87 (t, J(HH) 7.7 Hz, 2H, CH2), 3.48 (s, 3H, NMe), 3.64 (s, 3H, NMe), 4.04 (m, 4H, C5H4), 4.09 (s, 5H, C5H5), 12.66 (s, 1H, NH). 8-(1′-Acetyl-ferrocenylmethyl)theophylline (3). 8(Ferrocenylmethyl)theophylline 2a (3.78 g, 10 mmol) was dissolved in CH2Cl2 (600 mL). AlCl3 (6.65 g, 50 mmol) was added followed by the dropwise addition of acetic anhydride (0.94 mL, 1.02 g, 10 mmol). The reaction mixture was stirred for 4 d and then quenched with water. The orange organic layer was separated and dried (MgSO4). Filtration and removal of the solvent afforded an orange solid which was chromatographed (SiO2). Elution with Me2CO-CH2Cl2 (1:4) yielded a small amount of starting material 2a as a yellow band. The red product band was eluted with MeOH-CH2Cl2 (1:19) and evaporated to dryness to provide 3 as an orange solid (2.43 g, 58%). Mp 219-220 °C (dec). Anal. Found: C, 57.58; H, 4.88; N, 13.37%. C20H20N4O3Fe requires C, 57.16; H, 4.80; N, 13.33%. 1H NMR (500 MHz; CDCl3): 2.35 (s, 3H, CH3CO), 3.46 (s, 3H, NMe), 3.59 (s, 3H, NMe), 3.78 (s, 2H, CH2), 4.12 (m, 2H, C5H4), 4.23 (m, 2H, C5H4), 4.41 (t, 2H, C5H4), 4.61 (t, 2H, C5H4). 8-(2/3-Dimethylaminomethyl-ferrocenylmethyl)theophylline (2aa/2ab). 8-(Ferrocenylmethyl)theophylline 2a (1.0 g, 2.65 mmol) was added to a well-stirred solution of bis(dimethylamino)methane (0.51 g, 5 mmol) and H3PO4 (10 g) in 100 mL of glacial acetic acid. The resulting mixture was heated for 5 h in an oil bath maintained at 100 °C. A dark amber solution was obtained. This was diluted with water (100 mL) and then extracted with CH2Cl2 to remove the starting conjugate 2a. The aqueous layer was adjusted to pH7 with NaOH pellets and then extracted with CH2Cl2. The organic layer was dried (Na2SO4), filtered then evaporated to yield a brown oil soluble in CHCl3, CH2Cl2, EtOH and water. This oil was passed through a short column of basic Al2O3, eluting with MeOH/CH2Cl2 (1:9), to provide 2aa/ 2ab as an oil (0.1 g, 9%). 1H NMR (500 MHz; CDCl3): 2.31 (s, 6H, NMe2), 3.40 (s, 3H, NMe), 3.58 (s, 3H, NMe), 3.9-4.3 (12H, ferrocene ring protons + 2 × CH2). 8-(2/3-Dimethylaminomethyl-ferrocenylmethyl)theophylline Methiodide (2aa+/2ab+). Crude aminomethylated conjugate 2aa/2ab (0.1 g, 0.23 mmol) from the above reaction was dissolved in MeOH. Iodomethane (0.5 mL) was added and the resulting mixture was stirred for 2 h. After evaporation of the solvent, the residue was redissolved in the minimum volume of MeOH. The methiodide 2aa+/2ab+ was precipitated from this solution by the addition of Et2O. The precipitate was collected, washed with Et2O and dried to afford 0.11 g (83%) of a beige powder. Aminomethylation of Theophylline. (a) Reaction with Diethylamine/Formaldehyde (24). Formaldehyde solution (37%, 0.9 mL, 10 mmol) was added to a wellstirred solution of diethylamine (10 mmol) and theophylline (1.80 g, 10 mmol) in EtOH (30 mL). After 15 min,

Bioconjugate Chem., Vol. 15, No. 1, 2004 143

undissolved starting material was filtered off. The filtrate was evaporated to dryness to yield 7-(diethylaminomethyl)theophylline 5 as a white crystalline solid. The 1 H NMR spectrum of 5 in CDCl3 displayed a singlet at δ 5.2 for the methylene protons. (b) Reaction with Bis(dimethylamino)methane. Theophylline (1.80 g, 10 mmol) was added to a mixture of bis(dimethylamino)methane (2.04 g, 2.72 mL, 20 mmol), H3PO4 (10 g), and glacial acetic acid (150 mL). This mixture was heated at 100 °C on an oil bath for 5 h. Upon cooling, the reaction mixture was diluted with 200 mL of water and then made alkaline with NaOH pellets. Extraction with CH2Cl2 followed by separation, drying (MgSO4), and filtration of the organic layer gave a colorless solution. Removal of the solvent provided 7-(dimethylaminomethyl)theophylline 6 as a white solid (0.5 g, 21%). 1H NMR (500 MHz; CDCl3): 2.45 (s, 6H, NMe2), 3.37 (s, 3H, NMe), 3.56 (s, 3H, NMe), 5.12 (s, 2H, CH2). (c) Attempted Formation of the Methiodide. 7-(Dimethylaminomethyl)theophylline 6 (0.5 g, 1.14 mmol) was dissolved in MeOH and a large excess of iodomethane (1 mL) was added. The resulting solution was stirred for 2 h. Addition of Et2O produced no precipitate so the solvent was removed under reduced pressure to give a white solid which was washed with CH2Cl2. A white solid remained and this was identified as theophylline by 1H NMR spectroscopy. Apparatus. Cyclic voltammetry and chronoamperometry experiments were carried out in a two-chamber glass cell of working volume 0.5 cm3. The cell contained a 1 cm2 platinum gauze counter electrode and a KClsaturated calomel electrode as reference. All potentials are referred to SCE. The working electrode was a 4 mm diameter gold disk. An Oxford Electrodes potentiostat was employed. Solutions of Ferrocene Conjugates. The ferrocene derivatives were only sparingly soluble in the aqueous BES and phosphate buffer solutions. The solubility of the conjugates was enhanced through the use of either 1% tetrahydrofuran (THF) or 5% polyoxyethylene 9-lauryl ether. Generally, THF was employed in chronoamperometry experiments involving anti-theophylline antiserum whereas polyoxyethylene 9-lauryl ether was used during cyclic voltammetry of the conjugates. In the former case, concentrated solutions (0.2 mM) of the ferrocene derivatives were prepared by first dissolving them in THF then mixing with the BES buffer to the desired concentration (2 µM). In the latter case, concentrated solutions (0.25 mM) of the ferrocene derivatives were prepared by first dissolving them in warmed liquid polymer then mixing with the phosphate buffer to the desired percentage (5%). The aminomethylated methiodide conjugate 2aa+/2ab+ is water soluble so the use of either THF or polyoxyethylene 9-lauryl ether was unnecessary in the preparation of solutions for electrochemical experiments. Procedure. Solutions (2 µM) of ferrocene derivatives, including ferrocene-theophylline conjugates, were freshly prepared in the BES buffer solution containing a high level (0.1 M) of D-glucose such that the kinetics of glucose oxidase was saturated. The buffer solution of ferrocene derivative was added to the cell followed by rabbit antitheophylline antiserum (0-100 µL) and theophylline (0300 µM). All concentrations are final ones obtained in a total assay volume of 1 mL. The mixture was then stirred for 5 min at room temperature. Finally, glucose oxidase (35 µM) was added then the working electrode was replaced in the cell. A potential of +320 mV was applied and current-time transients were recorded. Catalytic

144 Bioconjugate Chem., Vol. 15, No. 1, 2004

currents were measured after 3 min and were corrected by subtraction of the background current obtained in the absence of enzyme and antisera. ACKNOWLEDGMENT

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BC034131B