Ferrocene labels for electrochemical assay and their use in analytical methods

Abstract

Compounds of general formula I are used as labels in an electrochemical assay: (I) in which: Fc and Fc′ are substituted or unsubstituted ferrocenyl moieties, X is a C1 to C6 alkylene chain which is optionally interrupted by —O— or —NH—; Y is a C1 to C6 alkylene chain which is optionally interrupted by —O— or —NH—; Z is a C1 to C12 alkylene chain which may optionally be substituted and/or may optionally be interrupted by —O—, —S—, cycloalkyl, —CO—, —CONR1-, —NR1CO— or —NR1- in which R1 represents hydrogen or C1 to C4 alkyl; and R is a linker group. Compounds I are used to make labelled substrates, as well as functionalised compounds for making the labelled substrates.

Claims

1. A method of detecting a nucleic acid, the method comprising: contacting the nucleic acid with a complementary nucleic acid probe under conditions to allow hybridization between said probe and an amplicon; and selectively degrading the probe, wherein the probe is labelled with the compound: ##STR00040##

2. The method of claim 1, further comprising measuring electrochemical activity of the compound labelling the probe, wherein said electrochemical activity is dependent either quantitatively or qualitatively on the degradation of the probe.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic representation of an electrochemical cell used in differential pulse voltammetry measurements described herein;

(2) FIG. 2 is a differential pulse voltammogram of Label A as manufactured in Example 1 below;

(3) FIG. 3 is a differential pulse voltammogram of Label B as manufactured in Example 2 below;

(4) FIG. 4 is a differential pulse voltammogram of Label C as manufactured in Example 3 below;

(5) FIG. 5 shows the voltammetric scans for both a Chlamydia positive and a negative sample according to Example 5(a) below;

(6) FIG. 6 is a mass spec analysis of a mononucleotide and a dinucleotide synthesised with the label A electrochemical label as described in Example 5(a) below;

(7) FIG. 7 is a mass spec analysis of the assay product of Example 5(a);

(8) FIG. 8 is a bar graph showing current measurements taken when a Chlamydia target is added directly to a PCR reaction at a range of concentrations and detected using a oligonucleotide probe as described in Example 5(b) below;

(9) FIG. 9 is a bar graph showing peak current at a range of concentrations when the Chlamydia target was added to a DNA extraction process and the output from the extraction process was amplified using PCR, then detected using an oligonucleotide probe labelled with Label A, as described in Example 5(c) below;

(10) FIG. 10 shows a comparison between electrochemical detection using the label A probe and a SYBR Green based qPCR assay for Norovirus;

(11) FIG. 11 shows a comparison between electrochemical detection using the label A probe and a SYBR Green based qPCR assay for Streptococcus equi;

(12) FIG. 12a is a voltammetric scan using label A coupled to a commercially available anti-goat IgG;

(13) FIG. 12b shows voltammetric scans carried out at time intervals using label A coupled to a commercially available anti-goat IgG;

(14) FIG. 13 shows voltammetric scans of microparticles according to Example 8 at various concentrations; and

(15) FIG. 14 is a differential pulse voltammogram of Label D as manufactured in Example 9 below.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

(16) With reference to FIG. 1, there is shown schematically an electrochemical cell 1 suitable for use in the cyclic voltammetry experiments described herein. The cell comprises a vessel 2, containing a background electrolyte solution 3, which is an aqueous 100 mM solution of sodium chloride. Immersed in the solution 3 is a printed carbon working electrode 5, a printed carbon counter electrode 6 and a silver/silver chloride reference electrode 7, all with silver connectors. The sample is spread on to the surface of the working electrode and voltammetry is performed by connecting the silver connectors to the appropriate leads on the potentiometer. By way of illustration, the sample may be prepared as follows: Ferrocenyl label precursor (2 ng) is dissolved in DMSO (1 mL). An aliquot of 10 μL is taken of this solution and is then further diluted in the buffer (500 μL). Then an aliquot (20 μL) is applied to the screen printed electrode to run the electrochemical scan.

(17) The following Examples illustrate the invention:

(18) Materials and Methods—Label Synthesis and Assays

(19) Ferrocene carboxylic acid was obtained from Sigma-Aldrich. Ferrocene carboxaldehyde was obtained from Sigma-Aldrich.

(20) 6-Aminohexanol was obtained from Sigma-Aldrich.

(21) Glycine was obtained from Sigma-Aldrich

(22) N,N-diisopropylethylamine was obtained from Sigma-Aldrich.

(23) 2-cyanoethyldiisopropylchlorophosphoramidite was obtained from Sigma-Aldrich. Papain solution at concentration 1 mg/mL was obtained from Sigma-Aldrich.

(24) Anti-goat IgG and Biotinylated goat IgG were obtained from Sigma.

(25) PCR methods were performed using a PTC-100 or PTC-200 Programmable Thermal Controller (MJ Research Inc.), or a PeqLab flat bed thermocycler

(26) Streptavidin coated microtitre wells were Sigma Screen™ high density wells.

(27) Materials and Methods—Electrochemical Detection

(28) The electrodes are ink based and are screen printed on to a polymer substrate (for example Mylar) followed by heat curing—produced by GM Name plate (Seattle, Wash.)

Example 1—Synthesis of N,N-diferrocenylmethyl-6-aminohexanol [Label A]

(29) ##STR00028##

(30) Ferrocene carboxaldehyde (2.1 g, 9.81 mmol) and 6-aminohexan-1-ol (0.5 g, 4.27 mmol) in dry THF (25 mL) were added to an oven dried flask. Sodium triacetoxyborohydride (2.3 g, 10.90 mmol) was added portionwise to the solution. The reaction was left overnight. The reaction was taken up in ethyl acetate (40 mL), the organic layer was washed with NaCO.sub.3 (sat; 20 mL), Brine (20 mL) and MilliQ water (20 mL). The organic fraction was then dried over Magnesium sulfate and the solvent removed in vacuo. The crude product is then columned using 9:1 solution B:solution A (solution A: ethyl acetate 95% TEA 5%, solution B: Petroleum ether 40-60 95%, TEA 5%) to elicit the pure product (dark orange solid). 85% Yield .sup.1H NMR (300 MHz, CDCl.sub.3) δ 4.18 (2H, s, Cp), 4.17 (2H, s, Cp), 4.13 (15H, s, Cp), 3.66 (4H, t, J=6.25 Hz, CH.sub.2), 3.48 (2H, s, CH.sub.2), 2.20 (2H, t, J=6, CH.sub.2), 1.59-1.31 (6H, m, CH.sub.2). .sup.13C NMR (75.5 Hz, CDCl.sub.3) δ 77.83, 77.40, 76.98, 70.58, 68.88, 63.36, 53.02, 52.17, 33.10, 27.43, 25.88. HRMS (ESI) calculated for C.sub.28H.sub.33N.sub.1O.sub.1Fe.sub.2 m/z 519.1430 found 519.1438.

(31) The electrochemistry of compound Label A is shown on the voltammogram of FIG. 2.

(32) The product label was found to have a redox potential of 0.275V.

Example 2—Synthesis of di-((dimethylamino)methylferrocenylmethyl)-6-aminohexanol (Label B)

(33) (a) Synthesis of Dimethylamino)methyl ferrocenecarboxaldehyde (Diaminomethyl)methylferrocene (1 g, 5 mmol) was dissolved in Et.sub.2O, n-butyl lithium (2.51 mL, 6.25 mmol) was added slowly and the reaction mixture was stirred at room temperature for 16 hrs.

(34) After 16 hrs, the reaction mixture was quenched with DMF (0.4 mL, 6.25 mmol) and stirred again at room temperature for 4 hrs. Water (15 mL) was then added to the reaction. The organic phase was then extracted with ether (2×25 mL). The combined organic phases were dried with magnesium sulphate, filtered and the solvent was removed under vacuum to afford the product in an 72% yield (dark red/brown oil). .sup.1H NMR (300 MHz, CDCl.sub.3) δ 9.81 (1H, s, CHO), 4.21 (2H, s, Cp), 4.14 (5H, s, Cp), 3.64 (2H, s, CH.sub.2), 2.08 (6H, s, NMe.sub.2) .sup.13C NMR (75.5 Hz, CDCl.sub.3) δ 193.2, 86.7, 83.4, 77.8, 77.5, 77.0, 76.62, 75.8, 71.8, 70.3, 70.2, 70.0, 68.4, 68.0, 59.2, 56.6, 44.8, 44.7. HRMS (ESI) calculated for C.sub.14H.sub.18N.sub.1O.sub.1Fe.sub.1 m/z 272.0737 found 272.0731 Ref: Biot, C., Glorian, G., Maciejewski, L. A., Brocard, J. S., Domarle, O., Blampain, G., Millet, P., Georges, A. J., Abessolo, H., Dive, D., Lebibi, J. J. Med. Chem. 1997, 40, 3715-3718.

(35) (b) Synthesis of Label B

(36) ##STR00029##

(37) (Dimethylamino)methyl ferrocenecarboxaldehyde (1.1 g, 4.04 mmol) was dissolved in dry THF (30 mL). 6-aminohexan-1-ol (0.25 g, 2.13 mmol) was added. Then sodium triacetoxyborohydride (1.3 g, 6.16 mmol) was added to the reaction mixture. The solution was stirred under nitrogen at room temperature overnight. Ethyl acetate (20 mL) and 1N NaOH (sat; 20 mL) were then added and the organic layer was then extracted with NaCO.sub.3 (25 mL), Brine (25 mL) and Milli Q filtered water (25 mL) then dried over MgSO.sub.4 and the solvent removed in vacuo to yield an orange oil (0.95, 75%). 1H NMR (300 MHz, CDCl.sub.3) δ 4.18 (2H, s, Cp), 4.17 (2H, s, Cp), 4.13 (15H, s, Cp), 3.66 (4H, t, J=6.25 Hz, CH.sub.2), 3.48 (2H, s, CH.sub.2), 2.20 (2H, t, J=6, CH.sub.2), 2.17 (12H, s, CH.sub.3) 1.59-1.31 (6H, m, CH.sub.2). HRMS (ESI) calcd for C.sub.34H.sub.49N.sub.3O.sub.1Fe.sub.2 m/z 627.3012 found 627.3126.

(38) The electrochemistry of the product compound is shown on the voltammogram in FIG. 3. The product label B was found to have a redox potential of 0.38V.

Example 3: Synthesis of 2-((diferrocenylmethyl)amino)-1-(4-(hydroxymethyl)piperidin-1-yl)ethanone (Label C)

(a) Synthesis of N,N-(diferrocenylmethyl)glycine

(39) ##STR00030##

(40) Ferrocene carboboxaldehyde (2.1 g) was added to a round bottomed flask containing dry THF (20 mL). Glycine (0.5 g) was added to the solution and the reaction was stirred under N.sub.2. Sodium triacetoxyborohydride (2.3 g) was added portionwise to the stirring solution. The reaction was stirred over night. The solution was then partitioned between ethyl acetate (40 mL) and 1M aqueous sodium hydroxide (40 mL). The organic fraction was washed with saturated aqueous NaHCO.sub.3 (sat; 20 mL), brine (40 mL) and water (40 mL). The organic fraction was dried using MgSO.sub.4 and the solvent was removed. The crude product was then columned (solvent A: petroleum ether 40-60:TEA 95:5, Solvent B: ethyl acetate:TEA 95:5). The product was an dark orange solid (80%). .sup.1H NMR (250 MHz, CDCl.sub.3) δ 4.099 (1H, s, CpH), 4.052 (1H, s, CpH), 4.022 (7H, s, FcCpH), 3.549 (4H, t, J=6.75, 2×CH.sub.2), 3.348 (2H, s, CH.sub.2), 1.979 (1H, s, 0H). .sup.13C NMR (75.5 Hz, CDCl.sub.3) δ 171.5, 78.0, 77.5, 77.1, 68.9, 67.4, 61.1. HRMS (ESI) calcd for C.sub.24H.sub.25N.sub.1O.sub.2Fe.sub.2 m/z: 477.3974 found 477.4213

(b) Synthesis of Label C from Diferrocenylglycine

(41) ##STR00031##

(42) Oxalyl chloride (0.87 mL) in dry DCM (2 mL) was added dropwise via a pressure equalising dropping funnel to a stirred solution of the di-ferrocenyl glycine derivative obtained in 3(a) above in dry DCM (100 mL) at 0° C. under N.sub.2. The reaction warmed to room temperature and stirred for 2 hrs. Then the solvent was removed and the acid chloride product was taken up in dry DCM (75 mL). 6-amino hexan-1-ol (0.56 g) in dry DCM (75 mL) was added dropwise via a dropping funnel at 0° C. under N.sub.2. The reaction was then stirred for 2 hrs while warming to room temperature. The solution was then washed with NaHCO.sub.3 (sat; 100 mL) and 1.0M HCL (100 mL). The organic fraction was dried over MgSO.sub.4 then the solvent was removed to yield the product (85%). An orange/yellow solid. .sup.1H NMR (250 MHz, CDCl.sub.3) δ 4.11 (12H, s, FcCp), 3.65 (4H, t, J=6.0 Hz, CH.sub.2), 3.55 (2H, s, CH.sub.2), 1.48-1.18 (5h, m, CH.sub.2). .sup.13C NMR (75.5 Hz, CDCl.sub.3) δ 173.32, 77.80, 77.39, 76.90, 62.10, 38.85, 35.45, 32.02, 30.67, 26.75, 25.54, 25.44. m/z: 576.

(43) The electrochemistry of the product compound Label Cis shown in Table 3 below and on the voltammogram of FIG. 4

(44) TABLE-US-00003 TABLE 3 Electrochemical activity of Label C Peak Position (mV) Peak Height 410 6.79e.sup.−6 425 8.47e.sup.−6 415 8.56e.sup.−6

Example 4: General Synthetic Procedure for Attaching Phosphoramidite Functional Group

(45) ##STR00032##

(46) The ferrocenyl derivative shown as a starting material in the above reaction scheme is illustrative, and may be replaced by a molar equivalent of any of the compounds made in Examples 1 to 3 above or Examples 9 to 13 below.

(47) N,N-diisopropylethylamine (0.4 mL, 8.4 mmol) was added to a stirred solution of the ferrocene derivative (2.1 mmol) in dry THF (25 mL) under a nitrogen atmosphere. 2-cyanoethyldiisopropylchlorophosphoramidite (0.2 ml, 3.15 mmol) was added dropwise and the resulting mixture was stirred for 15 mins. MilliQ filtered water (200 mL) was added and the solution was stirred for a further 30 mins. Ethyl Acetate-Triethylamine (1:1, 25 mL) was added, a precipitate formed. The mixture was washed with saturated NaCHCO.sub.3 (25 mL) and MilliQ filtered water (25 mL). The organic fraction was dried over MgSO.sub.4 and the solvent was removed under vacuo. The crude product was then purified by silica gel chromatography (petroleum ether:ethyl acetate 9:1).

(48) Using the above-described process with Label C as the ferrocenyl starting material, a phosphoramidite funtionalised compound of formula IX was obtained, having the characterising data listed below.

(49) ##STR00033##

(50) .sup.1H NMR (500 MHZ, CDCl.sub.3) δ 4.23 (2H, s, Cp), 4.18 (2H, s, Cp), 4.13 (15H, s, Cp), 3.90-3.82 (2H, m, CH.sub.2), 3.71-3.54 (4H, m, CH.sub.2), 3.44 (4H, s, CH.sub.2), 2.64 (2H, t, J=6, CH.sub.2), 2.35 (2H, t, J=6.5, CH.sub.2), 1.69-1.35 (85H, m, CH.sub.2, CH), 1.23 (12H, t, J=7, CH.sub.3). .sup.31P NMR (DEC) (202.5 Hz, CDCl.sub.3) δ 147.23. HRMS (ESI) calculated for C.sub.39H.sub.53N.sub.4O.sub.3Fe.sub.2P.sub.1 m/z: 768.0973 found 768.1254.

Example 5—Use of Label a Coupled to Oligonucleotide Probe

(51) Synthesis of oligonucleotide was carried out using standard oligonucleotide solid-phase synthesis techniques the nucleotides being added stepwise to the 5′ end of the oligonucleotide strand. Each addition to the oligonucleotide chain involves four reactions which are the de-blocking, coupling, capping and oxidation steps. Once the oligonucleotide sequence has been completed to the desired length the electrochemical label was added via a phosphoramidite linkage.

(52) Method

(53) The target sequence was amplified from a Chlamydia trachomatis target by a standard PCR method using 5′ and 3′ target specific primers and a uracil-DNA glycosylase (UDG) Step. PCR conditions are summarised in Table 1 below. When the PCR reaction was complete, a oligonucleotide probe (labelled with electrochemical Label A at the 5′ terminal) complementary to a sequence intermediate in position on the target between the 5′ and 3′ primers was added to the PCR reaction products and allowed to anneal to its target on the amplicon. T7 exonuclease (which is specific for double stranded nucleic acid) was added to the tube and incubated to allow it to digest dsRNA. Probe was digested by the T7 exonuclease to the extent that it was annealed to the PCR amplicon. Electrochemical detection was then carried out, showing a peak at a characteristic redox potential of 0.2V for the digest product nucleotide labelled with Label A.

(54) TABLE-US-00004 TABLE 4 Component Concentration PCR buffer 1X MgCl.sub.2 5 mM dUTP mix 1X Forward primer 0.04 μM Reverse primer 0.3 μM Taq 2.5 U UNG 0.5 U

(55) UNG protocol:

(56) 37° C.×10 minutes

(57) 94° C.×10 minutes

(58) PCR protocol:

(59) 94° C.×30 seconds

(60) 58° C.×45 seconds

(61) 72° C.×60 seconds

(62) Repeat steps×39 cycles (40 cycles in total)

(63) 72° C.×7 minutes

(64) Results

(65) FIG. 5 shows the peak height, as a current, at the known (label specific) redox potential for both a Chlamydia trachomatis positive sample (the strong peak) and negative sample (absence of peak).

(66) FIG. 6 shows a mass spec analysis of a mononucleotide and a dinucleotide synthesised with the label A electrochemical label. FIG. 7 is a mass spec analysis of the assay product, which shows exact correlation to a mononucleotide coupled to the label A electrochemical label, the electrochemical moiety detected in the assay.

(67) FIG. 8 shows the results of an experiment where the Chlamydia target is added directly to the PCR reaction at a range of concentrations. This is amplified using PCR, then detected using a label A oligonucleotide probe.

(68) FIG. 9 shows the result for an experiment where a range of concentrations of the Chlamydia target are added to a DNA extraction process (i.e. not added directly to the PCR) and the output from the extraction process is amplified using PCR, then detected using an oligonucleotide probe labelled with Label A.

Example 6—Comparison of Performance to qPCR

(69) a) FIG. 10 shows a comparison between electrochemical detection using the label A probe and a SYBR Green based qPCR assay for Norovirus. The materials and protocols are as follows:

(70) Bioline SensiMix dU Bioline 1×SYBR Green 0.3 μM forward primer 0.3 μM reverse primer 0.5 U UNG

(71) UNG protocol:

(72) 37° C.×10 minutes

(73) 60° C.×2 minutes

(74) qPCR protocol:

(75) 95° C.×10 minutes (taq activation step)

(76) 95° C.×15 seconds

(77) 45° C.×10 seconds

(78) 72° C.×10 seconds (SYBR acquisition)

(79) Repeat last 3 steps×39 cycles (40 cycles in total)

(80) 47° C.-95° C. in 1° C. increments (end-point melt to check for non-specific amplification)

(81) A decimalised inverse of the Ct value has been used for the qPCR results to provide a direct comparison to the electrochemical results.

(82) The data shows the limit of detection for the electrochemical assay to be 200 ag. The limit of detection for the qPCR assay is shown as between 2 and 20 fg. Below this level the Ct value is greater than 40-cycle cut-off, shown by the horizontal line as the inverse of a Ct value of 40. The qPCR negatives did not rise above the threshold. This demonstrates the advantageous sensitivity of the electrochemical assay using label A.

(83) b) FIG. 11 shows a comparison between electrochemical detection using the label A probe and a SYBR Green based qPCR assay for Streptococcus equi.

(84) The data shows that both the electrochemical assay and the qPCR assay are capable of detecting down to the lowest DNA concentration tested in this experiment (20 fg). The signal:noise ratio for the electrochemical detection at 20 fg was 4:1 in this experiment. 20 fg equates to 8 genomic copies of S. equi DNA.

Example 7—Label a Directly Bound to Protein

(85) a) FIG. 12a is a voltammetric scan using label A coupled directly to the primary amine of a commercially available anti-goat IgG, using an active NHS ester (N-hydroxysuccinimide ester.

(86) The following generalised reaction scheme illustrates attachment of the label to a free amine of, for example, a lysine residue in the anti-goat IgG.

(87) ##STR00034##

(88) Commercially available biotinylated goat IgG was immobilised onto a streptavidin coated microtitre well. The label A anti-goat IgG was then incubated in the well containing the immobilised goat IgG, this was then removed with washing. A papain solution was added to the well and incubated to allow digestion of the secondary antibody, with the resulting solution read electrochemically. The control in this experiment followed the same procedure, but the final incubation was carried out in buffer only, without papain.

(89) FIG. 12a shows that an electrochemical signal at the known oxidation potential for the label A is released when papain is present, but is not present in the absence of papain. This shows that the label A is directly bound to the antibody in this assay and signal is only observed when this antibody is digested, releasing the electrochemical label.

(90) b) The data in FIG. 12b uses the same model assay as in Example 7a) above, except that a papain digestion time course experiment is shown. In this experiment the control was the label A secondary antibody (anti-goat IgG) which was added to a well with no immobilised goat IgG antibody.

Example 8—Label A Bound to Microparticles

(91) A biotin molecule was coupled to label A. The biotinylation can be carried out in an automated oligonucleotide synthesiser or using standard laboratory conditions by reaction of ferrocenyl phosphoramidite label with N-hydroxysuccinimide (NHS) esters of biotin.

(92) Paramagnetic treptavidin particles were washed ×3 (phosphate buffer) and mixed with biotinylated label, followed by incubation for 1 hour at room temperature with mixing. The particles were washed ×2 (phosphate buffer) and washed ×1 (PCR buffer) They were resuspended in final buffer (PCR buffer)

(93) Following each wash step the supernatants were tested for electrochemical signal, and if necessary washing was repeated until the supernatants showed no indication of free electrochemical label.

(94) These particles were assayed at a range of concentrations to validate that the observed electrochemical signal was attributable to label A coupled to the magnetic particles. This involved magnetic capture of the particles and resuspension in a range of buffer volumes. The results are shown in FIG. 13.

Example 9—Synthesis of (N,N-diferrocenylmethyl-2-aminoethoxy) ethanol (Label D)

(95) ##STR00035##

(96) Ferrocene carboxaldehyde (2.1 g, 9.81 mmol) and (aminoethoxy) ethanol (0.5 g, 4.27 mmol) in dry THF (25 mL) were added to an oven dried flask. Sodium triacetoxyborohydride (2.3 g, 10.90 mmol) was added portionwise to the solution. The reaction was left overnight. The reaction was taken up in ethyl acetate (40 mL), the organic layer was washed with NaCO.sub.3 (sat; 20 mL), Brine (20 mL) and MilliQ water (20 mL). The organic fraction was then dried over magnesium sulfate and the solvent removed in vacuo. The crude product is then columned using 9:1 solution B: solution A (solution A: ethyl acetate 95% TEA 5%, solution B: Petroleum ether 40-60 95%, TEA 5%) to elicit the pure product label D (dark orange solid). 85% Yield .sup.1H NMR (300 MHz, CDCl.sub.3) δ 4.18 (2H, s, Cp), 4.17 (2H, s, Cp), 4.13 (15H, s, Cp), 3.66 (4H, t, J=6.25 Hz, CH.sub.2), 3.48 (2H, s, CH.sub.2), 2.20 (2H, t, J=6, CH.sub.2). .sup.13C NMR (75.5 Hz, CDCl.sub.3) δ 77.83, 77.40, 76.98, 70.58, 68.88, 63.36, 53.02, 52.17, 33.10, 27.43, 25.88. HRMS (ESI) calculated for C.sub.26H.sub.33N.sub.1O.sub.2Fe.sub.2 m/z 501.1430 found 501.1438.

(97) The electrochemistry of Label D is shown in the table below and on the voltammogram in FIG. 14:

(98) TABLE-US-00005 TABLE 5 Electrochemical activity of Label D Peak Position (mV) Peak Height 242 9.31e.sup.−6 245 9.38e.sup.−6 239 9.76e.sup.−6

Example 10—Synthesis of di-ferrocenyl glycine amino alcohol N,N-2-(diferrocenylmethylamino)acetyl-6-aminohexanol also named N-(6-hydroxylhexyl)-2-((diferrocenylmethyl) amino)-acetamide (Label E)

(99) ##STR00036##

(100) Oxalyl chloride (0.87 mL) in dry DCM (2 mL) was added dropwise via a pressure equalising dropping funnel to a stirred solution of di-ferrocenyl glycine (obtained as described in Example 3a) in dry DCM (100 mL) at 0° C. under N.sub.2. The reaction warmed to room temperature and was stirred for 2 hrs. Then the solvent was removed and the acid chloride product was taken up in dry DCM (75 mL). 6-aminohexan-1-ol (0.56 g) in dry DCM (75 mL) was added dropwise via a dropping funnel at 0° C. under N.sub.2. The reaction was then stirred for 2 hrs while warming to room temperature. The solution was then washed with NaHCO.sub.3 (sat; 100 mL) and 1.0M HCL (100 mL). The organic fraction was dried over MgSO.sub.4 then the solvent was removed to yield the product Label E (85%). An orange/yellow solid. .sup.1H NMR (250 MHz, CDCl.sub.3) δ 4.11 (12H, s, FcCp), 3.55 (4H, t, J=6.0 Hz, CH.sub.2), 3.31 (2H, s, CH.sub.2), 1.48-1.18 (12h, m, CH.sub.2). .sup.13C NMR (75.5 Hz, CDCl.sub.3) δ 173.32, 77.80, 77.39, 76.90, 62.10, 38.85, 35.45, 32.02, 30.67, 26.75, 25.54, 25.44. HRMS (ESI) calculated for C.sub.24H.sub.25N.sub.1O.sub.2Fe.sub.2 m/z: 576.0988 found 576.1264.

(101) The electrochemistry of Label E is illustrated in the data in the table below.

(102) TABLE-US-00006 TABLE 6 Electrochemical activity of Label E Peak Position Peak Height 504 6.61e.sup.−6 506 5.51e.sup.−6 507 3.28e.sup.−6

Example 11—6-(bis((1′-vinylferrocenyl)1-methylferrocenyl)amino)hexan-1-ol

(103) ##STR00037##

(104) 1′-Vinyl ferrocene carboxaldehyde (122 mg, 0.5 mmol) was dissolved in dry THF (5 cm.sup.3) and treated with 6-aminohexan-1-ol (29 mg, 0.25 mmol) and sodiumtrisacetoxyborohydride (205 mg, 1.25 mmol) successively. The solution was allowed to stir at room temperature overnight. After this time the reaction was quenched by addition of 10 cm.sup.3 saturated NaHCO.sub.3. The organic layer was separated, then the aqueous layer back extracted with ethyl acetate (3×10 cm.sup.3). Combined organic extracts were dried over magnesium sulfate, filtered then concentrated in vacuo to give a red solid. The product was purified by silica chromatography, eluting with 1:1 (ethyl acetate:hexane)+1% ammonium hydroxide to give the desired product as an orange oil 72 mg, in 50% yield.

(105) .sup.1H NMR (500 Mhz; CDCl.sub.3) δ.sub.H 6.38 (2H, dd, J 17.6, 10.7 ═CH), 5.30 (2H, dd, J 10.7, 1.5, ═CH.sub.2), 5.03 (2H, dd, J 10.7, 1.5, ═CH.sub.2), 4.25 (4H, t, J 1.8, CpH), 4.15 (4H, t, J 1.8, CpH), 4.07 (8H, s, CpH), 3.59 (2H, t, J 6.6, OCH.sub.2), 3.32 (4H, s, 2×F.sub.cCH.sub.2), 2.23, (2H, app t, J 7.4, NCH.sub.2), 1.49-1.55 (2H, m, CH.sub.2), 1.33-1.39 (2H, m, CH.sub.2), 1.22-1.33 (4H, m, CH.sub.2); .sup.13C NMR (125 Mhz; CDCl.sub.3) δ.sub.c 134.3, 111.3, 83.7, 83.6, 71.4, 69.2, 69.0, 67.2, 62.8, 52.1, 32.7, 27.1, 25.5; HRMS, m/z (ESI) 566.1825 (1.8%, [M+H], C.sub.32H.sub.39Fe.sub.2NO requires 566.1808); Electrode Potential: 298 mV.

Example 12: 6-(bis((1′-bromoferrocenyl).SUB.1.-methylferrocenyl)amino)hexan-1-ol

(106) ##STR00038##

(107) 1′-Bromo ferrocene carboxaldehyde (85 mg, 0.29 mmol) was dissolved in dry THF (3 cm.sup.3) and treated with 6-aminohexan-1-ol (25 mg, 0.144 mmol) and sodiumtrisacetoxyborohydride (59.3 mg, 0.36 mmol) successively. The solution was allowed to stir at room temperature overnight. After this time the reaction was quenched by addition of 5 cm.sup.3 saturated NaHCO.sub.3. The organic layer was separated, then the aqueous layer back extracted with ethyl acetate (3×10 cm.sup.3). Combined organic extracts were dried over magnesium sulfate, filtered then concentrated in vacuo to give a red solid. The product was purified by silica chromatography, eluting with 1:1 (ethyl acetate:hexane)+1% ammonium hydroxide to give the desired product as an yellow oil 17 mg, in 17% yield.

(108) .sup.1H NMR (500 Mhz; CDCl.sub.3) δ.sub.H 4.32, (4H, t, J 1.8, F.sub.cH), 4.21 (8H, s, F.sub.cH), 4.05 (4H, t, J 1.8, F.sub.cH), 3.63 (2H, t, J 6.5, OCH.sub.2), 3.46 (4H, s, F.sub.cCH.sub.2N), 2.31 (2H, t, J 7.1, NCH.sub.2), 1.45-1.58 (4H, m, CH.sub.2), 1.27-1.36 (4H, m, CH.sub.2); .sup.13C NMR (125 Mhz; CDCl.sub.3) δ.sub.c 78.4, 72.9, 70.8, 70.5, 68.6, 67.9, 63.1, 62.8, 52.1, 33.0, 27.3, 26.0; HRMS, m/z (ESI) 669.7929 (2.7%, [M+H], C.sub.28H.sub.34Fe.sub.2Br.sub.2NO requires 669.9705); Electrode Potential: 437 mV.

Example 13: 6-(bis((2-methylferrocenyl)methyl)amino)hexan-1-ol

(109) ##STR00039##

(110) Methylferrocenecarboxaldehyde (1 g, 5 mmol) was dissolved in dry THF (30 cm.sup.3). 6-aminohexan-1-ol (0.25 g, 2.13 mmol) was added. Then sodium triacetoxyborohydride (1.3 g, 6.16 mmol) was added to the reaction mixture. The solution was stirred under nitrogen at room temperature overnight. Ethyl acetate (20 cm.sup.3) and 1M NaOH (20 cm.sup.3) were then added and the organic layer was then extracted with saturated NaHCO.sub.3 (25 cm.sup.3), brine (25 cm.sup.3) and Milli Q filtered water (25 cm.sup.3) then dried over magnesium sulfate and the solvent removed in vacuo to yield an orange oil. The crude product is then columned using 9:1 solution B: solution A (solution A: ethyl acetate 95% TEA 5%, solution B: petroleum ether 40-60 95%, TEA 5%) to elicit the pure product (orange oil). (0.95, 65%). .sup.1H NMR (300 MHz; CDCl.sub.3) δ.sub.H 4.18 (2H, s, CpH), 4.17 (2H, s, CpH), 4.13 (15H, s, CpH), 3.66 (4H, t, J 6.25, CH.sub.2), 3.48 (2H, s, CH.sub.2), 2.36 (3H, s, CH.sub.3), 2.20 (2H, t, J 6.1, CH.sub.2), 1.59-1.31 (6H, m, CH.sub.2). .sup.13C NMR (75.5 Hz; CDCl.sub.3) δ.sub.c 77.8, 77.4, 76.9, 70.5, 68.9, 63.3, 53.0, 52.1, 33.1, 27.4, 25.8, 12.4; HRMS, m/z (ESI) 539.8156 (10%, [M+H], C.sub.30H.sub.47N.sub.1O.sub.1Fe.sub.2 requires 539.8232); Electrode potential: 330 mV.

(111) TABLE-US-00007 TABLE 7 Effect on electrode potential of substituents on ferrocenyl moieties of Label A Example Fc substituent Electrode potential 1 None 275 mV 2 Dimethylaminomethyl 380 mV 11 1′-vinyl 298 mV 12 1′-bromo 437 mV 13 2-methyl 330 mV

(112) The data in the above table illustrates how inclusion of a substituent on the ferrocenyl moieties and the selection of that substituent may be used to influence the electrode potential. This enables the electrochemical detection of compounds to be carried out under a variety of different conditions, for example selecting an optimum measurement potential or avoiding conditions under which measuring sensitivity may be compromised by interference with impurities that may be present. Furthermore, the use of labels with different electrode potentials allows for the development of multiplex reactions, in which more than one determination can be carried out in the same sample.