PHENAZINIUM MEDIATORS

Abstract

The present invention relates to a chemical compound or a salt or solvate thereof being an 1-amino-phenazine derivative and to uses thereof. The present invention further relates to a chemistry matrix and to a test element comprising the aforesaid chemical compound. Moreover, the present invention relates to a method for determining the amount of an analyte in a sample, comprising contacting said sample with a chemistry matrix according to the present invention, estimating the amount of electrons liberated or consumed by the chemistry matrix in the presence of said liquid sample, and thereby determining the amount of an analyte in a liquid sample.

Claims

1. A chemical compound or a salt or solvate thereof comprising the structure (I) ##STR00019## wherein X is —C(═O)—, —C(═S)—, or —S(═O).sub.2—, R.sup.1 is an organic side chain comprising at least 2 C-atoms if X is C(═O), and at least 1 C-atom if X is C(═S) or S(═O).sub.2, R.sup.2 is an organic side chain comprising at least 2 C-atoms, R.sup.3 is H or an organic side chain, and wherein at least one of R.sup.1, R.sup.2 and R.sup.3 is a hydrophilic side chain.

2. The chemical compound according to claim 1, wherein said hydrophilic side chain is a side chain comprising at least one hydrophilic functional group selected from the groups consisting of —C(═Y.sup.1)—OH, —C(OH)R.sup.11R.sup.12, —C(═Y.sup.1)—R.sup.11, —C(═Y.sup.1)—Y.sup.2—R.sup.11, —Y.sup.1—R.sup.11, —NH.sub.2, —NHR.sup.11, —NMe.sup.3+, —NH—C(═Y.sup.1)—R.sup.11, —S(O)R.sup.11, —SO.sub.2R.sup.11, —SO.sub.2—OH—, and —P(O)(OR.sup.11)(OR.sup.12) —O—P(O)(OR.sup.11)(OR.sup.12); with Y.sup.1 and Y.sup.2 being independently selected from O or S and with R.sup.11 and R.sup.12 being, independently of each other, selected from the group consisting of H and, unsubstituted or substituted, alkyl and aryl.

3. The chemical compound according to claim 2, wherein the at least one hydrophilic functional group is —C(═O)— or —C(═O)—OH.

4. The chemical compound according to claim 1, wherein R.sup.1 is alkyl with a contiguous chain of 3 to 8 C-atoms covalently bound to the C or S atom of group X, comprising at least one substituent independently selected from OH, OPO.sub.3.sup.2−, PO.sub.3.sup.2−, SO.sub.3.sup.−, and COO.sup.−.

5. The chemical compound according to claim 1, wherein R.sup.2 has the structure —(CH.sub.2).sub.n—CH.sub.3 with n being in the range of from 0 to 6.

6. The chemical compound according to claim 5, with n being 0, 1 or 2.

7. The chemical compound according to claim 6, wherein R.sup.2 is ethyl.

8. The chemical compound according to claim 1, having the structure (II) ##STR00020## wherein R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9and R.sup.10 are, independently of each other, selected from the group consisting of H; substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkinyl, aryl, heterocycloalkyl, heteroaryl, halogen; —NO.sub.2, —SO.sub.3.sup.−—CN, —CH═CH—COOH, and —Y.sup.3—R.sup.13 with Y.sup.3 being —O—, —C(═O)— or —N(R.sup.14)—, with R.sup.13 and R.sup.14 being, independently of each other, selected from the group consisting of unsubstituted or substituted, alkyl and aryl.

9. The chemical compound according to claim 6, wherein R.sup.3, R.sup.4, R.sup.6, R.sup.7, R.sup.8, R.sup.9and R.sup.10 are —H.

10. The chemical compound according to claim 1 comprising one of the following structures: ##STR00021## ##STR00022##

11. The chemical compound according to claim 1 consisting of one of the following structures: ##STR00023## ##STR00024##

12. A chemistry matrix comprising the chemical compound of claim 1.

13. A test element comprising the chemical compound of claim 1.

14. A test element comprising the chemistry matrix of claim 12.

15. A method for determining the amount of an analyte in a sample, comprising a) contacting said sample with a chemistry matrix according to claim 12, b) estimating the amount of redox equivalents liberated or consumed by the chemistry matrix in the presence of said liquid sample, and c) thereby determining the amount of an analyte in a liquid sample.

16. The method of claim 15, wherein the amount of redox equivalents liberated or consumed by the chemistry matrix in step b) is estimated by means of an optical or by an electrochemical sensor.

Description

[0088] In the Figures:

[0089] FIG. 1.) cyclic voltammograms of 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium (compound of formula (III)). A: Buffer pH 7.0 (blank current), B: 2 mM 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium in buffer, C: 2 mM 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium+1.2 mM ascorbic acid in buffer, D: 1.2 mM ascorbic acid in buffer.

[0090] FIG. 2A.) dose response curve of ascorbic acid interference; Chronoamperometry at −100 mV, pH 7.0; solution of 5 mM 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium, 35 mM cNAD, and 1.5 kU/g glucose dehydrogenase (GDH) was incubated with the glucose concentrations indicated. A: 0 mg/mL ascorbic acid, B: 30 mg/mL ascorbic acid, C: 100 mg/mL ascorbic acid; Linear: linear regression;

[0091] FIG. 2B.) dose response curve of ascorbic acid interference; Chronoamperometry at+650 mV, pH 7.0; solution of 5 mM 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium, 35 mM cNAD, and 1.5 kU/g glucose dehydrogenase (GDH) was incubated with the glucose concentrations indicated, A: 0 mg/mL ascorbic acid, B: 30 mg/mL ascorbic acid, C: 100 mg/mL ascorbic acid; Linear: linear regression.

[0092] FIG. 3.) Pot life of mediator formulations. Mediators 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium and 1-(3-Carboxypropoxy)-5-ethylphenazinium were compared in a pot life experiment. Pot life of 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium (A: 0 h, B: 48 h) and 1-(3-Carboxypropoxy)-5-ethylphenazinium (C:=0 h, D: 48 h) are shown; Linear: linear regression.

[0093] FIG. 4A.) Dose response curves for 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium (CPEP) and for 1-(3-Carboxypropoxy)-5-ethylphenazinium (CEPES) in glucose measurement strips. Shown are linear regression lines for five replicas in the absence of ascorbate (full lines) and in the presence of ascorbate (dashed lines). R.sup.2 for 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium was 0.9962 in the absence of ascorbate and was 0.9834 in the presence of ascorbate. R.sup.2 for 1-(3-Carboxypropoxy)-5-ethylphenazinium was 0.9728 in the absence of ascorbate and was 0.999 in the presence of ascorbate.

[0094] FIG. 4B.) Current offset caused by ascorbate with 1-(3-Carboxypropoxy)-5-ethylphenazinium (CEPES) in glucose measurement strips at low glucose concentrations; same data as in (A), detail view of values measured with CEPES at low glucose concentrations.

DETAILED DESCRIPTION OF THE EMBODIMENTS (EXAMPLES)

[0095] Key intermediates for the synthesis of compounds according to the invention are 1-amino phenazines, which can be synthesized by various methods (see Urleb, U. and Gobec, S., Science of Synthesis, 2004, 16, 913-943). 1-amino phenazine is then reacted with an acyl- or sulfonylchloride and alkylated, optionally after removal of a protecting group. For phenazinium salts with an aryl group on the phenazinium nitrogen, a different synthesis method can be used, see Kehrmann and Masslenikow; Chemische Berichte, 1911, 44, 2629.

Example 1

Synthesis of 1-amino-phenazine

[0096] ##STR00010##

[0097] A solution of sodium methanolate (25% in MeOH, 24.6 ml, 107 mmol) in 100 ml MeOH was cooled to −78° C. and a solution of bromine (2.10 ml, 40.9 mmol) in 10.0 ml MeOH was added over a period of 2 min. Under further cooling the solution was first stirred 5 min followed by the addition of phenazine-1-carboxamide (4.00 g, 17.9 mmol) in 200 ml dry methanol and 400 ml dry THF over a period of 1 h via dropping funnel. After the complete addition a clear orange solution was obtained that was warmed to room temperature and further stirred 2 h at 55° C. Following the mixture was cooled down to room temperature and stirred further 72 h. After evaporating under reduced pressure the residue was dissolved in methanol (300 ml) and aqueous NaOH (40%, 150 ml) and refluxed for 4 h at 90° C. Subsequently the solution was cooled down to 0° C. and set to pH 8.5 with concentrated HCl, obtaining a dark red suspension. After concentrating to about 200 ml under reduced pressure 500 ml water was added. The mixture was extracted three times with CHCl.sub.3. The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (n-hexane/ethyl acetate, 80:20->75:25) obtaining 2.86 g (82%) of the title compound as dark red solid.

Example 2

Synthesis of N-phenazine-1-yl-succinamic Acid Methyl Ester

[0098] ##STR00011##

[0099] 1-Aminophenazine (1.00 g, 5.12 mmol) was dissolved in 40.0 ml CH.sub.2Cl.sub.2 and N,N-diisopropylethylamine (957 μl, 5.63 mmol). After addition of 4-N,N-dimethylaminopyridine (31.3 mg, 0.256 mmol) the mixture was cooled to 0° C., followed by the addition of methyl 4-chloro-4-oxobutyrate (693 μl, 5.63 mmol) over a period of 5 min. The resulting solution was further stirred for 16 h at room temperature. After diluting with 50.0 ml CH.sub.2Cl.sub.2 the mixture was washed once with 50.0 ml aqueous NaOH (0.5%). The organic layer was dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (n-hexane/acetone 80:20) obtaining 1.45 g (92%) of the title compound as yellow solid.

Example 3

Synthesis of 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium trifluoroacetate

[0100] ##STR00012##

[0101] Ethyl trifluoromethanesulfonate (542 μl, 4.18 mmol) was added dropwise to a solution of N-Phenazin-1-yl-succinamic acid methyl ester (64.6 mg, 0.208 mmol) in 2.00 ml CH.sub.2Cl.sub.2. Subsequently the mixture was refluxed 3.5 h at 50° C. and stirred 16 h at room temperature. Then 50.0 ml CH.sub.2Cl.sub.2 and 2.00 ml NEt.sub.3 were added and the resulting solution was extracted two times with water. The combined aqueous layers were washed once with CHCl.sub.3 and lyophilized to obtain 42.0 mg crude product. This was purified by preparative HPLC (Chromolith, H.sub.2O/CH.sub.3CN gradient+0.1% TFA) resulting 28.9 mg (43%) of the title compound as dark purple crystals.

Example 4

Synthesis of N-Phenazin-1-yl-methanesulfonamide

[0102] ##STR00013##

[0103] 1-Amino-phenazine (50.0 mg, 0.256 mmol) was diluted in pyridine (1.00 ml) and cooled to 0° C. Under further cooling methanesulfonyl chloride (23.8 μl, 0.307 mmol) was added. The mixture was stirred 5 min at 0° C. and subsequently 16 h at room temperature. After concentrating under reduced pressure, the crude product was purified by silica gel chromatography (n-hexane/acetone 80:20) obtaining 66.0 mg (94%) of the title compound as yellow solid.

Example 5

Synthesis of 5-Ethyl-1-methanesulfonylamino-phenazinium trifluoracetate

[0104] ##STR00014##

[0105] N-Phenazin-1-yl-methanesulfonamide (20.0 mg, 0.073 mmol) was diluted in CHCl.sub.3 (2.00 ml) and ethyl trifluormethanesulfonate (1.00 ml, 7.70 mmol) was added turning the mixture red immediately. The mixture was refluxed at 70° C. for 7 h and stirred at room temperature for 16 h. Then N-Ethyldiisopropylamine (250 μl, 1.46 mmol) was added turning the color from dark red to brown. This mixture was further refluxed for 8 h and stirred at room temperature for 16 h. The crude product obtained after concentrating under reduced pressure was diluted in 10.0 ml CHCl.sub.3 and 10.0 ml water. The organic layer was extracted four times with water. The combined aqueous layers were reduced to dryness and purified over preparative HPLC (Chromolith; H.sub.2O/TFA-gradient+0.1% TFA) obtaining 2.2 mg (7%) as dark blue solid.

Example 6

Synthesis of Decanedioic Acid bis-phenazin-1-ylamide bistrifluoroacetate Salt

[0106] ##STR00015##

[0107] 1-Amino-phenazine (25.0 mg, 0.128 mmol) was dissolved in pyridine (1.30 ml) and cooled to 0° C. To this solution sebacoyl chloride (13.7 μl, 0.064 mmol) in 0.50 ml CH.sub.2Cl.sub.2 was added slowly over a period of 30 min. The resulting suspension was further stirred 48 h at room temperature. Following the mixture was diluted with triethylammonium acetate buffer (pH 7, 1M, 5.00 ml) and extracted three times with CH.sub.2Cl.sub.2. The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure.

[0108] The obtained crude product was suspended in 3.00 ml H.sub.2O/CH.sub.3CN (1:1+0.1% TFA) and filtered. The residue, mainly containing the title compound was used without further purification. Yield: 12.3 mg (34%) as yellow solid.

Example 7

Synthesis of Decanedioic Acid bis-[(5-ethyl-phenazin-1-yl)-amide

[0109] ##STR00016##

[0110] To a suspension of decanedioic acid bis-phenazin-1-ylamide ditrifluoroacetate salt (12.3 mg, 0.016 mmol) in CH.sub.2Cl.sub.2 (3.00 ml) was added diisopropylethylamine (37.4 μl, 0.22 mmol) and ethyl trifluoromethanesulfonate (300 μl, 2.31 mmol). The resulting brown solution was refluxed 3 h at 55° C. and further stirred for 16 h at room temperature. After evaporating under reduced pressure the obtained crude product was purified by preparative HPLC (XTerra, H.sub.2O/CH.sub.3CN gradient+0.1% TFA), yielding 0.9 mg (9%) of the title compound as dark purple crystals.

Example 8

Synthesis of Pentanedioic Acid bis-phenazin-1-ylamide

[0111] ##STR00017##

[0112] To a solution of 1-Amino phenazine (50.0 mg, 0.256 mmol) was added diisopropyl ethylamine (87.0 μl, 0.512 mmol) and a catalytically amount of dimethylaminopyridine. To the resulting red solution glutaryl chloride (16.3 μl, 0.128 mmol) was added and stirred for 16 h at room temperature. The obtained orange suspension was diluted with water and extracted two times with CH.sub.2Cl.sub.2. The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The resulting crude product was suspended in acidic acid and filtered. The obtained residue was further purified by silica gel chromatography (CHCl.sub.3/acetone, 9:1) yielding 15.8 mg (13%) of the title compound as yellow solid.

Example 9

Synthesis of 5-Ethyl-1-[4-(phenazin-1-ylcarbamoyl)-butyrylamino]-phenazinium trifluoroacetat

[0113] ##STR00018##

[0114] Pentanedioic acid bis-phenazin-1-ylamide diacetate salt (15.8 mg, 0.032 mmol) was suspended in CH.sub.2Cl.sub.2 (3.00 ml) and added with ethyl triflate (500 μl, 3.86 mmol). Diisopropyl ethylamine (48.9 μl, 0.288 mmol) was added to the resulting red brown suspension followed by refluxing for 1.5 h at 50° C. After 16 h stirring at room temperature the mixture was refluxed again for 7 h followed by further stirring at room temperature for 16 h. The resulting clear purple solution was concentrated under reduced pressure. The obtained crude product was suspended in 3.00 ml H.sub.2O/CH.sub.3CN (1:1+0.1% TFA) and filtered. The residue was further purified by preparative HPLC (XTerra, H.sub.2O/CH.sub.3CN gradient+0.1% TFA), yielding 1.0 mg (6%) of the title compound as redbrown solid.

Example 10

Redox Potential of 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium

[0115] The formal redox potential of a typical 1-Acylated amino phenazinium ethosulfate, 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium (compound of formula (Ill)), was measured vs. Ag/AgCl at a gold working electrode in a test strip. We obtained −236 mV vs. Ag/AgCl under physiological conditions (0.9% NaCl) by cyclic voltammetry, shown in FIG. 1. The cyclic voltammograms show, that a relatively low potential of −100 mV vs. Ag/AgCl is sufficient in order to oxidize the substance.

[0116] The quasi reversible oxidation and reduction of the mediator 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium occurs in a potential range between 0 mV and −500 mV vs. Ag/AgCl via two electron transfer. Ascorbic acid cannot be oxidized in this potential window and the current is similar to the blank current of the pure buffer solution. Adding ascorbic acid to the mediator 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium does not significantly change the anodic and cathodic currents and the redox potential is just shifted to −231 mV vs. Ag/AgCl. Therefore, the addition of ascorbic acid does not significantly reduce the redox mediator.

Example 11

ascorbate Interference With 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium

[0117] FIG. 2 shows the advantage of the low oxidation potential of −100 mV using the redox mediator 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium in comparison to the direct oxidation of cNADH at +650 mV. At the latter potential the ascorbic acid will be oxidized too and the blank current increases rapidly depending on the concentration of ascorbic acid. Therefore, the relatively low potential of −100 mV is very useful in order to avoid a direct oxidation of interfering substances and the blank current remains very close to zero even though the sample contains high concentrations of ascorbic acid. Currents were measured under conventional conditions (pH 7.0) in the presence of cNAD (35 mM), glucose dehydrogenase (1.5 kU/g) at various concentrations of ascorbate and glucose.

Example 12

Pot Life of 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium and 1-(3-Carboxypropoxy)-5-ethylphenazinium

[0118] Mediators 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium and 1-(3-Carboxypropoxy)-5-ethylphenazinium were compared in a pot life experiment. Performance of mediators was measured immediately after preparing the reaction mixture (t=0) and after 48 h. As shown in FIG. 3, both redox mediators show no significant decrease in current after 48 hours of pot life. Therefore, both mediators seem to be very stable in a formulation. The mediator 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium shows higher currents than the mediator 1-(3-Carboxypropoxy)-5-ethylphenazinium over the entire range of glucose concentrations.

Example 13

Ascorbate Interference With Other 1-amino-penazine Derivatives

[0119] 1-Acetylamino-5-methyl-phenazinium trifluormethanesulfonate, 1-Acetylamino-5-ethyl-phenazinium trifluoroacetate, and 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium (formula III) were compared regarding their reactivity towards ascorbate. To this end, each 0.23 mM of the respective compound was incubated at room temperature in 0.1 M triethylammonium acetate buffer (pH 7) in the presence of a 5 fold molar excess of ascorbate. Decrease of absorption at 517 nm (1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium) or 512 nm (other two compounds) was recorded over time. Whereas with 1-Acetylamino-5-methyl-phenazinium, the absorption decreased by 12% per minute, the decrease slowed to 7% per minute for 1-Acetylamino-5-ethyl-phenazinium, and to less than 5% for 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium.

Example 14

Performance and Ascorbate Interference With 1-hydroxy-phenazine Derivatives

[0120] 1-(3-Carboxypropoxy)-5-ethylphenazinium and 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium were compared regarding their performance as redox mediators in glucose test strips in the absence and in the presence of ascorbate. To this end, dose response curves were recorded with glucose concentrations of 0 mg/dL,10 mg/dL (0.5 mM), 30 mg/dL (1.5 mM), and 80 mg/dL (4.0 mM) and in the presence of 1.48 mM of either redox mediator. Ascorbic acid, if present, was used at a concentration of 15 mg/dL (0.85 mM). As shown in FIG. 4A), the dose response at a given glucose concentration is higher with 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium (CPEP) as compared to 1-(3-Carboxypropoxy)-5-ethylphenazinium (CEPES). Also, the slope dose response is approximately twofold for 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium as compared to 1-(3-Carboxypropoxy)-5-ethylphenazinium (34 nA*dL/mg vs. 16.7 nA*dL/mg). Moreover, ascorbate only has a small effect on the currents measured in the presence of 1-(3-Carboxy-propionylamino)-5-ethyl-phenazinium. In contrast, ascorbate causes a current offset at low glucose concentrations, in particular below 30 mg/dL when 1-(3-Carboxypropoxy)-5-ethylphenazinium is used as a redox mediator (FIG. 4B)).