Redoxindicators

Abstract

The present invention relates to a chemical compound or a salt or solvate thereof being a phenazine-, phenanthridine-, phenanthroline-, quinoline-, quinoxaline-, acridine- isoquinoline-, pyrazine- or pyridine-derivative comprising a conjugated -system and a -acceptor group, and to uses thereof. The present invention further relates to a chemistry matrix and to a test element comprising the chemical compound of the present invention. The present invention also relates to a method of determining the amount of an analyte in a test sample, comprising contacting said test sample with a chemical compound, with a chemistry matrix, or with a test element of the invention and estimating the amount of redox equivalents liberated or consumed by the chemical compound, by the chemical compound comprised in said chemistry matrix, or by the chemical compound comprised in said test element, in the presence of said test sample, thereby determining the amount of an analyte in said test sample. Moreover, the present invention relates to a system comprising a test element according to the present invention and a device comprising a sensor for measuring the amount of redox equivalents liberated or consumed.

Claims

1. A tricyclic chemical compound or a salt or solvate thereof, said tricyclic chemical compound comprising a tricyclic heterocyclic group covalently bound to a -acceptor group, having the general structure of formula (I) ##STR00056## wherein X is CH or N, R.sup.1, R.sup.2, R.sup.3, R.sup.4 are independently selected from hydrogen; alkyl, in an embodiment lower alkyl; unsubstituted or substituted aryl, in an embodiment ArR.sup.9 or phenyl; halide; nitro; sulfonate; CN; COOH; OR.sup.9; SR.sup.9; SSR.sup.9; C(O)OR.sup.9; C(O)NHR.sup.9; NHC(O)R.sup.9; C(O)NH.sub.2; with R.sup.9 selected from; alkyl, in an embodiment lower alkyl; or unsubstituted or substituted aryl, in an embodiment phenyl; and/or wherein R.sup.a and R.sup.a+1, with a=1, 2, or 3, together form a bridge to form a 5- to 6-membered cycloalkyl, aryl, heterocycloalkyl or heteroaryl ring; R.sup.5 and R.sup.6 are independently selected organic side chains, in an embodiment methyl, in a further embodiment ethyl, in a further embodiment phenyl, n is an integer between 0 and 5, in an embodiment 0, 1, or 2, m is an integer selected from 0 and 1, R.sup.7 is H or an organic side chain, R.sup.8 is an organic side chain, or wherein R.sup.8 and R.sup.7 together form a bridge to form an, optionally substituted, 5- to 6-membered heterocycloalkyl or heteroaryl ring.

2. The tricyclic chemical compound or a salt or solvate thereof of claim 1, wherein (i) X is N and wherein the -system is covalently bonded to C1, C2, C3, or C4 of the heteroaromatic system as indicated in formula (I), or (ii) X is CH and wherein the -system is covalently bonded to C2 or C4 of the heteroaromatic system as indicated in formula (I).

3. The tricyclic chemical compound or a salt or solvate thereof of claim 1 or 2, (i) X is N and wherein the -system is covalently bonded to C1 of the heteroaromatic system as indicated in formula (I), or (ii) X is CH and wherein the -system is covalently bonded to C2 of the heteroaromatic system as indicated in formula (I).

4. The tricyclic chemical compound or a salt or solvate thereof of any one of claims 1 to 3, wherein the -acceptor group is selected from the list consisting of (i) a side chain comprising the structure of formula (II) ##STR00057## wherein Y is N(Me)-, S, Se, O, or C(Me).sub.2-, (ii) a side chain comprising the structure of formula (III) ##STR00058## (iii) a side chain comprising the structure of formula (IV) ##STR00059## and (iv) a side chain comprising the structure of formula (V) ##STR00060## wherein in each of formulas (II) to (V), R.sup.10 is alkyl or cycloalkyl, in an embodiment methyl.

5. The tricyclic chemical compound or a salt or solvate thereof of any one of claims 1 to 4, wherein said chemical compound or a salt or solvate thereof is a compound undergoing a bathochromic shift upon reduction.

6. The tricyclic chemical compound or a salt or solvate thereof of any one of claims 1 to 5, wherein in said chemical compound or salt or solvate thereof the -acceptor group is not reduced by reduced coenzymes NADH, FADH, or PQQH.

7. A chemistry matrix comprising a redox cofactor and a chemical compound or a salt or solvate thereof, said chemical compound comprising a heterocyclic group (Het) covalently bound to a -acceptor group (Acc), having the general structure of formula (VI) ##STR00061## wherein Het comprises a structure selected from formulas VII to XV: ##STR00062## wherein R.sup.11 is an organic side chain, in an embodiment methyl, in a further embodiment ethyl, in a further embodiment phenyl, wherein the -(vinylene).sub.1Acc group is attached to one of the carbon atoms indicated as C*, wherein 1 is an integer between 0 and 5, in an embodiment 0, 1, or 2, and wherein Acc is selected from (CO)Aryl, (CC(CN).sub.2), and an acceptor group of the general formula (XVI) ##STR00063## wherein R.sup.12 is an organic side chain, in an embodiment methyl, in a further embodiment ethyl, in a further embodiment phenyl, k is an integer selected from 0 and 1, R.sup.13 is H or an organic side chain, R.sup.14 is an organic side chain, or wherein R.sup.13 and R.sup.14 together form a bridge to form a 5- to 6-membered heterocycloalkyl or heteroaryl ring.

8. The chemistry matrix of claim 7, wherein said chemical compound comprises or consists of a structure of any one of (XVII) to (XXVII), (XXX) to (XXXII), and (LII): ##STR00064## ##STR00065## ##STR00066##

9. The chemistry matrix of claim 7 or 8, wherein said chemical compound is a compound according to any one of claims 1 to 6.

10. The chemistry matrix of any one of claims 7 to 9, further comprising an oxidoreductase enzyme.

11. The chemistry matrix of any one of claims 7 to 10, wherein said redox cofactor is nicotine adenine dinucleotide phosphate (NADP), pyrroloquinoline quinone (PQQ), flavine adenine dinucleotide (FAD), or, in an embodiment, nicotine adenine dinucleotide (NAD) or carbaNAD, or, in an embodiment, a reduced form of any of the aforesaid redox cofactors.

12. The chemistry matrix of any one of claims 7 to 11, wherein said chemical compound is a compound undergoing a bathochromic shift upon reduction.

13. The chemistry matrix of any one of claims 7 to 12, wherein in said chemical compound the -acceptor group is not reduced by reduced coenzymes NADH, FADH, or PQQH.

14. A test element comprising the chemical compound of any one of claims 1 to 6 and/or the chemistry matrix of any one of claims 7 to 13.

15. Use of a tricyclic chemical compound according to any one of claims 1 to 6 or of a chemical compound as structurally defined in any one of claims 7 to 9, of a chemistry matrix according to any one of claims 7 to 13, or of a test element according to claim 14 in an analytical or diagnostic test.

16. Use of a tricyclic chemical compound or a salt or solvate thereof according to any one of claims 1 to 6 or of a chemical compound as structurally defined in any one of claims 7 to 9 for analyzing the viability or metabolism of cells.

17. Use of a tricyclic chemical compound or a salt or solvate thereof according to any one of claims 1 to 6 or of a chemical compound as structurally defined in any one of claims 7 to 9 for producing a chemistry matrix or for producing a test element.

18. A method for determining the amount of an analyte in a test sample, comprising a) contacting said test sample with a tricyclic chemical compound according to any one of claims 1 to 6, with a chemical compound as structurally defined in any one of claims 7 to 9, with a chemistry matrix according to any one of claims 7 to 13, or with a test element according to claim 14, b) estimating the amount of redox equivalents liberated or consumed by the tricyclic chemical compound, by the chemical compound comprised in said chemistry matrix, or by the chemical compound comprised in said test element, in the presence of said test sample, and c) thereby determining the amount of an analyte in said test sample.

19. A system for determining the amount of an analyte in a sample, comprising a) a test element according to claim 14 and b) a device comprising a sensor for measuring the amount of redox equivalents liberated or consumed in said test element.

Description

[0111] In the figures:

[0112] FIG. 1 Representative time-dependent UV/Vis spectra upon treating the indicator 1-methyl-3-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)ethenyl]quinolinium iodide trifluoromethanesulfonate (0.5 mM) with NADH (20 M) in 50 mM phosphate buffer pH 7, showing a change in absorbance with max at 535 nm after adding NADH. The arrow indicates increasing time after addition of NADH; NADH: no NADH added. x-axis: wavelength (, (nm)), y-axis: absorbance.

[0113] FIG. 2 Determination of NADH with the indicator 1-methyl-6-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)vinyl] quinolinium iodide trifluoromethanesulfonate. Absorbance change at 516 nm after 10 min vs. NADH concentration. x-axis: Absorption change at 516 nm, y-axis: NADH concentration (mM).

[0114] FIG. 3 Representative UV/Vis spectra of the kinetics upon treating of two solutions of the indicator 1-methyl-3-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)ethenyl]quinolinium iodide trifluoromethanesulfonate with NADH (disodium salt, 20 M) or sodium ascorbate (asc, 20 M) in phosphate buffer pH 7, respectively. x-axis: time (min), y-axis: absorbance.

[0115] FIG. 4 Representative time-dependent UV/Vis spectra upon treating a mixture of 1-methyl-6-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)vinyl]quinolinium iodide trifluoromethanesulfonate (0.5 mM) and NAD/GlucDH2 with glucose (385 M) in 100 mM phosphate buffer pH 7, showing a change in absorbance with max at 516 nm run after adding glucose. The arrow indicates increasing time after glucose addition; glc: no glucose added. x-axis: wavelength () (nm), y-axis: absorbance.

[0116] FIG. 5 Representative UV/Vis spectra of the kinetics upon treating the mixture of 1-methyl-6-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)vinyl]quinolinium iodide trifluoromethanesulfonate (0.5 mM) and NAD/GlucDH2 with glucose (0-385 M) in 100 mM phosphate buffer pH 7. The arrow indicates increasing glucose concentrations (119 M, 244 M, and 385 M, respectively); glc: no glucose added. x-axis: time (min), y-axis: absorbance.

[0117] FIG. 6 Calibration plot corresponding to Abs of the determination of glucose with the mixture of 1-methyl-6-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)vinyl]quinolinium iodide trifluoromethanesulfonate (0.5 mM) and NAD/GlucDH2 in 100 mM phosphate buffer pH 7 recorded at 517 nm after 20 min. Each point in the figure is a mean of three replicates, error bars represent the standard deviation. x-axis: glucose concentration (glc (M)), y-axis: Abs.

[0118] FIG. 7 Representative UV/Vis spectra of the kinetics upon treating the mixture of 1-methyl-6-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)vinyl]quinolinium iodide trifluoromethanesulfonate (0.5 mM) and carbaNAD/GlucDH2 with glucose (0-385 M) in 100 mM phosphate buffer pH 7. The arrow indicates increasing glucose concentrations (119 M, 244 M, and 385 M, respectively); glc: no glucose added. x-axis: time (min), y-axis: absorbance.

[0119] FIG. 8 Calibration plot corresponding Abs of the determination of glucose with the mixture of 1-methyl-6-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)vinyl]quinolinium iodide trifluoromethanesulfonate (0.5 mM) and carbaNAD/GlucDH2 in 100 mM phosphate buffer pH 7 at 517 nm recorded after 20 min. Each point in the figure is a mean of three replicates, error bars represent the standard deviation. x-axis: glucose concentration (M), y-axis: Abs.

[0120] FIG. 9 Effects of the initial concentration of NADH on the change in absorbance of the reduced form of 1-methyl-3-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)ethenyl]quinolinium iodide trifluoromethanesulfonate recorded at 535 nm. Each point in the figure is a mean of three replicates; error bars represent the standard deviation. x-axis: initial NADH concentration ([NADH].sub.0 (M)), y-axis: absorption change at 535 nm (Abs).

[0121] FIG. 10 Representative fluorescence emission spectra upon treating the indicator 1-methyl-3-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)ethenyl]quinolinium iodide trifluoromethanesulfonate (20 M) with NADH (4 M) in 10 mM PIPES pH 7, showing a change in fluorescence intensity with max at 560 nm after adding NADH using excitation at 535 nm; NADH: no NADH added. x-axis: emission ( (nm)), y-axis: fluorescence intensity.

[0122] FIG. 11 Calibration plot correlating fluorescence changes of the indicator 1-methyl-3-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)ethenyl]quinolinium iodide trifluoromethanesulfonate (20 M) with NADH concentration (0-4 M). Measurements were performed in 10 mM PIPES pH 7 at 560 nm and were recorded after 30 min incubation, using excitation at 535 nm. x-axis: initial NADH concentration ([NADH].sub.0 (M)), y-axis: fluorescence intensity.

EXAMPLES

Example 1

1-Methyl-6-((E)-3-oxo-3-phenyl-propenyl)-quinolinium methosulfate (MOPPQ)

1.1 Synthesis

1.1.1 Synthesis, Step 1: Synthesis of (E)-1-phenyl-3-quinolin-6-yl-propenone (XIV)

[0123] ##STR00016##

[0124] To a solution of 6-Quinolinecarbaldehyde (XXXIII) (500 mg, 3.18 mmol) in 50.0 ml EtOH and 6.40 ml NaOH (10% in H.sub.20) Acctophenone (XXXIV) (0.371 ml, 3.18 mmol) was added. The mixture was stirred at room temperature for 16 h and subsequently concentrated under reduced pressure. The remaining crude product was purified by silica gel chromatography (n-hexane/acetone; 75:25) obtaining 117.4 mg (14%) of the title compound.

1.1.2. Synthesis, Step 2: Synthesis of 1-methyl-6-((E)-3-oxo-3-phenyl-propenyl)-quinolinium methosulfate (MOPPQ, (XXII))

[0125] ##STR00017##

[0126] To a solution of (E)-1-phenyl-3-quinolin-6-yl-propenone (XXXV) (80.0 mg, 0.309 mmol) in 3.00 ml acetone dimethylsulfate (0.587 ml, 6.17 mmol) was added. The mixture was stirred 16 h at room temperature. The obtained suspension was filtered and the remaining precipitate was washed 3 times with acetone. The crude product was purified by preparative HPLC (H.sub.2O/CH.sub.3CN) obtaining 22.0 mg (18%) of the title compound (XXII).

1.2 Measurements

1.2.1 MOPPQ (XXII), Approximate Evaluation of the Reaction Rates with NADH and Ascorbate

[0127] Two solutions of the redoxindicator 1-methyl-6-((E)-3-oxo-3-phenyl-propenyl)-quinolinium methosulfate (5.00 mg, 0.013 mmol) in 1.00 ml water were each treated with an excess of NADH (disodium salt) or sodium ascorbate, respectively. The solution treated with NADH changed rapidly from nearly colorless to orange. After a few minutes, a larger amount of an orange precipitate was obtained, probably the insoluble dihydrochinoline. In contrast, the solution treated with ascorbate showed only a pale orange color. Even after 16 h no precipitate was found. Thus, turnover rates of MOPPQ with NADH are much higher than with ascorbate.

Example 2

9-ethoxyphenazine-1-carbaldehyde

2.1 Synthesis, Step 1: Synthesis of (9-ethoxyphenazin-1-yl)methanol

[0128] ##STR00018##

[0129] A suspension of 9-ethoxyphenazine-1-carboxylic acid (XXXVI) (1.50 g, 5.59 mmol) (synthesized from N-(2,6-difluorophenyl)-3-nitroanthranilic acid by a method in Rewcastle, G. W.; Denny, W. A. Synth. Commun. 1987, 17, 1171) in DMF (15 mL) was treated with 1,1-carbonyldiimidazole (CDI) (1.85 g, 11.40 mmol), and the mixture was stirred at 50 C. for 1 h. After cooling, the mixture was diluted with DCM/petroleum ether (1:1) to complete precipitation of the imidazolide, which was collected, washed with petroleum ether, dried, dissolved in THF (200 mL) then slowly added to a solution of NaBH.sub.4 in H.sub.2O (50 mL). After stirring for 1 h, the mixture was neutralized by dropwise addition of concd HCl and then extracted with EtOAc. The organic layer was washed with aqueous Na.sub.2CO.sub.3, water, dried (Na.sub.2SO.sub.4), and evaporated, to give (9-ethoxyphenazin-1-yl)methanol (XXXVII) (1.20 g).

[0130] .sup.1H NMR (CHLOROFORM-d, 400 MHz): [ppm]=8.16 (dd, J=8.8, 1.3 Hz, 1H), 7.81 (dd, J=8.8, 1.3 Hz, 1H), 7.79 (dd, J=8.8, 6.8 Hz, 1H), 7.76 (dd, J=8.8, 7.3 Hz, 1H), 7.68 (dd, J=6.8, 1.0 Hz, 1H), 7.06 (dd, J=7.3, 1.3 Hz, 1H), 5.36 (s, 2H), 5.26 (br. s., 1H), 4.34 (q, J=7.0 Hz, 2H), 1.66 (t, J=6.9 Hz, 3H)

[0131] .sup.13C NMR (CHLOROFORM-d, 101 MHz): [ppm]=154.4, 144.2, 143.7, 141.1, 139.1, 135.1, 131.0, 130.6, 128.8, 127.7, 120.9, 107.6, 64.8, 64.6, 14.7

[0132] LC-MS m/z 255.2 ([M+H].sup.+)

2.2 Synthesis, Step 2: Synthesis of 9-ethoxyphenazine-1-carbaldehyde

[0133] ##STR00019##

[0134] A mixture of (9-ethoxyphenazin-1-yl)methanol (XXXVII) (750 mg, 2.95 mmol), activated manganese (IV) oxide (5.65 g, 58.5 mmol) and DCM (50 mL) was stirred at room temperature under an argon atmosphere for 3 h. After this time, the reaction was filtered through a pad of silica gel and concentrated. The residue was dissolved in EtOAc and the solution obtained was washed with aqueous Na.sub.2CO.sub.3, water, dried (Na.sub.2SO.sub.4), evaporated and dried under vacuum for 24 h to give 9-ethoxyphenazine-1-carbaldehyde (XXXVIII) (530 mg) as a yellow solid.

[0135] .sup.1H NMR (CHLOROFORM-d, 400 MHz): [ppm]=11.63 (s, 1H), 8.52 (dd, J=8.7, 1.4 Hz, 1H), 8.48 (dd, J=7.1, 1.5 Hz, 1H), 7.99 (dd, J=8.3, 7.3 Hz, 1H), 7.78-7.87 (m, 2H), 7.14 (dd, J=6.8, 1.8 Hz, 1H), 4.39 (q, J=7.0 Hz, 2H), 1.70 (t, J=7.1 Hz, 3H)

[0136] .sup.13C NMR (CHLOROFORM-d, 101 MHz): [ppm]=191.3, 154.7, 144.4, 142.6, 141.1, 137.1, 135.7, 132.1, 131.6, 130.2, 130.0, 121.2, 108.4, 65.1, 14.7

[0137] LC-MS m/z 253.1 ([M+H].sup.+)

Example 3

Synthesis of 2-[(E)-2-(9-ethoxyphenazin-1-yl)vinyl]-1,3,3-trimethyl-3H-indolium iodide (XL)

[0138] ##STR00020##

[0139] A mixture of 9-ethoxyphenazine-1-carbaldehyde (XXXVIII) (120 mg, 0.40 mmol) and 1,2,3,3-tetramethyl-3H-indolium iodide (XXXIX) (99 mg, 0.33 mmol) in ethanol (10 ml) was heated to reflux under an argon atmosphere for 3 h in the presence of piperidine (10 L, 0.10 mmol). The reaction mixture was allowed to cool slowly to room temperature, and a red precipitate was filtered off, washed with cold ethanol, then with diethyl ether and dried. 120 mg, red powder was obtained.

[0140] .sup.1H NMR (DMSO-d.sub.6, 500 MHz): [ppm]=9.58 (d, J=16.7 Hz, 1H), 8.94 (d, J=6.9 Hz, 1H), 8.49 (d, J=8.5 Hz, 1H), 8.48 (d, J=16.7 Hz, 1H), 8.19 (dd, J=8.4, 7.4 Hz, 1H), 7.93-8.02 (m, 3H), 7.85 (d, J=8.5 Hz, 1H), 7.64-7.76 (m, 2H), 7.40 (d, J=7.6 Hz, 1H), 4.41 (q, J=6.8 Hz, 2H), 4.28 (s, 3H), 1.96 (s, 6H), 1.62 (t, J=6.9 Hz, 3H)

[0141] .sup.13C NMR (DMSO-d.sub.6, 101 MHz): [ppm]=182.0, 154.1, 147.3, 144.0, 143.7, 142.6, 141.9, 139.5, 135.5, 134.0, 132.5 (2C), 132.4, 131.1, 129.8, 129.2, 123.1, 120.4, 116.1, 115.6, 109.0, 64.5, 52.4, 34.9, 25.9 (2C), 14.8

[0142] LC-MS m/z 408.0 (M.sup.+)

Example 4

Synthesis of (9E)-10,10-dimethyl-9-(quinolin-3-ylmethylene)-7,8,9,10-tetrahydro-6H-pyrido[1,2-a]indolium hexafluorophosphate (XLII)

[0143] ##STR00021##

[0144] A mixture of quinoline-3-carbaldehyde (XXXIII) (500 mg, 3.18 mmol) and 10,10-dimethyl-7,8,9,10-tetrahydro-6H-pyrido[1,2-a]indolium hexafluorophosphate (XLI) (922 mg, 2.67 mmol) (synthesized from 2,3,3-trimethyl-3H-indole by a method in Mushkalo, I. L.; Turova, L. S.; Murovanaya, N. V. Dopov. Akad. Nauk Ukr. RSR, Ser. B: Geol., Khim. Biol. Nauki 1979, 1022) in ethanol (25 ml) was heated to reflux under an argon atmosphere for 16 h in the presence of piperidine (105 L, 1.06 mmol). The reaction mixture was allowed to cool slowly to room temperature, and a precipitate was filtered off, washed with cold ethanol, then with diethyl ether and dried. 1.0 g, yellow powder was obtained.

[0145] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): [ppm]=9.26 (d, J=2.0 Hz, 1H), 8.83 (s, 1H), 8.30 (s, 1H), 8.17 (d, J=7.8 Hz, 1H), 8.11 (d, J=8.3 Hz, 1H), 7.90-7.98 (m, 3H), 7.74 (t, J=7.5 Hz, 1H), 7.64-7.70 (m, 2H), 4.46 (t, J=5.6 Hz, 2H), 3.12 (t, J=5.2 Hz, 2H), 2.21 (t, J=5.6 Hz, 2H), 1.89 (s, 6H)

[0146] .sup.13C NMR (DMSO-d.sub.6, 101 MHz): [ppm]=181.03, 152.74, 147.89, 144.80, 144.54, 141.34, 139.27, 132.27, 130.51, 129.79, 129.47, 129.22, 128.33, 128.12, 127.99, 127.29, 123.39, 115.58, 53.16, 46.16, 26.15 (2C), 24.18, 19.73

[0147] LC-MS m/z 339.1 (M.sup.+)

Example 5

Synthesis of Indolium Salts

[0148] The indolium salts listed in Table 1 are obtained analogously to example 3-4

TABLE-US-00001 TABLE 1 LC-MS m/z, Structure (M.sup.+) Remarks [00022] 263.1 .sup.1H NMR (DMSO-d.sub.6, 400 MHz): [ppm] = 9.30 (d, J = 1.8 Hz, 1 H), 8.75 (dd, J = 4.8, 1.5 Hz, 1 H), 8.65 (dt, J = 8.1, 1.8 Hz, 1 H), 8.44 (d, J = 16.7 Hz, 1 H), 7.87-7.99 (m, 2 H), 7.82 (d, J = 16.7 Hz, 1 H), 7.65 (s, 3 H), 4.19 (s, 3 H), 1.81 (s, 6 H) .sup.13C NMR (DMSO-d.sub.6, 101 MHz): [ppm] = 181.8, 152.9, 151.6, 149.3, 143.8, 141.8, 136.1, 130.3, 129.8, 129.1, 124.2, 122.9, 115.6, 115.0, 52.5, 34.9, 25.0 (2C) [00023] 261.1 (M.sup.2+) .sup.1H NMR (DMSO-d.sub.6, 400 MHz): [ppm] = 9.10 (d, J = 2.3 Hz, 2 H), 8.60 (dd, J = 8.5, 2.4 Hz, 2 H), 8.39 (d, J = 16.4 Hz, 2 H), 7.87-7.99 (m, 6 H), 7.84 (d, J = 16.7 Hz, 2 H), 7.61-7.72 (m, 4 H), 4.19 (s, 6 H), 1.80 (s, 12 H) .sup.13C NMR (DMSO-d.sub.6, 101 MHz): [ppm] = 181.7, 152.4, 147.7, 145.1, 143.9, 141.8, 138.6, 130.1, 130.0, 129.1, 128.7, 122.9, 115.7 (4C), 52.5, 35.0, 24.9 (4C) [00024] 313.1 .sup.1H NMR (DMSO-d.sub.6, 400 MHz): [ppm] = 9.66 (d, J = 2.0 Hz, 1 H), 9.23 (d, J = 1.8 Hz, 1 H), 8.62 (d, J = 16.4 Hz, 1 H), 8.08-8.15 (m, 2 H), 7.99 (d, J = 16.7 Hz, 1 H), 7.86-7.99 (m, 3 H), 7.76 (ddd, J = 8.0, 6.9, 1.0 Hz, 1 H), 7.63-7.70 (m, 2 H), 4.25 (s, 3 H), 1.86 (s, 6 H) .sup.13C NMR (DMSO-d.sub.6, 101 MHz): [ppm] = 181.7, 150.6, 149.5, 148.7, 143.8, 141.8, 138.5, 132.2, 129.8, 129.3, 129.1, 129.0, 127.9, 127.7, 127.1, 122.9, 115.5, 114.8, 52.4, 34.9, 25.1 (2C) [00025] 313.17 .sup.1H NMR (DMSO-d.sub.6, 600 MHz): [ppm] = 9.04 (dd, J = 4.1, 1.7 Hz, 1 H), 8.83 (d, J = 1.8 Hz, 1 H), 8.63 (dd, J = 8.9, 2.0 Hz, 1 H), 8.62 (d, J = 16.4 Hz, 1 H), 8.48 (dd, J = 8.4, 1.2 Hz, 1 H), 8.17 (d, J = 8.9 Hz, 1 H), 7.90- 7.98 (m, 2 H), 7.87 (d, J = 16.4 Hz, 1 H), 7.64-7.70 (m, 3 H), 4.23 (s, 3 H), 1.85 (s, 6 H) .sup.13C NMR (DMSO-d.sub.6, 151 MHz): [ppm] = 181.8, 152.9, 151.7, 149.3, 143.7, 141.8, 137.2, 133.7, 132.6, 129.9, 129.6, 129.0, 128.0, 127.8, 122.9, 122.7, 115.4, 114.4, 52.4, 34.8, 25.1 [00026] 313.2 .sup.1H NMR (DMSO-d.sub.6, 400 MHz): [ppm] = 9.53 (s, 1 H), 9.34 (s, 1 H), 8.94 (d, J = 16.4 Hz, 1 H), 8.44 (d, J = 8.3 Hz, 1 H), 8.31 (d, J = 8.1 Hz, 1 H), 7.97-8.09 (m, 2 H), 7.90 (d, J = 16.4 Hz, 1 H), 7.83-7.95 (m, 2 H), 7.64-7.73 (m, 2 H), 4.25 (s, 3 H), 1.88 (s, 6 H) .sup.13C NMR (DMSO-d.sub.6, 101 MHz): [ppm] = 182.0, 156.0, 146.3, 143.7, 143.5, 141.9, 132.9, 132.4, 129.9, 129.1, 128.8, 128.5, 127.7, 125.5, 123.0, 122.9, 116.9, 115.7, 52.7, 35.1, 25.2 (2C) [00027] 314.1 .sup.1H NMR (DMSO-d.sub.6, 400 MHz): [ppm] = 9.41 (d, J = 16.7 Hz, 1 H), 9.18 (d, J = 1.8 Hz, 1 H), 9.14 (d, J = 1.8 Hz, 1 H), 8.91 (dd, J = 7.5, 0.9 Hz, 1 H), 8.39 (dd, J = 8.3, 1.0 Hz, 1 H), 8.16 (d, J = 16.9 Hz, 1 H), 8.12 (t, J = 8.1 Hz, 1 H), 7.95-8.01 (m, 1 H), 7.89-7.95 (m, 1 H), 7.65-7.71 (m, 2 H), 4.24 (s, 3 H), 1.87 (s, 6 H) .sup.13C NMR (DMSO-d.sub.6, 101 MHz): [ppm] = 181.9, 146.9, 146.3, 146.1, 143.6, 142.4, 141.9, 140.4, 134.0, 132.0, 130.5, 130.3, 129.8, 129.2, 122.9, 115.7, 115.6, 52.4, 34.9, 25.7 [00028] 364.18 .sup.1H NMR (DMSO-d.sub.6, 600 MHz): [ppm] = 9.46 (d, J = 16.6 Hz, 1 H), 8.99 (d, J = 6.9 Hz, 1 H), 8.54 (dd, J = 8.7, 1.1 Hz, 1 H), 8.50 (d, J = 16.6 Hz, 1 H), 8.39-8.43 (m, 1 H), 8.33- 8.37 (m, 1 H), 8.21 (dd, J = 8.7, 7.2 Hz, 1 H), 8.06-8.14 (m, 2 H), 8.00-8.04 (m, 1 H), 7.94-7.99 (m, 1 H), 7.68- 7.73 (m, 2 H), 4.28 (s, 3 H), 1.95 (s, 6 H) .sup.13C NMR (DMSO-d.sub.6, 151 MHz): [ppm] = 182.0, 146.9, 143.6, 143.2, 142.8, 142.1, 141.9, 140.9, 134.2, 133.3, 132.3, 132.0 (2C), 130.8, 129.8, 129.7, 129.4, 129.1, 123.0, 116.3, 115.6, 52.4, 34.9, 25.7 (2C) [00029] 364.1806 .sup.1H NMR (DMSO-d.sub.6, 500 MHz): [ppm] = 9.13 (d, J = 1.9 Hz, 1 H), 8.77 (dd, J = 9.5, 1.9 Hz, 1 H), 8.70 (d, J = 16.4 Hz, 1 H), 8.39 (d, J = 9.5 Hz, 1 H), 8.27- 8.33 (m, 2 H), 8.02-8.07 (m, 2 H), 7.99 (d, J = 16.4 Hz, 1 H), 7.95-7.99 (m, 1 H), 7.90-7.95 (m, 1 H), 7.62-7.73 (m, 2 H), 4.27 (s, 3 H), 1.88 (s, 6 H) .sup.13C NMR (DMSO-d.sub.6, 126 MHz): [ppm] = 181.7, 150.6, 144.3, 144.0, 143.6, 143.5, 142.7, 141.9, 136.7, 134.4, 132.4, 131.8, 130.2, 129.9, 129.6, 129.4, 129.1, 128.9, 123.0, 116.1, 115.6, 52.5, 35.0, 25.0 (2C)

Example 6

Synthesis of 1-methyl-3-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)ethenyl]quinolinium iodide trifluoromethanesulfonate (XXIV)

[0149] ##STR00030##

[0150] To a solution of 1,3,3-trimethyl-2-[(E)-2-(quinolin-3-yl)ethenyl]-3H-indolium iodide (XLV) (100 mg, 0.23 mmol) in 8 mL of dry DCM was added TfOMe (103 L, 0.91 mmol) in dry DCM (2 mL) dropwise under an argon atmosphere. After the mixture was stirred for 16 h at room temperature, a precipitate was filtered off, washed with DCM (510 ml) and then with diethyl ether, and dried. 127 mg, yellow powder was obtained.

[0151] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): [ppm]=10.16 (s, 1H), 9.93 (s, 1H), 8.61 (d, J=8.6 Hz, 1H), 8.59 (d, J=16.7 Hz, 1H), 8.50 (d, J=7.8 Hz, 1H), 8.37-8.44 (m, 1H), 8.17 (t, J=7.6 Hz, 1H), 8.05 (d, J=16.7 Hz, 1H), 8.00-8.03 (m, 1H), 7.92-7.97 (m, 1H), 7.68-7.76 (m, 2H), 4.70 (s, 3H), 4.26 (s, 3H), 1.85 (s, 6H)

[0152] .sup.13C NMR (DMSO-d.sub.6, 101 MHz): [ppm]=181.3, 150.9, 146.4, 144.8, 144.1, 141.9, 138.5, 137.2, 131.4, 131.1, 130.4, 129.3, 128.7, 128.3, 123.1, 122.3 (TfO.sup.), 119.7, 119.1 (TfO.sup.), 117.4, 116.0, 52.7, 46.1, 35.3, 24.7 (2C)

[0153] LC-MS m/z 327.2 ([MH].sup.+), 345.1 ([M+HO.sup.].sup.+)

[0154] UV-Vis (50 mM phosphate buffer pH=7): max 512 (weak), 384, 311 nm; after reduction with NADH: 535, 528sh nm;

[0155] Solubility in water: 10 mM

Example 7

Synthesis of Diquaternary Salts

[0156] The diquaternary salts listed in Table 2 are obtained by reacting indolium salts (from examples 3-5) with various triflate alkylating agents analogously to example 6. The temperature and time of reaction can usually be varied over wide ranges. The product is crystallized from a suitable solvent and, if required, the anion can be changed by conventional procedures, for example by the use of ion exchange resins.

TABLE-US-00002 TABLE 2 Structure Remarks [00031] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): [ppm] = 9.63 (s, 1 H), 9.26 (d, J = 8.3 Hz, 1 H), 9.09 (d, J = 6.1 Hz, 1 H), 8.41 (d, J = 16.7 Hz, 1 H), 8.32 (dd, J = 8.1, 6.3 Hz, 1 H), 8.01 (s, 1 H), 7.97 (d, J = 16.9 Hz, 1 H), 7.90-7.94 (m, 1 H), 7.67-7.75 (m, 2 H), 4.42 (s, 3 H), 4.23 (s, 3 H), 1.81 (s, 6 H) .sup.13C NMR (DMSO-d.sub.6, 101 MHz): [ppm] = 181.3, 146.9, 146.7, 144.1, 143.7, 143.4, 141.8, 133.9, 130.5, 129.3, 127.8, 123.0, 122.3 (TfO.sup.), 119.1 (TfO.sup.), 118.6, 116.1, 52.8, 48.4, 35.4, 24.5 (2C) LC-MS m/z 277.1 ([M H].sup.+), 295.2 ([M + HO.sup.].sup.+) UV-Vis (50 mM phosphate buffer pH = 7): max 364, 290, 251 nm; after reduction with NADH: 546, 530sh. nm; Solubility in water: 114 mM [00032] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): [ppm] = 8.78 (d, J = 2.5 Hz, 2 H), 8.39 (dd, J = 9.6, 2.5 Hz, 2 H), 8.25 (d, J = 15.9 Hz, 2 H), 7.76- 7.87 (m, 4 H), 7.58 (quind, J = 7.4, 1.1 Hz, 4 H), 7.31 (d, J = 16.2 Hz, 2 H), 6.63 (d, J = 9.6 Hz, 2 H), 4.04 (s, 6 H), 3.54 (s, 6 H), 1.74 (s, 12 H) .sup.13C NMR (DMSO-d.sub.6, 101 MHz): [ppm] = 181.1, 161.6, 150.0, 149.2, 143.1, 141.9, 136.6, 128.9, 128.8, 122.8, 122.3 (TfO.sup.), 119.7, 119.1 (TfO.sup.), 114.7, 114.6, 108.5, 51.6, 37.6, 33.9, 25.6 (4C) UV-Vis (water): max 369, 310sh, 218 nm; after reduction with NADH: 650, 538 nm; Solubility in water: 15 mM [00033] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): [ppm] = 10.17 (s, 1 H), 9.94 (s, 1 H), 8.71 (d, J = 8.8 Hz, 1 H), 8.59 (d, J = 16.7 Hz, 1 H), 8.51 (d, J = 8.1 Hz, 1 H), 8.39 (t, J = 8.0 Hz, 1 H), 8.16 (t, J = 7.5 Hz, 1 H), 8.06 (d, J = 16.7 Hz, 1 H), 8.02 (s, 1 H), 7.92- 7.97 (m, 1 H), 7.70- 7.75 (m, 2 H), 5.14 (q, J = 7.1 Hz, 2 H), 4.27 (s, 3 H), 1.86 (s, 6 H), 1.72 (t, J = 7.2 Hz, 3 H) .sup.13C NMR (DMSO-d.sub.6, 101 MHz): [ppm] = 181.3, 150.2, 146.3, 144.9, 144.0, 141.8, 137.5, 137.4, 131.8, 130.9, 130.4, 129.3, 129.2, 128.5, 123.0, 122.3 (TfO.sup.), 119.3, 119.1 (TfO.sup.), 117.4, 116.0, 53.9, 52.7, 35.3, 24.7 (2C), 15.0 LC-MS m/z 341.2 ([M H].sup.+), 359.1 ([M + HO.sup.].sup.+) UV-Vis (50 mM phosphate buffer pH = 7): max 512 (weak), 384, 311, 241 nm; after reduction with NADH: 535, 528sh nm; Solubility in water: 4 mM [00034] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): [ppm] = 9.91 (s, 1 H), 9.54 (s, 1 H), 8.68 (d, J = 8.8 Hz, 1 H), 8.61 (d, J = 8.1 Hz, 1 H), 8.37 (t, J = 7.8 Hz, 1 H), 8.23 (s, 1 H), 8.14 (t, J = 7.8 Hz, 1 H), 8.00 (d, J = 7.6 Hz, 1 H), 7.95 (d, J = 7.1 Hz, 1 H), 7.72 (quin, J = 6.7 Hz, 2 H), 5.15 (q, J = 7.1 Hz, 2 H), 4.52 (t, J = 5.3 Hz, 2 H), 3.07-3.20 (m, J = 5.1, 5.1 Hz, 2 H), 2.18- 2.30 (m, J = 4.8, 4.8 Hz, 2 H), 1.90 (s, 6 H), 1.69 (t, J = 7.1 Hz, 3 H) .sup.13C NMR (DMSO-d.sub.6, 101 MHz): [ppm] = 180.1, 151.7, 147.2, 147.2, 144.3, 141.0, 139.7, 137.0, 136.5, 131.8, 130.6, 130.6, 130.2, 129.2, 129.2, 128.1, 123.0, 119.0, 115.6, 53.6, 53.0, 46.2, 25.4 (2C), 23.1, 19.2, 15.2 LC-MS m/z 367.1 ([M H].sup.+), 385.1 ([M + HO.sup.].sup.+) UV-Vis (50 mM phosphate buffer pH = 7): max 361, 237, 212 nm; after reduction with NADH: 550, 530sh nm (Cl.sup. form); Solubility in water: 0.3 mM (Cl.sup. form) [00035] .sup.1H NMR (DMSO-d.sub.6, 600 MHz): [ppm] = 9.56 (d, J = 5.7 Hz, 1 H), 9.26 (d, J = 8.4 Hz, 1 H), 9.16 (s, 1 H), 9.04 (dd, J = 9.4, 1.9 Hz, 1 H), 8.68 (d, J = 9.3 Hz, 1 H), 8.64 (d, J = 16.5 Hz, 1 H), 8.26 (dd, J = 8.4, 5.7 Hz, 1 H), 8.01 (d, J = 16.6 Hz, 1 H), 7.96-8.01 (m, 1 H), 7.92-7.96 (m, 1 H), 7.67-7.73 (m, 2 H), 4.69 (s, 3 H), 4.26 (s, 3 H), 1.85 (s, 6 H) (Cl.sup. form) .sup.13C NMR (METHANOL-d.sub.4, 151 MHz): [ppm] = 184.2, 152.6, 150.7, 150.8, 149.7, 145.5, 143.4, 141.9, 137.4, 135.8, 134.5, 132.0, 130.9, 124.3, 124.2, 121.3, 118.4, 116.9, 54.7, 46.7, 36.0, 25.9 (2C) (Cl.sup. form) LC-MS m/z 164.10 (M.sup.2+) UV-Vis (50 mM phosphate buffer pH = 7): max 377, 317 nm; after reduction with NADH: 517 nm; Solubility in water: 15 mM [00036] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): [ppm] = 10.10 (s, 1 H), 9.35 (s, 1 H), 8.86 (d, J = 16.4 Hz, 1 H), 8.67 (d, J = 8.6 Hz, 1 H), 8.60 (d, J = 8.3 Hz, 1 H), 8.42 (t, J = 7.6 Hz, 1 H), 8.19 t, J = 7.6 Hz, 1 H), 8.02-8.08 (m, 1 H), 7.95- 8.00 (m, 1 H), 7.89 (d, J = 16.2 Hz, 1 H), 7.69-7.77 (m, 2 H), 4.56 (s, 3 H), 4.28 (s, 3 H), 1.88 (s, 6 H) LC-MS m/z 327.2 ([M H].sup.+), 345.1 ([M + HO.sup.].sup.+) UV-Vis (water): max 371 nm; after reduction with NADH: 520sh, 492 nm; Solubility in water: 25 mM [00037] .sup.1H NMR (DMSO-d.sub.6, 400 MHz): [ppm] = 10.19 (s, 1 H), 9.41 (s, 1 H), 8.85 (d, J = 16.4 Hz, 1 H), 8.67 (d, J = 8.6 Hz, 1 H), 8.60 (d, J = 8.3 Hz, 1 H), 8.43 (t, J = 7.7 Hz, 1 H), 8.20 (t, J = 7.6 Hz, 1 H), 8.03-8.09 (m, 1 H), 7.95- 7.99 (m, 1 H), 7.91 (d, J = 16.4 Hz, 1 H), 7.70-7.79 (m, 2 H), 4.83 (q, J = 7.2 Hz, 2 H), 4.29 (s, 3 H), 1.88 (s, 6 H), 1.75 (t, J = 7.3 Hz, 3 H) LC-MS m/z 341.2 ([M H].sup.+), 359.1 ([M + HO.sup.].sup.+) [00038] .sup.1H NMR (METHANOL-d.sub.4, 400 MHz): [ppm] = 9.76 (d, J = 3.0 Hz, 1 H), 9.57 (d, J = 2.3 Hz, 1 H), 9.51 (d, J = 16.7 Hz, 1 H), 9.12 (d, J = 7.6 Hz, 1 H), 8.82 (d, J = 8.8 Hz, 1 H), 8.52 (dd, J = 8.7, 7.7 Hz, 1 H), 8.17 (d, J = 16.9 Hz, 1 H), 7.95 (s, 1 H), 7.86 (s, 1 H), 7.69-7.77 (m, 2 H), 4.88 (s, 3 H), 4.34 (s, 3 H), 1.95 (s, 6 H) UV-Vis (water): max 408, 347, 247 nm; after reduction with NADH: 535sh nm; Solubility in water: 9 mM [00039] .sup.1H NMR (MeOD, 400 MHz): [ppm] = 9.70 (d, J = 16.7 Hz, 1 H), 9.13 (d, J = 6.3 Hz, 1 H), 9.05 (d, J = 8.8 Hz, 1 H), 8.90 (d, J = 9.1 Hz, 1 H), 8.79-8.87 (m, 1 H), 8.56-8.74 (m, 2 H), 8.38-8.45 (m, 1 H), 8.29 (d, J = 16.4 Hz, 1 H), 7.93-8.00 (m, 1 H), 7.85-7.92 (m, 1 H), 7.67-7.80 (m, 2 H), 5.15 (br. s., 3 H), 4.38 (s, 3 H), 2.03 (s, 6 H) LC-MS m/z 189.60 (M.sup.2+) UV-Vis (50 mM phosphate buffer pH = 7): max 481, 394, 377sh., 261 nm; after reduction with NADH: 806sh., 740 nm; Solubility in water: 4 mM [00040] LC-MS m/z 423.1 (M.sup.+) HRMS m/z 422.2237 ([M H].sup.+) UV-Vis (50 mM phosphate buffer pH = 7): max 516sh., 442, 390, 278 nm; after reduction with NADH: 746 nm; Solubility in water: 7 mM [00041] LC-MS m/z 379.3 (M.sup.+) HRMS m/z 378.1970 ([M H].sup.+) UV-Vis (water) after reduction with NADH: max 740 nm;

Example 8

Reaction of an Indicator with NADH

[0157] The following were mixed in a cuvette: 50 L of 10 mM solution of an indicator (1-methyl-3-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)ethenyl]quinolinium iodide trifluoromethanesulfonate) in distilled water, 950 L of 50 mM phosphate buffer pH 7, 2 L of 10 mM NADH solution in distilled water. A UV/Vis spectrum (400-1000 nm) was recorded every 7.5 sec for a 1 min after addition of NADH (resolution 2 nm) (FIG. 1). No further color change could be measured after 52.5 sec.

[0158] UV/Vis spectral properties of further indicators according to the invention listed in Table 3.

TABLE-US-00003 TABLE 3 Redoxindicators. UV/Vis studies UV/Vis oxidized UV/Vis reduced form (nm) form (nm) 50 mM phosphate 50 mM phosphate Indicator buffer pH = 7 buffer pH = 7 [00042] 364, 290, 251 546, 530sh. [00043] 512 (weak), 384, 311 535, 528sh [00044] 384, 311, 241 535, 528sh [00045] 361, 237, 212 550, 530sh. [00046] 377, 317 517 [00047] 481, 394, 377sh., 261 806sh., 740 [00048] 516sh., 442, 390, 278 746

Example 9

Determination of NADH

[0159] The following were mixed in a cuvette: 100 L of 10 mM solution of an indicator (1-methyl-6-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)vinyl]quinolinium iodide trifluoromethanesulfonate) in distilled water, 1000 L of 50 mM phosphate buffer pH 7, 20-80 L of 10 mM NADH solution in distilled water. Change in absorbance at 516 nm was recorded after 10 min (FIG. 2).

Example 10

1-Methyl-3-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)ethenyl]quinolinium iodide trifluoromethanesulfonate, Evaluation of the Reaction Rates with NADH and Ascorbate

[0160] Two solutions of the redoxindicators 1-methyl-3-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)ethenyl]quinolinium iodide trifluoromethanesulfonate (0.005 mmol) in 1.00 ml of 50 mM phosphate buffer pH 7 were each treated with 2 L of 10 mM NADH (disodium salt) or sodium ascorbate solutions in distilled water, respectively. The absorbance at 535 nm was measured in relation to time (FIG. 3). The solution treated with NADH changed rapidly from nearly colorless to pink. In contrast, the solution treated with ascorbate did not change color. Thus, turnover rates of 1-methyl-3-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)ethenyl]quinolinium iodide trifluoromethanesultrifluoromethanesulfonate with NADH are much higher than with ascorbate.

Example 11

Determination of Glucose with GlucDH2/an Indicator

[0161] 11.1 Measurement 1: Change in Absorbance Upon Reaction with Glucose and GlucDH2/NAD

[0162] The following were mixed in a cuvette: 500 L of 1.0 mM solution of an indicator (1-methyl-6-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)vinyl]quinolinium iodide trifluoromethanesulfonate) in 100 mM phosphate buffer pH 7, 500 L of glucose dehydrogenase 2 (GlucDH2) solution (in 100 mM phosphate buffer pH 7 containing 4.0 mM NAD.sup.+) at a concentration of 491 U/ml, 40 L of 10 mM solution of glucose in distilled water. A UV/Vis spectrum (300-1000 nm) was recorded every 1 min for a 10 min after addition of glucose (resolution 1 nm) (FIG. 4).

11.2 Measurement 2: Kinetics of Reaction with Glucose and GlucDH2/NAD

[0163] The reaction mixture was the same as in Measurement 1, except for 10-40 L samples of 10 mM solution of glucose in distilled water were used. The absorbance at 517 nm was recorded every 10 sec for a 20 min after addition of glucose (FIG. 5, 6).

11.3 Measurement 3: Kinetics of Reaction with Glucose and GlucDH2/carbaNAD

[0164] Conditions were the same as in Measurement 2, with carbaNAD replacing NAD. The absorbance at 517 nm was recorded every 10 sec for a 20 min after addition of glucose (FIG. 7, 8).

Example 12

1-Methyl-3-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)ethenyl]quinolinium iodide trifluoromethanesulfonate, Evaluation of Extinction Coefficient () Under Pseudo-First-Order Reaction Conditions

[0165] The following were mixed in a cuvette: 100 L of 10 mM solution of 1-methyl-3-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)ethenyl]quinolinium iodide trifluoromethanesulfonate in distilled water, 900 L of 50 mM phosphate buffer pH 7, 1-3 L of 10 mM NADH in distilled water. Change in absorbance at 535 nm was recorded after 5 min, when no further color change could be measured (FIG. 3, 9).

[0166] FIG. 9 shows that concentration of the reduced form of 1-methyl-3-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)ethenyl]quinolinium iodide trifluoromethanesulfonate is proportional to the initial NADH concentration. Using the equation

AbS.sub.535 nm=[NADH].sub.0l(1),

an value for the reduced form of 1-methyl-3-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)ethenyl]quinolinium iodide trifluoromethanesulfonate of 69.3 mM.sup.1cm.sup.1 was calculated from the slope of the trend line. This value is nine times greater than those of the reduced form of MTT (Czerlinski, G. H.; et al; Journal of Biochemical and Biophysical Methods 1988, 15, 241).

Example 13

Cyclic Voltammetry (CV) Studies of the Redoxindicators

[0167] The CVs of the compounds synthesized in example 6-7 were recorded vs. the Ag/AgCl reference electrode in a phosphate buffer pH 7 (Table 4).

TABLE-US-00004 TABLE 4 Redoxindicators. CV studies Reduction potential Structure (mV) [00049] 564 [00050] 534 [00051] 524 [00052] 634 [00053] 424 [00054] 454 [00055] 104, 224

[0168] E/mV vs Ag/AgCl, 0.9% NaCl at pH 7, phosphate buffer, Au working electrode, scan rate 100 mVs.sup.1

Example 14

Fluorometric Determination of NADH

[0169] 13.1 Measurement 1: Change in Fluorescence Intensity Upon Reaction with NADH

[0170] The following were mixed in a cuvette: 2 L of 10 mM solution of an indicator (1-methyl-3-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)ethenyl]quinolinium iodide trifluoromethanesulfonate) in distilled water, 1000 L of 10 mM PIPES buffer pH 7, 0.5-4 L of 1 mM NADH solution in distilled water. The mixture was incubated for 30 min at room temperature. Fluorescence (550-800 nm) was recorded using excitation at 535 nm. (FIG. 10, 11).

Example 15

1-Methyl-3-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)ethenyl]quinolinium iodide trifluoromethanesulfonate, Evaluation of an Application for Cell Viability Testing

[0171] Viable HEK-293 cells and 4% paraformaldehyde killed HEK-293 cells were incubated with 1-methyl-3-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)ethenyl]quinolinium iodide trifluoromethanesulfonate (20 M) in 10 mM PBS pH 7 at 37 C. Confocal fluorescence microscope images were recorded at 611 nm using excitation at 561 nm. The image of a viable HEK-293 cell showed cellular uptake of 1-methyl-3-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)ethenyl]quinolinium iodide trifluoromethanesulfonate; simultaneously, the fluorescence intensity was enhanced, which is consistent with the fact that NADH was generated by the cell metabolism. The resulting cells retained viability for at least 24 h. In contrast, the paraformaldehyde treated HEK-293 cells did not show enhancement in fluorescence intensity. Therefore, fluorescence intensity upon treatment the HEK-293 cells with 1-methyl-3-[(E)-2-(1,3,3-trimethyl-3H-indolium-2-yl)ethenyl]quinolinium iodide trifluoromethanesulfonate is much higher in viable cells than in dead cells.

[0172] The work leading to this invention has received funding from the European Research Council under the European Union's Seventh Framework Program (FP7/2007-2013)/ERC grant agreement no 264772 (CHEBANA).