Transition metal complexes containing tetradentate nitrogen donor ligands and electrochemical biosensors containing the same

Abstract

The present invention relates to novel transition metal complexes containing tetradentate nitrogen donor ligands for use in a variety of devices, including electrochemical sensors, and to devices containing the same, preferably electrochemical sensors.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority based on Korean Patent Application No. 10-2020-0189139, filed on December 31, 2020, and all contents disclosed in the documents of this Korean patent application are incorporated herein by reference.

[0002] The present invention relates to transition metal complexes containing tetradentate nitrogen donor ligands useful as electron transfer mediators and electrochemical biosensors containing the same. [Background technology]

[0003] Diabetes is a disease that occurs when high blood sugar levels persist for a long period of time, leading to complications such as cardiovascular disease, stroke, and kidney disease. High blood sugar levels require insulin injections to lower them, but if too much insulin is injected, hypoglycemia can occur, which can lead to shock or death. To prevent this, diabetics must continually monitor their blood sugar levels using a blood glucose sensor to maintain appropriate blood sugar levels.

[0004] Recently, blood glucose sensors that use the Continuous Glucose Monitoring System (CGMS) have been extensively researched and commercialized. CGMS sensors are inserted into the subcutaneous tissue and continuously measure glucose levels using interstitial fluid rather than blood. While it is difficult to accurately ascertain changes in blood glucose levels with fingertip blood sampling, which requires testing five to six times a day, CGMS has the advantage of being able to check changes and trends in blood glucose levels throughout the day. This allows patients to quickly recognize symptoms of hyperglycemia or hypoglycemia, further improving their blood glucose management.

[0005] A biosensor is a device that selectively senses a biological sample and converts it into a specific signal. In particular, enzyme-based biosensors using an electrochemical method are preferred for their improved selectivity, miniaturization, and measurement accuracy.

[0006] Electrochemical blood glucose biosensors are broadly divided into first-generation and second-generation types. First-generation blood glucose sensors were first developed by Clark and Lyon and measure blood glucose levels based on the changes in the oxygen concentration reduced and the hydrogen peroxide concentration generated through an enzymatic oxidation-reduction reaction. Second-generation blood glucose sensors transfer electrons generated by the enzymatic oxidation-reduction reaction to the electrode via an electron transfer mediator. Second-generation sensors have many advantages over first-generation sensors, including less error due to oxygen concentration and a more efficient and faster electron transfer reaction via the mediator. For these reasons, second-generation sensor methods that include electron transfer mediators are used in CGMS blood glucose sensors.

[0007] Electrochemical enzyme-based blood glucose biosensors generally consist of an enzyme, an electron transfer mediator, and an electrode. First, glucose is oxidized to gluconolactone by the enzyme, and then the reduced enzyme is oxidized and donates electrons to the electron transfer mediator. The reduced mediator is then oxidized and transfers electrons to the electrode. This series of processes allows blood glucose levels to be confirmed through an electrical signal.

[0008] Enzymes commonly used in blood glucose sensors include glucose oxidase (GOx) and glucose dehydrogenase (GDH). GOx selectively oxidizes glucose and is widely used in blood glucose sensors due to its low cost and high stability. However, because GOx is significantly affected by oxygen during the oxidation process, GOx-based blood glucose sensors are known to produce inaccurate readings at high altitudes or low atmospheric pressure, resulting in higher-than-actual blood glucose levels. Unlike GOx, GDH has the advantage of being able to selectively oxidize glucose regardless of oxygen concentration. In particular, GDH, which contains flavin adenine dinucleotide (FAD), is not affected by oxygen and has excellent thermal stability, making it ideal for blood glucose sensors. However, a thick protein membrane surrounding the FAD active site makes it difficult for GDH to directly transfer electrons to the electrode surface. To resolve this issue, the role of an electron transfer mediator that facilitates the electron transfer reaction between the enzyme and the electrode surface is crucial.

[0009] The electrochemical properties of the electron transfer mediator are crucial factors that affect the rapid electron transfer, selectivity, and sensitivity of blood glucose sensors. The potential of the mediator can avoid interference from in vivo interfering substances and thermodynamically prevent redox shuttling, a phenomenon in which the mediator between adjacent electrodes repeatedly undergoes oxidation / reduction reactions, causing current errors. Therefore, for an electron transfer mediator to function efficiently, it is ideal to have an appropriately low oxidation-reduction potential (-0.2V to 0V vs. Ag / AgCl). Furthermore, both the oxidized and reduced species must be chemically stable and non-toxic in the body.

[0010] Electron transfer mediators that have been widely studied to date include ferrocene derivatives, ferricyanide, tetrathiafulvalene, and transition metal complexes. Recently, complexes with iron, ruthenium, and osmium as central metals have been widely studied as electron transfer mediators. However, there is still a need for new electron transfer mediators that are highly efficient, stable, and non-toxic in the body.

[0011] Under these circumstances, the present inventors have conducted extensive research into transition metal complexes useful as electron transfer mediators for electrochemical biosensors. As a result, they have found that when tris(2-pyridylmethyl)amine (TPMA) is introduced as a tetradentate ligand, it exhibits superior stability compared to conventional monodentate or bidentate ligands, and that transition metal complexes prepared from these ligands can be easily synthesized and exhibit stable electrochemical characteristics, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0012] It is an object of the present invention to provide novel transition metal complexes for use as electron transfer mediators which contain tetradentate nitrogen donor ligands.

[0013] Another object of the present invention is to provide an electrochemical biosensor comprising a redox polymer containing the transition metal complex.

[0014] A further object of the present invention is to provide a method for producing the transition metal complex. [Means for solving the problem]

[0015] In accordance with one aspect of the present invention, there are provided transition metal complexes containing tetradentate nitrogen donor ligands that are useful as electron transfer mediators.

[0016] According to one aspect of the present invention, there is provided a method for producing the transition metal complex.

[0017] According to one aspect of the present invention, there is provided a device comprising the transition metal complex as an electron transfer mediator.

[0018] According to one aspect of the present invention, there is provided a sensing membrane for an electrochemical biosensor, which comprises an enzyme capable of oxidizing and reducing a liquid biological sample; and the transition metal complex as an electron transfer mediator. [Effects of the Invention]

[0019] When used in an electrochemical sensor, the transition metal complexes containing the tetradentate nitrogen donor ligands according to the present invention significantly improve the performance of the electrochemical sensor. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 illustrates various synthetic methods for preparing tetradentate ligands for preparing transition metal complexes of the present invention. [Figure 2] This is a reaction scheme showing the synthesis of the [Os(TPMA)Cl2]+(PF6-) complex and various derivatives that can be synthesized from it. [Figure 3] FIG. 1 shows the crystal structure of [Os(TPMA)Cl](PF). [Figure 4] This is a reaction scheme showing the synthesis of the [Os(TPMA)(L)1-2]n+(PF6-)n complex and various derivatives that can be synthesized from it. [Figure 5] FIG. 1 shows changes in mass signals observed on ESI-MS of hydroxymethyl TPMA osmium complex. [Figure 6] 1 is a graph showing CV data for [Os(TPMA)Cl]+(X-) (18). [Figure 7a] FIG. 1 shows the structure and oxidation / reduction potential of TPMA-based osmium complexes. [Figure 7b]FIG. 1 shows the structure and oxidation / reduction potential of TPMA-based osmium complexes. [Figure 7c] FIG. 1 shows the structure and oxidation / reduction potential of TPMA-based osmium complexes. [Figure 8a] This graph compares the potentials of the [Os(TPMA)Cl2]+(PF6-) complexes with different substituents measured by the cyclic current-voltage method. Figure 8a compares the potentials of the complexes with those of the EDG complexes (18, 21, and 23). To compare the potentials of the complexes regardless of concentration, the current was corrected to that of 18, and the measurements were performed at 10 mV / s in a 0.1 M TPMP solution in CH3CN. [Figure 8b] Figure 8b shows a comparison of the potentials of the [Os(TPMA)Cl]+(PF6-) complexes with different substituents measured by cyclic current-voltage spectroscopy. Figure 8b compares the potentials of the complexes with those of the EWG complexes (18, 19, 20, 22). To compare the potentials of the complexes regardless of their concentration, the current was corrected to that of 18, and the measurements were performed at 10 mV / s in a 0.1 M TPMP solution in CH3CN. [Figure 9] 1 is a graph showing CV data of [Os(TPMA)Cl]+(PF6-) Complex (19). [Figure 10] 1 is a graph showing CV data of [Os(TPMA)Cl2]+(PF6-) Complex (20). [Figure 11] 1 is a graph showing CV data of [Os(TPMA)Cl]+(PF6-) Complex (21). [Figure 12] 1 is a graph showing CV data of [Os(TPMA)Cl2]+(PF6-) Complex (22). [Figure 13] 1 is a graph showing CV data of [Os(TPMA)Cl]+(PF6-) Complex (23). [Figure 14] 1 is a graph showing CV data of [Os(TPMA)(L)1-2]n+(PF6-)n Complex (24). [Figure 15] 1 is a graph showing CV data of [Os(TPMA)(L)1-2]n+(PF6-)n Complex (25). [Figure 16] 1 is a graph showing CV data of [Os(TPMA)(L)1-2]n+(PF6-)n Complex (26). [Figure 17] 1 is a graph showing CV data of [Os(TPMA)(L)1-2]n+(PF6-)n Complex (27). [Figure 18] 1 is a graph showing CV data of [Os(TPMA)(L)1-2]n+(PF6-)n Complex (28). [Figure 19] 1 is a graph showing CV data of [Os(TPMA)(L)1-2]n+(PF6-)n Complex (29). [Figure 20] 1 is a graph showing CV data of [Os(TPMA)(L)1-2]n+(PF6-)n Complex (30). [Figure 21] 1 is a graph showing CV data of [Os(TPMA)(L)1-2]n+(PF6-)n Complex (31). [Figure 22] 1 is a graph showing CV data of [Os(TPMA)(L)1-2]n+(PF6-)n Complex (32). [Figure 23] 1 is a graph showing CV data of [Os(TPMA)(L)1-2]n+(PF6-)n Complex (33). [Figure 24] 1 is a graph showing CV data of [Os(TPMA)(L)1-2]n+(PF6-)n Complex (34). [Figure 25] 1 is a graph showing CV data of [Os(TPMA)(L)1-2]n+(PF6-)n Complex (35). DETAILED DESCRIPTION OF THE INVENTION

[0021] One embodiment of the present invention provides transition metal complexes containing tetradentate nitrogen donor ligands useful as electron transfer mediators.

[0022] For example, the transition metal complex is represented by the following formula 1 or 2: [Chemical formula 1] [ka] [Chemical formula 2] [ka]

[0023] In the above Chemical Formula 1 or Chemical Formula 2, M is one selected from the group consisting of Fe, Co, Ru, Os, Rh and Ir; C a , C b and C c are each independently a heterocyclic compound containing one or more nitrogen atoms, preferably linked to an amine group and a methylene group at the 2-position of the ring compound; L m1 and L m2 are each independently a coordinated monodentate ligand; L b is a nitrogen- or oxygen-containing bidentate ligand; m is a negative or positive charge representing -1 to -5 or 1 to 5; R 1 , R 2 and R 3 are each independently a linker having a reactive group introduced therein that can be linked to a polymer, or a functional group such as an electron donating group (EDG) or an electron withdrawing group (EWG) for adjusting the oxidation / reduction potential; X is a counter ion, preferably a counter ion selected from the group consisting of F, Cl, Br, I, and PF6; n means the number of counter ions and is 1 to 5.

[0024] For example, the transition metal complex may include a tetradentate ligand represented by the following Formula 3: [Chemical formula 3] [ka]

[0025] In the above formula, C a , C b and C c are each independently a heterocyclic compound containing one or more nitrogen atoms; R 1 , R 2 and R 3 are independently a linker with a reactive group that can be linked to a polymer, and a functional group of an electron donating group (EDG) or an electron withdrawing group (EWG) to adjust the oxidation / reduction potential.

[0026] In one embodiment, the tetradentate ligand of Formula 3 can be one of the ligands having the following structure: [ka]

[0027] Preferably, the heterocyclic compound is linked to an amine group and a methylene group at the 2-position, and three nitrogen atoms of the three heterocyclic rings and one central nitrogen atom connecting the three heterocyclic rings to each other are linked to a transition metal M.

[0028] Specifically, R1, R2, and R3 in Chemical Formulas 1 to 3 can each independently be -H; -F; -Cl; -Br; -I; -NO2; -CN; -CO2H; -SO3H; -NHNH2; -SH; -OH; -NH2; -CH2OH; -CONHCH2CH2NH2; or substituted or unsubstituted alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, alkylamino, dialkylamino, alkanylamino, arylcarboxamido, hydrazino, alkylhydrazino, hydroxyamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl.

[0029] Specifically, the L m1 and L m2 are monodentate ligands and may each independently be -H, -F, -Cl, -Br, -I, -NO2, -NCCH3, -CO, -OH2, -NH3, or a heterocyclic compound containing one or more nitrogen atoms.

[0030] Specifically, the L b -L b is a bidentate ligand and can be catechol, acetylacetone, 2-picolinic acid, 2-pyridinecarboxamide, 2,2'-bipyridine, or 2,2'-bithiazole.

[0031] In one specific embodiment, the heterocyclic compound may be at least one selected from the group consisting of imidazole, pyridine, pyrimidine, pyrazole, isoxazole, oxazole, thiazole, benzothiazole, benzimidazole, benzoxazole, and diazafluorenone.

[0032] In a specific embodiment, when the alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, alkylamino, dialkylamino, alkanylamino, arylcarboxamide, hydrazino, alkylhydrazino, hydroxyamino, alkoxyamino, alkylthio, alkenyl, aryl, alkyl, and 3-membered heterocycle are substituted, they may be substituted with one or more, preferably 1 to 3, selected from the group consisting of -F, -Cl, -Br, -I, -OH, oxo, an alkyl group having 1 to 3 carbon atoms, and an alkoxy group having 1 to 3 carbon atoms.

[0033] In one specific embodiment, the transition metal complex of Formula 1 or 2 according to the present invention may be one of the transition metal complexes shown in Table 1 below: [Table 1] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0034] In yet another aspect, the present invention provides a method for producing a transition metal complex useful as the electron transfer mediator.

[0035] The transition metal complex useful for the electron transfer mediator according to the present invention can be prepared using a transition metal salt, preferably an osmium salt. In one specific example, the transition metal salt is an ammonium salt of a halogenated transition metal represented by the following formula 4: [Chemical formula 4] [(NH4)2MX6]

[0036] In the above formula, M is one selected from the group consisting of Fe, Co, Ru, Os, Rh and Ir; X is F, Cl, Br or I.

[0037] Preferably, the ammonium salt of a transition metal halide of formula 4 is ammonium hexachloroosmate of formula 5, which is commercially available. [Chemical formula 5] [(NH4)2OsCl6]

[0038] In one embodiment, the method for preparing a transition metal complex according to the present invention can be synthesized using a tris-pyridinemethylamine-based ligand and an ammonium salt of a transition metal halide. As a specific example, in the case of a complex containing osmium, the method can include the following steps: a) introducing a tetradentate tris-pyridinemethylamine-based ligand represented by Chemical Formula 3 into an ammonium salt of an osmium halide represented by Chemical Formula 5 to synthesize an osmium complex represented by Chemical Formula 6; and b) introducing one or two monodentate or bidentate ligands selected from the group consisting of NN ligands, N-ligands, NO ligands and OO ligands into the osmium complex of formula 6;

[0039] For example, the transition metal complex may include a tetradentate ligand represented by the following Formula 3: [Chemical formula 3] [ka]

[0040] In the above formula, C a , C b and C c are each independently a heterocyclic compound containing one or more nitrogen atoms; R 1 , R 2 and R 3 are each independently a linker having a reactive group introduced therein that can be linked to a polymer.

[0041] The first step is to introduce a tris-pyridinemethylamine-based ligand (Chemical Formula 3) into an osmium salt (Chemical Formula 5) in a tetravalent ion state to synthesize an osmium complex (Chemical Formula 6) in a trivalent ion state. [Chemical formula 6] [ka]

[0042] The second step is a step of introducing one or two monodentate or bidentate ligands selected from the group consisting of N-N, N-, NO-, and OO-ligands into the osmium complex synthesized in the first step to synthesize an osmium complex represented by any one of Chemical Formulas 7 to 11. Preferably, in the second step, one N-N ligand, two N-ligands, one N-ligand, one NO-ligand, and one OO-ligand can be introduced into the synthesized osmium complex of Chemical Formula 6. [Chemical formula 7] [ka] [Chemical formula 8] [ka] [Chemical formula 9] [ka] [Chemical formula 10] [ka] [Chemical formula 11] [ka]

[0043] As an example, the N ligand can be -NO2, -NCCH3, -NH3, or a heterocyclic compound containing one or more nitrogen atoms.

[0044] As an example, the NN ligand can be 2-pyridinecarboxamide, 2,2'-bipyridine, 2,2'-bithiazole or 2-pyridylmethylamine.

[0045] By way of example, the NO ligand may be 2-picolinic acid, 2-aminophenol, or 2-hydroxymethylpyridine.

[0046] As an example, the OO ligand can be catechol or acetylacetone.

[0047] The electron transfer mediator transition metal complex according to the present invention plays a role in transferring electrons obtained by the reduction (glucose oxidation) of an oxidoreductase, and can be used in the form of an oxidation-reduction polymer linked to a polymer matrix corresponding to a polymer backbone, such as one or more selected from the group consisting of poly(vinylpyridine) (PVP), poly(vinylimidazole) (PVI), and polyallyl glycidyl ether (PAGE).

[0048] Therefore, a further aspect of the present invention relates to an oxidation-reduction polymer comprising the transition metal complex for the electron transfer mediator and a polymer backbone.

[0049] For example, the oxidation-reduction polymer may include a linker structure connecting the polymer backbone and the organic electron transfer mediator.

[0050] Another aspect of the present invention relates to a sensing film for an electrochemical biosensor, which comprises an enzyme capable of oxidizing and reducing a liquid biological sample and an electron transfer mediator including the transition metal complex.

[0051] Oxidoreductase is a general term for enzymes that catalyze oxidation-reduction reactions in living organisms. In the present invention, it refers to an enzyme that reacts with and reduces a target substance to be measured, such as a target substance in the case of a biosensor. The reduced enzyme reacts with an electron transfer mediator, and the target substance is quantified by measuring a signal, such as a change in current, generated during this reaction. The oxidoreductase that can be used in the present invention is one or more enzymes selected from the group consisting of various dehydrogenases, oxidases, esterases, etc. Depending on the oxidation-reduction or target substance to be detected, an enzyme that uses the target substance as a substrate can be selected from the above enzyme group and used.

[0052] More specifically, the oxidoreductase may be one or more selected from the group consisting of glucose dehydrogenase, glutamate dehydrogenase, glucose oxidase, cholesterol oxidase, cholesterol esterase, lactate oxidase, ascorbic acid oxidase, alcohol oxidase, alcohol dehydrogenase, bilirubin oxidase, and the like.

[0053] Meanwhile, the oxidoreductase may contain a cofactor that serves to store the hydrogen removed by the oxidoreductase from the target substance to be measured (e.g., the target substance), and the cofactor may be, for example, one or more selected from the group consisting of flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), pyrroloquinoline quinone (PQQ), etc.

[0054] For example, when measuring the glucose concentration in blood, glucose dehydrogenase (GDH) can be used as the oxidoreductase, and the glucose dehydrogenase can be flavin adenine dinucleotide-glucose dehydrogenase (FAD-GDH) containing FAD as a cofactor and / or nicotinamide adenine dinucleotide-glucose dehydrogenase containing FAD-GDH as a cofactor.

[0055] Specific examples of the usable oxidoreductase include FAD-GDH (e.g., EC 1.1.99.10, etc.), NAD-GDH (e.g., EC 1.1.1.47, etc.), PQQ-GDH (e.g., EC 1.1.5.2, etc.), glutamate dehydrogenase (e.g., EC 1.4.1.2, etc.), glucose oxidase (e.g., EC 1.1.3.4, etc.), cholesterol oxidase (e.g., EC 1.1.3.6, etc.), cholesterol esterase (e.g., EC 3.1.1.13, etc.), lactate oxidase (e.g., EC 1.1.3.2, etc.), ascorbate oxidase (e.g., EC 1.10.3.3, etc.), alcohol oxidase (e.g., EC 1.1.3.13, etc.), alcohol dehydrogenase (e.g., EC 1.1.1.1, etc.), bilirubin oxidase (e.g., EC 1.3.3.5, etc.) and the like.

[0056] Most preferably, the oxidoreductase is a glucose dehydrogenase that can maintain 70% or more of its activity in a buffer solution at 37°C for one week.

[0057] The sensing membrane according to the present invention may contain 20 to 700 parts by weight, for example, 60 to 700 parts by weight or 30 to 340 parts by weight, of the oxidation-reduction polymer based on 100 parts by weight of the oxidation-reductase. The content of the oxidation-reduction polymer can be adjusted appropriately depending on the activity of the oxidation-reductase.

[0058] Furthermore, the sensing film according to the present invention may further include carbon nanotubes to enhance the film performance. Specifically, when carbon nanotubes are used together with a transition metal complex, especially osmium, the electron transfer rate increases, thereby further enhancing the performance of the sensing film.

[0059] The sensing membrane according to the present invention may further include a cross-linking agent.

[0060] Meanwhile, the sensing membrane according to the present invention may further contain one or more additives selected from the group consisting of surfactants, water-soluble polymers, quaternary ammonium salts, fatty acids, thickeners, etc., to serve as a dispersant when dissolving the reagent, an adhesive when preparing the reagent, a stabilizer for long-term storage, etc.

[0061] The surfactant may serve to ensure that the composition is evenly spread and dispensed at a uniform thickness on the electrode when dispensed. The surfactant may be at least one selected from the group consisting of Triton X-100, sodium dodecyl sulfate, perfluorooctane sulfonate, sodium stearate, etc. The reagent composition according to the present invention may contain the surfactant in an amount of 3 to 25 parts by weight, for example, 10 to 25 parts by weight, based on 100 parts by weight of the oxidoreductase, to ensure that the composition is evenly spread and dispensed at a uniform thickness on the electrode when dispensed. For example, when an oxidoreductase with an activity of 700 U / mg is used, the surfactant may be added in an amount of 10 to 25 parts by weight based on 100 parts by weight of the oxidoreductase. If the activity of the oxidoreductase is higher than this, the surfactant content may be adjusted to a lower amount.

[0062] The water-soluble polymer may function as a polymer support in the reagent composition, helping to stabilize and disperse the enzyme. The water-soluble polymer may be at least one selected from the group consisting of polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), polyperfluorosulfonate, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), carboxymethyl cellulose (CMC), cellulose acetate, and polyamide. The reagent composition according to the present invention may contain the water-soluble polymer in an amount of 10 to 70 parts by weight, for example, 30 to 70 parts by weight, based on 100 parts by weight of the redox enzyme, to adequately and appropriately stabilize and disperse the redox enzyme. For example, when an oxidoreductase with an activity of 700 U / mg is used, the water-soluble polymer may be contained in an amount of 30 to 70 parts by weight based on 100 parts by weight of the oxidoreductase. If the activity of the oxidoreductase is higher than this, the content of the water-soluble polymer may be adjusted to be lower than this.

[0063] The water-soluble polymer may have a weight-average molecular weight of about 2,500 g / mol to 3,000,000 g / mol, for example, about 5,000 g / mol to 1,000,000 g / mol, in order to effectively aid in stabilizing and dispersing the support and enzyme.

[0064] The thickener serves to firmly attach the reagent to the electrode. The thickener may be at least one selected from the group consisting of Natrosol, diethylaminoethyl-dextran hydrochloride (DEAE-Dextran hydrochloride), and the like. The electrochemical sensor according to the present invention may contain the thickener in an amount of 10 to 90 parts by weight, for example, 30 to 90 parts by weight, based on 100 parts by weight of the oxidoreductase, to firmly attach the oxidation-reduction polymer according to the present invention to the electrode. For example, when an oxidoreductase having an activity of 700 U / mg is used, the thickener may be contained in an amount of 30 to 90 parts by weight based on 100 parts by weight of the oxidoreductase. If the activity of the oxidoreductase is higher than this, the amount of the thickener may be adjusted to be lower.

[0065] In yet another aspect, the present invention provides a device, preferably an insertable device, more specifically a device insertable into the human body, comprising such an organic electron transfer mediator. The device may also preferably be an electrochemical biosensor, more preferably an electrochemical glucose (blood sugar) sensor.

[0066] Specifically, there is no limitation on the type of the electrochemical biosensor, but it may preferably be a continuous blood glucose monitoring sensor.

[0067] The continuous glucose monitoring sensor of the present invention may include, for example, an electrode, an insulator, a substrate, a sensing layer containing the oxidation-reduction polymer and the redox enzyme, a diffusion layer, and a protection layer. The electrodes may include two types of electrodes, such as a working electrode and a counter electrode, or three types of electrodes, such as a working electrode, a counter electrode, and a reference electrode. In one embodiment, the biosensor of the present invention may be an electrochemical biosensor fabricated by coating a substrate having at least two, preferably two or three, electrodes with a reagent composition containing the oxidation-reduction polymer containing the organic electron transfer mediator of Formula 1 and an enzyme capable of oxidizing and reducing a liquid biological sample, followed by drying. For example, a planar electrochemical biosensor is provided, in which a working electrode and a counter electrode are provided on opposite sides of a substrate, a sensing layer containing the oxidation-reduction polymer having the organic electron transfer mediator of the present invention is laminated on the working electrode, and an insulator, a diffusion layer, and a protection layer are laminated, in order, on both sides of the substrate on which the working electrode and counter electrode are provided.

[0068] In a specific embodiment, the substrate may be made of one or more materials selected from the group consisting of PET (polyethylene terephthalate), PC (polycarbonate), and PI (polyimide).

[0069] The working electrode may be a carbon, gold, platinum, silver, or silver / silver chloride electrode.

[0070] In addition, in the case of an electrochemical biosensor having two electrodes, the counter electrode also serves as the reference electrode, so a gold, platinum, silver, or silver / silver chloride electrode can be used as the counter electrode, and in the case of an electrochemical biosensor having three electrodes including a reference electrode, a gold, platinum, silver, or silver / silver chloride electrode can be used as the reference electrode, and a carbon electrode can be used as the counter electrode.

[0071] The diffusion membrane may be made of Nafion, cellulose acetate, or silicone rubber, and the protective membrane may be made of silicone rubber, polyurethane, or polyurethane copolymer, but is not limited thereto.

[0072] As a non-limiting example, in the case of a two-electrode system, the counter electrode also serves as the reference electrode, so silver chloride or silver can be used, and in the case of a three-electrode system, the reference electrode can be silver chloride or silver, and the counter electrode can be a carbon electrode.

[0073] Although the embodiments of the present invention illustrate a biosensor for measuring glucose as an example of an applicable electrochemical biosensor, by varying the type of enzyme contained in the reagent composition of the present invention, the present invention can be applied to biosensors for quantifying various substances such as cholesterol, lactate, creatinine, hydrogen peroxide, alcohol, amino acids, and glutamate. [Example]

[0074] The present invention will be described in more detail below with reference to the following examples, but the following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0075] Experimental materials Commercially purchased solvents and reagents were used without further purification. For the purification of metal complexes, neutral alumina (Aldrich) was used, filtered using a 10 mL pipette. For the chloride zwitterion exchange of complexes, Dowex 1x4 chloride form (50-100 mesh) (Aldrich) was used.

[0076] 1 H-NMR and 13C-NMR spectra were obtained using a Varian Inova 400 ( 1 400MHz for H, 13 All chemical shifts were observed using the peak of tetramethylsilane (δ 0.00) or deuterated chloroform ( 1 H NMR δ 7.26 vs. CDCl3 13 C NMR δ 77.16 vs. CDCl3), deuterated dimethylacetamide ( 1 H NMR δ 2.50 vs. DMSO, 13 The C NMR was determined proportionally to δ 39.52 relative to DMSO. Mass spectra were obtained at the Organic Chemistry Research Center of Sogang University using a ThermoFisher Scientific LTQ XL model ESI-Iontrap (low resolution) and ESI-orbitrap (high resolution) mass spectrometer.

[0077] Example 1. Synthesis of TPMA Ligand TPMA ligands bearing various functional groups were synthesized using the synthetic pathway shown in Figure 1. Bis-2-pyridylmethylamine (1) and tris-2-pyridylmethylamine (10) were synthesized by reductive amination between 2-pyridylaldehyde and 2-pyridylmethylamine. The other ligands (11, 12, 15, and 16) were synthesized by slightly modifying a previously published method (Kojima, T.; Fukuzumi, S. Chem. Eur. J. 2007, 13, 8212-8222). 2-Chloromethylpyridine hydrochloride bearing various functional groups at the 4-position was synthesized by Sn2 reaction with bis-2-pyridylmethylamine (1) in a 1:1 ratio or Sn2 reaction with 2-pyridylmethylamine in a 1:2 ratio. Furthermore, ligand 13 was synthesized by ester reduction, and ligand 14 was synthesized via aminolysis with excess ethylenediamine. Most of the synthesized TPMA ligands have three pyridine rings, and therefore are strongly basic and cannot be purified by column chromatography using silica. Instead, they were purified by column chromatography using basic or neutral alumina.

[0078] 1-1.Bis(2-pyridiylmethyl)amine(1) [ka]

[0079] 2-Pyridylaldehyde (3.0 g, 27 mmol) and 2-aminomethylpyridine (3.0 g, 27 mmol) were placed in a round-bottom flask and methanol (25 mL) was added. The mixture was stirred at room temperature for 3 hours, and then NaBH4 (3.1 g, 81 mmol) was slowly added at 0 °C. After the addition, the mixture was stirred at room temperature for 4 hours. Once the reaction was complete, 10% aqueous hydrochloric acid was added to terminate the reaction. The solution was concentrated using a rotary evaporator, and saturated aqueous NaCO3 was added to adjust the pH to 9. After transferring to a separatory funnel, the aqueous layer was extracted three times with CHCl2. The combined organic layer was then added with anhydrous MgSO4 to remove water, and the desiccant was filtered off using a glass filter under reduced pressure. The organic solvent was removed using a rotary evaporator. This gave product 1 (5.4 g, 98%) as a yellow oil. 1 H NMR (400MHz, CDCl3) δ8.57(d, J=4.8Hz, 2H), 7.65(dd, J=7.2, 4.8Hz, 2H), 7.37(d, J=7.6Hz, 2H), 7.17(d, J=5.2, 7.2Hz, 2H), 3.99(s, 4H)

[0080] 1-2.2,4-Diethoxycarbonylpyridine(2) [ka]

[0081] 2,4-Pyridinedicarboxylic acid (5 g, 30 mmol) was dissolved in ethanol (250 mL) in a round-bottom flask. Concentrated sulfuric acid (3.28 mL) was added to the reaction flask, a reflux condenser was attached, and the mixture was stirred at 80°C for 24 hours under reflux. After cooling the reaction flask to room temperature, the ethanol was removed using a rotary evaporator. A saturated aqueous solution of NaCO3 was added, and the mixture was transferred to a separatory funnel. The aqueous layer was extracted three times with CHCl2. Anhydrous MgSO4 was then added to the combined organic layer to remove water, and the desiccant was filtered off using a glass filter under reduced pressure. The organic solvent was then removed using a rotary evaporator. This afforded a white solid product 2 (6.5 g, 97%). 1 H NMR (400MHz, CDCl3) δ8.92(d, J=5.6Hz, 1H), 8.65(s, 1H), 8.04(d, J=6.0Hz, 1H), 4.52 (q, J=6.4Hz, 2H), 4.46(q, J=5.6Hz, 2H), 1.48(t, J=6.0Hz, 3H), 1.43(t, J=6.0Hz, 3H)

[0082] 1-3.4-Ethoxycarbonyl-2-hydroxymethylpyridine(3) [ka]

[0083] The starting material (2) (6.0 g, 27 mmol) and NaBH4 (0.664 g, 35 mmol) were placed in a round-bottom flask and dissolved in ethanol (50 mL). CaCl2 (3.0 g, 27 mmol) dissolved in ethanol was slowly added to the reaction flask at 0 °C. After the addition, the mixture was stirred at 0 °C for 2 hours and 30 minutes. After confirming complete completion of the reaction, concentrated sulfuric acid was added to terminate the reaction. The white precipitate was filtered using a glass filter, and the solution was concentrated using a rotary evaporator. A saturated aqueous solution of NaCO3 was added, and the mixture was transferred to a separatory funnel. The aqueous layer was extracted three times with CHCl2. The combined organic layer was then added to anhydrous MgSO4 to remove the water, and the desiccant was filtered off using a glass filter under reduced pressure. The organic solvent was removed using a rotary evaporator. This gave the pale yellow solid product 3 (3.1 g, 64%). 1 H NMR (400MHz, CDCl3) δ8.96(d, J=5.6Hz, 1H), 8.42(s, 1H), 8.34(d, J=5.6Hz, 1H), 5.22(s, 2H), 4.53(q, J=7.2Hz, 2H), 1.47(t, J=6.8Hz, 3H)

[0084] 1-4.4-Ethoxycarbonyl-2-chloromethylpyridine hydrochloride(4) [ka]

[0085] The starting material (3) (2.9 g, 16 mmol) was placed in a round-bottom flask and dissolved in CHCl (30 mL). Thionyl chloride (9.5 g, 80 mmol) diluted in CHCl (15 mL) was slowly added to the reaction flask. After the addition, the mixture was stirred overnight at room temperature. Once the reaction was complete, the solvent was removed using a rotary evaporator, yielding the white solid product 4 (3.616 g, 90%). 1H NMR (400MHz, CDCl3) δ8.82(d, J=6.0Hz, 1H), 8.55(s, 1H), 8.35(d, J=6.4Hz, 1H), 5.24(s, 2H), 4.54(q, J=7.6Hz, 2H), 1.48(t, J=7.2Hz, 3H)

[0086] 1-5.4-Methoxy-2-hydroxymethylpyridine(5) [ka]

[0087] 4-Methoxypyridine-2-carboxylate (4.95 g, 29 mmol) and NaBH4 (1.45 g, 44 mmol) were placed in a round-bottom flask and dissolved in ethanol (40 mL). CaCl2 (3.28 g, 29 mmol) dissolved in ethanol was slowly added to the reaction flask at 0 °C. After the addition, the mixture was stirred at -5 °C for 2 hours and 30 minutes. After confirming complete completion of the reaction, concentrated sulfuric acid was added to terminate the reaction. The white precipitate was filtered using a glass filter, and the solution was concentrated using a rotary evaporator. A saturated aqueous solution of NaCO3 was added, and the mixture was transferred to a separatory funnel. The aqueous layer was extracted three times with CHCl2. The combined organic layer was then added to anhydrous MgSO4 to remove water, and the desiccant was filtered off using a glass filter under reduced pressure. The organic solvent was removed using a rotary evaporator. This gave the white solid product 5 (2.14 g, 53%). 1 H NMR (400MHz, CDCl3) δ8.37(d, J=4.0Hz, 1H), 6.78(s, 1H), 6.74(d, J=3.2Hz, 1H), 4.71(s, 2H), 3.86(s, 3H)

[0088] 1-6.4-Methoxy-2-chloromethylpyridine hydrochloride(6) [ka]

[0089] The starting material (5) (1.60 g, 11 mmol) was placed in a round-bottom flask and dissolved in CHCl (20 mL). Thionyl chloride (6.83 g, 57 mmol) diluted in CHCl (10 mL) was slowly added to the reaction flask. After the addition, the mixture was stirred overnight at room temperature. Once the reaction was complete, the solvent was removed using a rotary evaporator, yielding the white solid product 6 (2.11 g, 95%). 1 H NMR (400MHz, CD3CN) δ8.42(d, J=6.8Hz, 1H), 7.43(s, 1H), 7.31(d, J=6.4Hz, 1H), 4.98(s, 2H), 4.06(s, 3H)

[0090] 1-7.2-(Hydroxymethyl)-4-pyridinecarboxamide(7) [ka]

[0091] The starting material (3) (1.60 g, 11 mmol) was placed in a round-bottom flask and dissolved in ethanol (10 mL). 30% aqueous ammonia (30 mL) was slowly added to the reaction flask. After the addition was complete, the mixture was stirred at room temperature for 24 hours. After confirming that the reaction was complete, the solvent was removed using a rotary evaporator. This was then used in the next reaction without further purification. This gave a white solid product 7 (0.78 g, 90%). 1 H NMR (400MHz, DMSO) δ8.60(d, J=4.0Hz, 1H), 8.42(d, J=4.0Hz, 1H, NH 1 ), 7.89(s, 1H), 7.63(d, J=4.0Hz, 1H), 7.53(d, J=4.0Hz, 1H, NH 2 ), 4.61(s, 2H)

[0092] 1-8.4-Cyano-2-chloromethylpyridine(8) [ka]

[0093] The starting material (7) (94 mg, 0.55 mmol) was placed in a round-bottom flask and dissolved in distilled and purified DMF (3 mL). Thionyl chloride (325 mg, 2.8 mmol) diluted in DMF (6 mL) was slowly added to the reaction flask at 0 °C. After the addition was complete, the mixture was stirred at room temperature for 12 hours. The reaction was terminated by neutralizing with saturated aqueous NaCO3. The product in the flask was transferred to a separatory funnel, and the aqueous layer was extracted three times with ethyl acetate. The organic layer was then washed with water until all the DMF was removed. Anhydrous MgSO4 was added to the combined organic layer to remove the water, and the desiccant was filtered off using a glass filter under reduced pressure. The organic solvent was removed using a rotary evaporator. Once the reaction was confirmed to be complete, the solvent was removed using a rotary evaporator. This gave product 8 as a brown oil (695 mg, 70%). 1 H NMR (400MHz, CDCl3) δ7.76(d, J=4.8Hz, 1H), 7.76(s, 1H), 7.50(d, J=4.8Hz, 1H), 4.73(s, 2H), 13 C NMR (100 MHz, CDCl) δ 158.28, 150.32, 124.46, 124.29, 121.47, 116.12 and 45.63

[0094] 1-9,2-thiazoylmethylamine(9) [ka]

[0095] A round-bottom flask was charged with NH2OH·HCl (2.3 g, 30 mmol), 2-thiazolecarboxaldehyde (2.5 g, 20 mmol), and NaOH (2.6 g, 60 mmol), and ethanol (20 mL) and distilled water (4 mL) were added to dissolve the mixture. A reflux condenser was attached to the reaction flask, and the mixture was refluxed at 80 °C for 30 minutes with stirring. After cooling to room temperature, the mixture was acidified to pH 4 by adding 2 N hydrochloric acid. The product was transferred to a separatory funnel, and the aqueous layer was extracted twice with Et2O. The combined organic layer was then added to anhydrous MgSO4 to remove the water, and the desiccant was filtered off under reduced pressure using a glass filter. The organic solvent was removed using a rotary evaporator, yielding a white solid intermediate product. This intermediate product was dissolved in ethanol (30 mL) and 30% aqueous ammonia (60 mL). Zinc dust (10.1 g, 200 mmol) and ammonium acetate (1.3 g, 20 mmol) were added sequentially, and a reflux condenser was attached. The mixture was refluxed at 80°C for 30 minutes with stirring. After cooling to room temperature, the mixture was filtered through a glass filter. The filtered solution was diluted with water and transferred to a separatory funnel, followed by three extractions with CHCl. ​​Anhydrous MgSO was then added to the combined organic layer to remove water, and the desiccant was filtered off under reduced pressure through a glass filter. The organic solvent was removed using a rotary evaporator. This yielded product 9 (0.8 g, 35%) as a yellow oil. 1 H NMR (400MHz, DMSO) δ7.68(d, J=3.2Hz, 1H), 7.55(d, J=3.2Hz, 1H), 3.98(s, 2H), 2.32(br, 2H)

[0096] 1-10.Tris(2-pyridiylmethyl)amine(10) [ka]

[0097] The starting material (1) (1.05 g, 5.3 mmol) and 2-pyridylaldehyde (0.63 g, 5.3 mmol) were placed in a round-bottom flask and methanol (15 mL) was added. The mixture was stirred at room temperature for 2 hours, and then NaBH4 (0.61 g, 16 mmol) was slowly added at 0 °C. After the addition, the mixture was stirred at room temperature for 12 hours. After confirming complete reaction, the reaction was terminated by adding 10% aqueous hydrochloric acid. The solution was concentrated using a rotary evaporator, and saturated aqueous NaCO3 was added to adjust the pH to 9. After transferring to a separatory funnel, the aqueous layer was extracted three times with CHCl2. The combined organic layer was then added anhydrous MgSO4 to remove water, and the desiccant was filtered off using a glass filter under reduced pressure. The organic solvent was removed using a rotary evaporator. The product was then purified by recrystallization from EtO to obtain colorless crystalline product 10 (1.2 g, 78%). 1 H NMR (400MHz, CDCl3) δ8.53(d, J=4.4Hz, 3H), 7.65(dd, J=5.0, 7.6Hz, 3H), 7.58(d, J=7.6Hz, 3H), 7.15(dd, J=5.6, 7.2Hz, 3H), 3.88(s, 6H)

[0098] 1-11.2-(Bis(4,4'-ethoxycarbonyl-2,2'-pyridinyl)methylamino)methylpyridine(11) [ka]

[0099] A round-bottom flask was charged with starting material (4) (1.0 g, 4.25 mmol), 2-pyridylmethylamine (0.21 g, 1.93 mmol), Na2CO3 (4.5 g, 42.5 mmol), and acetonitrile (20 mL). A reflux zone was installed in the reaction flask, and the mixture was refluxed at 80 °C with stirring for 12 hours. After the reaction was completed, the mixture was cooled to room temperature and the excess Na2CO3 was removed using a glass filter. The solution was concentrated using a rotary evaporator, and the resulting dark red oil was purified by column chromatography on basic alumina using a 2:5 hexane:EtOAc eluent to obtain product 11 (0.55 g, 65%) as a red oil. 1 H NMR (400MHz, CDCl3) δ8.68(d, J=5.2Hz, 2H), 8.54(d, J=5.0Hz, 1H), 8.10(s, 2H), 7.70(d, 2H), 7.69(dd, 1H), 7.60(d , J=3.6Hz, 1H), 7.16(dd, J=6.0, 7.2Hz, 1H), 4.42(q, J=7.2Hz, 4H), 3.99(s, 2H), 3.93(s, 4H), 1.42(t, J=7.2Hz, 6H)

[0100] 1-12.2-(Bis(2-pyridinyl)methylamino)methyl-4-ethoxycarbonylpyridine(12) [ka]

[0101] A round-bottom flask was charged with starting material (1) (0.35 g, 1.77 mmol), starting material (4) (0.5 g, 1.94 mmol), Na2CO3 (2.2 g, 19.4 mmol), and acetonitrile (15 mL). A reflux zone was installed in the reaction flask, and the mixture was refluxed at 80 °C with stirring for 12 hours. After the reaction was completed, the mixture was cooled to room temperature and the excess Na2CO3 was removed using a glass filter. The solution was concentrated using a rotary evaporator, and the resulting dark red oil mixture was purified by column chromatography on basic alumina using acetonitrile as a eluent to obtain product 12 (0.53 g, 82%) as a dark orange oil. 1 H NMR (400MHz, CDCl3) δ8.68(d, J=4.8Hz, 1H), 8.54(d, J=4.0Hz, 2H), 8.12(s, 1H), 7.69(d, J=4.0Hz, 1H), 7.65(dd, 2H), 7. 60(d, J=7.6Hz, 2H), 7.15(dd, J=6.0, 7.2Hz, 2H), 4.42(q, J=6.8Hz, 2H), 3.96(s, 2H), 3.89(s, 4H), 1.42(t, J=6.8Hz, 3H)

[0102] 1-13.2-(Bis(2-pyridinyl)methylamino)methyl-4-hydroxymethylpyridine(13) [ka]

[0103] The starting material (12) (100 mg, 0.28 mmol) was dissolved in ethanol (10 mL) in a round-bottom flask. NaBH4 (21 mg, 0.84 mmol) was slowly added at 0 °C, and the mixture was stirred at room temperature for 12 hours. After 12 hours, saturated aqueous ammonium chloride solution was added to terminate the reaction. The white precipitate was filtered using a glass filter, and the solution was concentrated using a rotary evaporator. Saturated aqueous NaCO3 solution was added, and the mixture was transferred to a separatory funnel. The aqueous layer was extracted three times with CHCl2. Anhydrous MgSO4 was then added to the combined organic layer to remove water, and the desiccant was filtered off using a glass filter under reduced pressure. The organic solvent was removed using a rotary evaporator. This gave product 13 (40 mg, 45%) as a pale yellow oil. 1 H NMR (400MHz, CDCl3) δ8.51(d, J=4.8Hz, 2H), 8.47(d, J=5.2Hz, 1H), 7.64(dd, J=3.6, 4.0Hz, 2H), 7.59(d, J=5.2 Hz, 2H), 7.55(s, 1H), 7.16(d, J=5.2Hz, 1H), 7.13(dd, J=3.2, 3.2Hz, 2H), 4.73(s, 2H), 3.88(s, 2H), 3.86(s, 4H)

[0104] 1-14.2-(Bis(2-pyridinyl)methylamino)methyl-4-(2-aminoethyl)pyridine carboxamide(14) [ka]

[0105] The starting material (12) (1.35 g, 3.7 mmol) was dissolved in a minimum amount of CHCl in a round-bottom flask and slowly added to purified ethylenediamine (22.5 g, 370 mmol). The reaction flask was set up in a reflux zone and stirred at 80°C for 16 hours. After the reaction was completed, the mixture was cooled to room temperature and 30 mL of water was added. The product in the flask was transferred to a separatory funnel, and the aqueous layer was extracted three times with CHCl. ​​The combined organic layer was then added to anhydrous MgSO to remove the water, and the desiccant was filtered off under reduced pressure using a glass filter. The organic solvent was removed using a rotary evaporator, yielding product 14 (1.20 g, 86%) as a pale yellow oil. 1 H NMR (400MHz, CDCl3) δ8.62(d, J=4.0Hz, 1H), 8.54(d, J=8.0Hz, 2H), 8.23(s, 1H), 7.64(dd, J=8.0Hz, 2H), 7.55(d, J=4.0Hz, 1H), 7.49 (d, J=8.0Hz, 2H), 7.43(br, 1H), 7.15(dd, J=6.0, 6.0Hz, 2H), 3.93(s, 2H), 3.87(s, 4H), 3.55(t, J=8.0Hz, 2H), 2.98(t, J=8.0Hz, 2H)

[0106] 1-15.2-(bis(4,4'-methoxy-2,2'-pyridinyl)methylamino)methylpyridine(15) [ka]

[0107] A round-bottom flask was charged with starting material (6) (1.0 g, 5.18 mmol), 2-pyridylmethylamine (0.28 g, 2.59 mmol), Na2CO3 (2.7 g, 25.9 mmol), and acetonitrile (20 mL). A reflux zone was installed in the reaction flask, and the mixture was refluxed at 80 °C with stirring for 12 hours. After the reaction was completed, the mixture was cooled to room temperature and the excess Na2CO3 was removed using a glass filter. The solution was concentrated using a rotary evaporator, and the resulting dark red oily mixture was purified by column chromatography on neutral alumina using acetonitrile as a eluent to obtain product 15 (0.54 g, 60%) as an orange oily mixture. 1 H NMR (400MHz, CDCl3) δ8.55(d, J=4.8Hz, 1H), 8.34(d, J=5.6Hz, 2H), 7.64(dd, J=4.8, 4.4Hz, 1H), 7.58(d, J=4.0 Hz, 1H), 7.21(s, 2H), 7.15(dd, J=5.2, 4.4Hz, 1H), 6.68(d, J=3.2Hz, 2H), 3.90(s, 2H), 3.85(s, 4H)3.84(s, 6H)

[0108] 1-16.2-(bis(4,4'-cyano-2,2'-pyridinyl)methylamino)methylpyridine(16) [ka]

[0109] A round-bottom flask was charged with starting material (8) (54 mg, 0.345 mmol), 2-pyridylmethylamine (17 mg, 0.157 mmol), Na2CO3 (166 mg, 1.57 mmol), and acetonitrile (10 mL). A reflux zone was installed in the reaction flask, and the mixture was refluxed at 80 °C with stirring for 12 hours. After the reaction was completed, the mixture was cooled to room temperature and the excess Na2CO3 was removed using a glass filter. The solution was concentrated using a rotary evaporator, and the resulting dark red oily mixture was purified by column chromatography on neutral alumina using acetonitrile as a developing solvent to obtain product 16 (36 mg, 68%) as a yellow oil. 1 H NMR (400MHz, CDCl3) δ8.73(d, J=5.2Hz, 2H), 8.58(d, J=4.4Hz, 1H), 7.80(s, 2H), 7.70(dd, J=6.8, 5.2Hz, 1H), 7.46(d, J=5.2Hz, 1H), 7.41(d, J=4.4Hz, 2H), 7.20(dd, J=7.2, 4.4Hz, 1H), 3.99(s, 4H), 3.91(s, 2H)

[0110] 1-17.2-(bis(4,4'-ethoxycarbonyl-2,2'-pyridinyl)methylamino)methylthiazole(17) [ka]

[0111] A round-bottom flask was charged with starting material (4) (0.45 g, 1.929 mmol), starting material (9) (0.1 g, 0.877 mmol), Na2CO3 (2.04 g, 19.3 mmol), and acetonitrile (15 mL). A reflux zone was installed in the reaction flask, and the mixture was refluxed at 80 °C with stirring for 36 hours. After the reaction was completed, the mixture was cooled to room temperature and the excess Na2CO3 was removed using a glass filter. The solution was concentrated using a rotary evaporator, and the resulting orange oily mixture was purified by column chromatography using neutral alumina and acetone as a developing solvent to obtain product 17 (0.22 g, 57%) as a yellow oil.1 H NMR (400MHz, CDCl3) δ8.69(d, J=7.2Hz, 2H), 8.18(s, 2H), 7.73(d, J=4.0Hz, 1H), 7.72(d, J=7.2Hz, 2H), 7.31(d, J=4.0Hz, 2H), 4.43(q, J=7.2Hz, 4H), 4.16(s, 2H), 4.04(s, 4H), 1.43(t, J=7.2Hz, 6H)

[0112] Example 2. Synthesis of TPMA-based osmium complexes [Os(TPMA)Cl] from the ligand synthesized in Example 1 (1-1 to 1-17) + (PF6 - We synthesized osmium complexes and their derivatives. All of the complexes were synthesized from ammonium hexachloroosmate. The osmium salt, which is in a tetravalent ion state, is reduced to the osmium complex, which is in a trivalent ion state. After the reaction, a precipitate is obtained in an aqueous solution of ammonium hexafluorophosphate (NH4PF6), and all of the complexes initially have PF6 as a zwitterion. The synthesis reaction scheme is shown in Figure 2.

[0113] [Os(TPMA)Cl2] + (PF6 - ) complexes all have paramagnetic properties, 1 This could not be observed by H-NMR. This is because the unpaired electrons in the paramagnetic substance interact with the nuclear spins, shortening the relaxation time. Therefore, the synthesis results and oxidation state of the produced osmium complex were determined by ESI-MS. [Os(TPMA)Cl2] + (PF6 - The crystal structure of the (18) complex was confirmed by single crystal X-ray diffraction analysis, and is shown in Figure 3.

[0114] [Os(TPMA)Cl2] synthesized in an additional step + (PF6- Various TPMA-based osmium complexes were synthesized by reacting the complexes with various monodentate or bidentate ligands. Monodentate ligands such as 1-methylimidazole and pyridine, and bidentate ligands such as 4-methoxy-2-pyridylcarboxamide, picolinic acid, catechol, and acetylacetone resulted in [Os(TPMA)Cl2]. + (PF6 - ) and undergo an exchange reaction with the chlorine (Cl) ligand to form [Os(TPMA)(L) 1~2 ] + (PF6 - ) complexes were synthesized. The synthesis reaction scheme is shown in Figure 4.

[0115] The synthesized [Os(TPMA)(L) 1~2 ] n+ (PF6 - ) n The system complex is [Os(TPMA)Cl2] + (PF6 - ) complexes, all of which have paramagnetic properties, 1 The synthesis results and oxidation state of the material were determined by ESI-MS. [Os(TPMA)(L) 1~2 ] n+ (PF6 - ) n In the case of oxygen-coordinated compounds (27-33) such as catechol, acetylacetone, and picolinic acid, the reaction was carried out in the presence of a base such as triethylamine (TEA) or sodium hydroxide (NaOH).

[0116] Among the synthesized TPMA-based complexes, in the case of complexes having a hydroxymethyl (-CH2OH) group in the TPMA ligand, such as 21, 31, and 32, a characteristic mass signal corresponding to the removal of the hydroxyl (-OH) group was confirmed by ESI-MS. This is thought to be because the highly reactive pyridylmethyl (-PyCH2) group is easily ionized and is stable, resulting in the observation of a mass signal corresponding to the removal of the hydroxyl group (Figure 5).

[0117] The monodentate ligand 1-methylimidazole was added to [Os(TPMA)Cl2] + (PF6 - In the case of complexes 26, 34, and 35, which were reacted with 2-10 equivalents of 1-methylimidazole in ethylene glycol at 130-180 °C for 3-12 hours, high yields were obtained. However, the monodentate 1-methylimidazole complex [Os(TPMA)(imi)Cl] n+ (PF6 - ) n In the case of the complex, when only one equivalent of 1-methylimidazole was reacted, no reaction occurred at all, but when two equivalents were added, two imidazole ligands were coordinated, making it impossible to obtain this selectively.

[0118] All the synthesized TPMA-based osmium complexes were initially prepared using PF6 - It is obtained in zwitterionic form and Cl - Using ion exchange resin [Os(TPMA)Cl2] + (Cl - ) or [Os(TPMA)(L) 1~2 ] n+ (Cl - ) n Most of the TPMA-based osmium complexes showed large differences in solubility depending on the zwitterionic form. - When in the zwitterionic form, Cl exhibits good solubility in acetonitrile and acetone, but poor solubility in water and methanol. - When in the zwitterionic form, it exhibited good solubility in water and methanol, but was poorly soluble in organic solvents such as acetonitrile and acetone.

[0119] 2-1.[Os(TPMA)Cl2] + (PF6 - ) Complex(18) [ka]

[0120] Ammonium hexachloroosmate (30 mg, 0.068 mmol) and starting material (10) (20 mg, 0.068 mmol) were placed in a glass culture tube, and ethylene glycol (2 mL) was added to form a mixed solution. Argon gas was then bubbled through the tube for 10 minutes to create an argon atmosphere, and the mixture was then refluxed at 140°C for 6 hours. The reaction mixture was then slowly added dropwise to a saturated solution of distilled water containing excess ammonium hexafluorophosphate. The precipitate formed in the solution was filtered and washed with distilled water and excess diethyl ether to confirm that the zwitterion was PF6. - The green solid product 18 was obtained (35 mg, 73%). - To convert it to PF6 - The product was dissolved in a small amount (~1 mL) of acetonitrile and then added Cl - A mixture of ion exchange resin and excess distilled water (25 mL) was prepared and stirred overnight. - The resin is filtered, and the filtrate is collected and the solvent removed using vacuum distillation to obtain the zwitterion Cl. - The product was obtained. ESI-MS (Low resolution): Calculated for cation [M] + C 18 H 18 Cl2N4Os:552.05 Found:552.16[M] +

[0121] 2-2.[Os(TPMA)Cl2] + (PF6 - ) Complex(19) [ka]

[0122] Ammonium hexachloroosmate (129 mg, 0.293 mmol) and starting material (11) (120 mg, 0.293 mmol) were placed in a glass culture tube, and ethylene glycol (3 mL) was added to form a mixed solution. Argon gas was then bubbled through the tube for 10 minutes to create an argon atmosphere, and the mixture was then refluxed at 140°C for 6 hours. The reaction mixture was then slowly added dropwise to a saturated solution of distilled water containing excess ammonium hexafluorophosphate. The precipitate formed in the solution was filtered and washed with distilled water and excess diethyl ether to confirm that the zwitterion was PF6. - The brown solid product 19 was obtained (220 mg, 95%). - To convert it to PF6 - The product was dissolved in a small amount (~1 mL) of acetonitrile and then added Cl - A mixture of ion exchange resin and excess distilled water (25 mL) was prepared and stirred overnight. - The resin is filtered, and the filtrate is collected and the solvent removed using vacuum distillation to obtain the zwitterion Cl. - The product was obtained. ESI-MS (Low resolution): Calculated for cation [M] + C 24 H 26 Cl2N4O4Os:696.09 Found:696.25[M] +

[0123] 2-3.[Os(TPMA)Cl2] + (PF6 - ) Complex(20) [ka]

[0124] Ammonium hexachloroosmate (327 mg, 0.744 mmol) and starting material (12) (270 mg, 0.744 mmol) were placed in a glass culture tube, and ethylene glycol (3 mL) was added to form a mixed solution. Argon gas was then bubbled through the tube for 10 minutes to create an argon atmosphere, and the mixture was then refluxed at 140°C for 6 hours. The reaction mixture was then slowly added dropwise to a saturated solution of distilled water containing excess ammonium hexafluorophosphate. The precipitate formed in the solution was filtered and washed with distilled water and excess diethyl ether to confirm that the zwitterion was PF6. - The brown solid product 20 was obtained (521 mg, 91%). - To convert it to PF6 - The product was dissolved in a small amount (~1 mL) of acetonitrile and then added Cl - A mixture of ion exchange resin and excess distilled water (25 mL) was prepared and stirred overnight. - The resin is filtered, and the filtrate is collected and the solvent removed using vacuum distillation to obtain the zwitterion Cl. - The product was obtained. ESI-MS (Low resolution): Calculated for cation [M] + C 21 H 22 C l2 N4O2Os:624.07 Found:624.25[M] +

[0125] 2-4.[Os(TPMA)Cl2] + (PF6 - ) Complex(21) [ka]

[0126] Ammonium hexachloroosmate (62 mg, 0.141 mmol) and starting material (13) (45 mg, 0.141 mmol) were placed in a glass culture tube, and ethylene glycol (3 mL) was added to form a mixed solution. Argon gas was then bubbled through the tube for 10 minutes to create an argon atmosphere, and the mixture was then refluxed at 140°C for 6 hours. The reaction mixture was then slowly added dropwise to a saturated solution of distilled water containing excess ammonium hexafluorophosphate. The precipitate formed in the solution was filtered and washed with distilled water and excess diethyl ether to confirm that the zwitterion was PF6. - The brown solid product 21 was obtained (51 mg, 50%). - To convert it to PF6 - The product was dissolved in a small amount (~1 mL) of acetonitrile and then added Cl - A mixture of ion exchange resin and excess distilled water (25 mL) was prepared and stirred overnight. - The resin is filtered, and the filtrate is collected and the solvent removed using vacuum distillation to obtain the zwitterion Cl. - The product was obtained. ESI-MS (Low resolution): Calculated for cation [M] + C 19 H 20 Cl2N4OOs:582.06 Found:566.2500[M-OH] +

[0127] 2-5.[Os(TPMA)Cl2] + (PF6 - ) Complex(22) [ka]

[0128] Ammonium hexachloroosmate (326 mg, 0.742 mmol) and starting material (14) (280 mg, 0.742 mmol) were placed in a glass culture tube, and ethylene glycol (3 mL) was added to form a mixed solution. Argon gas was then bubbled through the tube for 10 minutes to create an argon atmosphere, and the mixture was then refluxed at 140°C for 16 hours. The reaction mixture was then slowly added dropwise to a saturated solution of distilled water containing excess ammonium hexafluorophosphate. The precipitate formed in the solution was filtered and washed with distilled water and excess diethyl ether to confirm that the zwitterion was PF6. - The brown or black solid product 22 was obtained (355 mg, 61%). - To convert it to PF6 - The product was dissolved in a small amount (~1 mL) of acetonitrile and then added Cl - A mixture of ion exchange resin and excess distilled water (25 mL) was prepared and stirred overnight. - The resin is filtered, and the filtrate is collected and the solvent removed using vacuum distillation to obtain the zwitterion Cl. - The product was obtained. ESI-MS (High resolution): Calculated for cation [M] + C 21 H 24 Cl2N6OOs:638.10 Found:638.0998[M] + , 319.5538[M] 2+

[0129] 2-6.[Os(TPMA)Cl2] + (PF6 - ) Complex(23) [ka]

[0130] Ammonium hexachloroosmate (129 mg, 0.293 mmol) and starting material (15) (120 mg, 0.293 mmol) were placed in a glass culture tube, and ethylene glycol (3 mL) was added to form a mixed solution. Argon gas was then bubbled through the tube for 10 minutes to create an argon atmosphere, and the mixture was then refluxed at 140°C for 6 hours. The reaction mixture was then slowly added dropwise to a saturated solution of distilled water containing excess ammonium hexafluorophosphate. The precipitate formed in the solution was filtered and washed with distilled water and excess diethyl ether to confirm that the zwitterion was PF6. - The brown solid product 23 was obtained (220 mg, 95%). - To convert it to PF6 - The product was dissolved in a small amount (~1 mL) of acetonitrile and then added Cl - A mixture of ion exchange resin and excess distilled water (25 mL) was prepared and stirred overnight. - The resin is filtered, and the filtrate is collected and the solvent removed using vacuum distillation to obtain the zwitterion Cl. - The product was obtained. ESI-MS (Low resolution): Calculated for cation [M] + C 20 H 22 Cl2N4O2Os:612.07 Found:612.16[M] +

[0131] 2-7.[Os(TPMA)(L) 1~2 ] n+ (PF6 - ) n Complex(24) [ka]

[0132] The starting material (18) (60 mg, 0.086 mmol) and 4-methoxy-2-pyridinecarboxamide (65 mg, 0.430 mmol) were placed in a glass culture tube, and ethylene glycol (3 mL) was added to form a mixed solution. Argon gas was then bubbled through the tube for 10 minutes to create an argon atmosphere, and the mixture was then refluxed at 130°C for 12 hours. The reaction mixture was slowly added dropwise to a saturated solution of distilled water containing excess ammonium hexafluorophosphate. The precipitate formed in the solution was filtered and washed with distilled water and excess diethyl ether to confirm that the zwitterion was PF6. - The brown solid product 24 was obtained (32 mg, 40%). - To convert it to PF6 - The product was dissolved in a small amount (~1 mL) of acetonitrile and then added Cl - A mixture of ion exchange resin and excess distilled water (25 mL) was prepared and stirred overnight. - The resin is filtered, and the filtrate is collected and the solvent removed using vacuum distillation to obtain the zwitterion Cl. - The product was obtained. ESI-MS (Low resolution): Calculated for cation [M] + C 25 H 26 N6O2Os:634.17 Found:634.2500[M] + , 317.1667[M] 2+

[0133] 2-8.[Os(TPMA)(L) 1~2 ] n+ (PF6 - ) n Complex(25) [ka]

[0134] The starting material (18) (30 mg, 0.043 mmol) and N-methylimidazole (35 mg, 0.430 mmol) were placed in a glass culture tube, and ethylene glycol (1.5 mL) was added to form a mixed solution. Argon gas was then bubbled through the tube for 10 minutes to create an argon atmosphere, and the mixture was then refluxed at 130 °C for 3 hours. The reaction mixture was then slowly added dropwise to a saturated solution of distilled water containing excess ammonium hexafluorophosphate. The precipitate formed in the solution was filtered and washed with distilled water and excess diethyl ether to confirm that the zwitterion was PF6. - The brown solid product 25 was obtained (34 mg, 85%). - To convert it to PF6 - The product was dissolved in a small amount (~1 mL) of acetonitrile and then added Cl - A mixture of ion exchange resin and excess distilled water (25 mL) was prepared and stirred overnight. - The resin is filtered, and the filtrate is collected and the solvent removed using vacuum distillation to obtain the zwitterion Cl. - The product was obtained. ESI-MS (Low resolution): Calculated for cation [M] + C 26 H 30 N8Os:646.22 Found:323.08[M] 2+

[0135] 2-9.[Os(TPMA)(L) 1~2 ] n+ (PF6 - ) n Complex(26) [ka]

[0136] The starting material (18) (100 mg, 0.143 mmol) and pyridine (11 mg, 0.143 mmol) were placed in a glass culture tube, and ethylene glycol (4 mL) was added to form a mixed solution. Argon gas was then bubbled through the tube for 10 minutes to create an argon atmosphere, and the mixture was then refluxed at 180°C for 15 hours. The reaction mixture was slowly added dropwise to a saturated solution of distilled water containing excess ammonium hexafluorophosphate. The precipitate formed in the solution was filtered and washed with distilled water and excess diethyl ether to confirm that the zwitterion was PF6. - The brown solid product 26 was obtained (31 mg, 29%). - To convert it to PF6 - The product was dissolved in a small amount (~1 mL) of acetonitrile and then added Cl - A mixture of ion exchange resin and excess distilled water (25 mL) was prepared and stirred overnight. - The resin is filtered, and the filtrate is collected and the solvent removed using vacuum distillation to obtain the zwitterion Cl. - The product was obtained. ESI-MS (Low resolution): Calculated for cation [M] + C 23 H 23 ClN5Os:596.13 Found:596.3333[M] +

[0137] 2-10.[Os(TPMA)(L) 1~2 ] n+ (PF6 - ) n Complex(27) [ka]

[0138] The starting material (18) (100 mg, 0.143 mmol), 4-bromopicolinic acid (173 mg, 0.861 mmol), and triethylamine (145 mg, 1.430 mmol) were placed in a glass culture tube, and distilled water (3 mL) was added to prepare a mixed solution. Argon gas was then bubbled through the tube for 10 minutes to create an argon atmosphere, and the mixture was then refluxed at 100°C for 3 hours. The reaction mixture was then slowly added dropwise to a saturated solution of distilled water containing excess ammonium hexafluorophosphate. The precipitate formed in the solution was filtered and washed with distilled water and excess diethyl ether to confirm that the zwitterion was PF6. - The brown solid product 27 was obtained (89 mg, 75%). - To convert it to PF6 - The product was dissolved in a small amount (~1 mL) of acetonitrile and then added Cl - A mixture of ion exchange resin and excess distilled water (25 mL) was prepared and stirred overnight. - The resin is filtered, and the filtrate is collected and the solvent removed using vacuum distillation to obtain the zwitterion Cl. - The product was obtained. ESI-MS (Low resolution): Calculated for cation [M] + C 24 H 21 BrN5O2Os:682.05 Found:682.1667[M] +

[0139] 2-11.[Os(TPMA)(L) 1~2 ] n+ (PF6 - ) n Complex(28) [ka]

[0140] The starting material (18) (30 mg, 0.043 mmol), catechol (47 mg, 0.430 mmol), and K2CO3 (59 mg, 0.430 mmol) were placed in a glass culture tube, and distilled water (1.5 mL) was added to prepare a mixed solution. Argon gas was then bubbled through the tube for 10 minutes to create an argon atmosphere, and the mixture was then refluxed at 100 °C for 12 hours. The reaction solution was then slowly added dropwise to a saturated solution of distilled water containing excess ammonium hexafluorophosphate. The precipitate formed in the solution was filtered and washed with distilled water and excess diethyl ether to confirm that the zwitterion was PF6. - The product 28 was obtained as a black solid (19.6 mg, 62%). - To convert it to PF6 - The product was dissolved in a small amount (~1 mL) of acetonitrile and then added Cl - A mixture of ion exchange resin and excess distilled water (25 mL) was prepared and stirred overnight. - The resin is filtered, and the filtrate is collected and the solvent removed using vacuum distillation to obtain the zwitterion Cl. - The product was obtained. ESI-MS (Low resolution): Calculated for cation [M] + C 24 H 22 N4O2Os:590.14 Found:590.2500[M] +

[0141] 2-12.[Os(TPMA)(L) 1~2 ] n+ (PF6 - ) n Complex(29) [ka]

[0142] The starting material (18) (200 mg, 0.287 mmol), 4-bromopicolinic acid (70 mg, 0.569 mmol), and triethylamine (29 mg, 0.287 mmol) were placed in a glass culture tube, and ethylene glycol (3 mL) was added to form a mixed solution. Argon gas was then bubbled through the tube for 10 minutes to create an argon atmosphere, and the mixture was then refluxed at 140°C for 3 hours. The reaction solution was then slowly added dropwise to a saturated solution of distilled water with excess ammonium hexafluorophosphate. The precipitate formed in the solution was filtered and washed with distilled water and excess diethyl ether to confirm that the zwitterion was PF6. - The brown solid product 29 was obtained (113 mg, 53%). - To convert it to PF6 - The product was dissolved in a small amount (~1 mL) of acetonitrile and then added Cl - A mixture of ion exchange resin and excess distilled water (25 mL) was prepared and stirred overnight. - The resin is filtered, and the filtrate is collected and the solvent removed using vacuum distillation to obtain the zwitterion Cl. - The product was obtained. ESI-MS (High resolution): Calculated for cation [M] + C 24 H 22 N5O2Os:604.14 Found:604.13824[M] +

[0143] 2-13.[Os(TPMA)(L) 1~2 ] n+ (PF6 - ) n Complex(30) [ka]

[0144] The starting material (18) (30 mg, 0.043 mmol), acetylacetone (43 mg, 0.430 mmol), and NaOH (8 mg, 0.215 mmol) were placed in a glass culture tube, and distilled water (1.5 mL) was added to prepare a mixed solution. Argon gas was then bubbled through the tube for 10 minutes to create an argon atmosphere, and the mixture was then refluxed at 100°C for 4 hours. The reaction mixture was then slowly added dropwise to a saturated solution of distilled water containing excess ammonium hexafluorophosphate. The precipitate formed in the solution was filtered and washed with distilled water and excess diethyl ether to confirm that the zwitterion was PF6. - The product 30 was obtained as a black solid (16 mg, 50%). - To convert it to PF6 - The product was dissolved in a small amount (~1 mL) of acetonitrile and then added Cl - A mixture of ion exchange resin and excess distilled water (25 mL) was prepared and stirred overnight. - The resin is filtered, and the filtrate is collected and the solvent removed using vacuum distillation to obtain the zwitterion Cl. - The product was obtained. ESI-MS (Low resolution): Calculated for cation [M] + C 23 H 25 N4O2Os:581.16 Found:581.3333[M] + , 290.6666[M] 2+

[0145] 2-14.[Os(TPMA)(L) 1~2 ] n+ (PF6 - ) n Complex(31) [ka]

[0146] The starting material (21) (100 mg, 0.137 mmol), picolinic acid (67 mg, 0.548 mmol), and triethylamine (56 mg, 0.548 mmol) were placed in a glass culture tube, and ethylene glycol (3 mL) was added to form a mixed solution. Argon gas was then bubbled through the tube for 10 minutes to create an argon atmosphere, and the mixture was then refluxed at 140 °C for 4 hours. The reaction solution was then slowly added dropwise to a saturated solution of distilled water with excess ammonium hexafluorophosphate. The precipitate formed in the solution was filtered and washed with distilled water and excess diethyl ether to confirm that the zwitterion was PF6. - The brown solid product 31 was obtained (54 mg, 51%). - To convert it to PF6 - The product was dissolved in a small amount (~1 mL) of acetonitrile and then added Cl - A mixture of ion exchange resin and excess distilled water (25 mL) was prepared and stirred overnight. - The resin is filtered, and the filtrate is collected and the solvent removed using vacuum distillation to obtain the zwitterion Cl. - The product was obtained. ESI-MS (Low resolution): Calculated for cation [M] + C 25 H 24 N5O3Os:634.15 Found:618.3333[M-OH] +

[0147] 2-15.[Os(TPMA)(L) 1~2 ] n+ (PF6 - ) n Complex(32) [ka]

[0148] The starting material (21) (100 mg, 0.137 mmol), 4-methylpicolinic acid (76 mg, 0.550 mmol), and triethylamine (56 mg, 0.550 mmol) were placed in a glass culture tube, and ethylene glycol (3 mL) was added to form a mixed solution. Argon gas was then bubbled through the tube for 10 minutes to create an argon atmosphere, and the mixture was then refluxed at 140 °C for 4 hours. The reaction solution was then slowly added dropwise to a saturated solution of distilled water with excess ammonium hexafluorophosphate. The precipitate formed in the solution was filtered and washed with distilled water and excess diethyl ether to confirm that the zwitterion was PF6. - The brown solid product 32 was obtained (58 mg, 53%). - To convert it to PF6 - The product was dissolved in a small amount (~1 mL) of acetonitrile and then added Cl - A mixture of ion exchange resin and excess distilled water (25 mL) was prepared and stirred overnight. - The resin is filtered, and the filtrate is collected and the solvent removed using vacuum distillation to obtain the zwitterion Cl. - The product was obtained. ESI-MS (Low resolution): Calculated for cation [M] + C 26 H 26 N5O3Os:648.17 Found:632.4167[M-OH] +

[0149] 2-16.[Os(TPMA)(L) 1~2 ] n+ (PF6 - ) n Complex(33) [ka]

[0150] The starting material (22) (40 mg, 0.051 mmol), 4-methylpicolinic acid (28 mg, 0.204 mmol), and triethylamine (15 mg, 153 mmol) were placed in a glass culture tube, and ethylene glycol (3 mL) was added to form a mixed solution. Argon gas was then bubbled through the tube for 10 minutes to create an argon atmosphere, and the mixture was then refluxed at 120°C for 5 hours. The reaction mixture was then slowly added dropwise to a saturated solution of distilled water containing excess ammonium hexafluorophosphate. The precipitate formed in the solution was filtered and washed with distilled water and excess diethyl ether to confirm that the zwitterion was PF6. - The brown solid product 33 was obtained (12 mg, 28%). - To convert it to PF6 - The product was dissolved in a small amount (~1 mL) of acetonitrile and then added Cl - A mixture of ion exchange resin and excess distilled water (25 mL) was prepared and stirred overnight. - The resin is filtered, and the filtrate is collected and the solvent removed using vacuum distillation to obtain the zwitterion Cl. - The product was obtained. ESI-MS (High resolution): Calculated for cation [M] + C 28 H30N7O3Os:704.20 Found:704.2019[M] + , 352.6044[M] 2+

[0151] 2-17.[Os(TPMA)(L) 1~2 ] n+ (PF6 - ) n Complex(34) [ka]

[0152] The starting material (22) (40 mg, 0.051 mmol) and N-methylimidazole (42 mg, 0.510 mmol) were placed in a glass culture tube, and ethylene glycol (2 mL) was added to form a mixed solution. Argon gas was then bubbled through the tube for 10 minutes to create an argon atmosphere, and the mixture was then refluxed at 130 °C for 3 hours. The reaction mixture was slowly added dropwise to a saturated solution of distilled water containing excess ammonium hexafluorophosphate. The precipitate formed in the solution was filtered and washed with distilled water and excess diethyl ether to confirm that the zwitterion was PF6. - The brown solid product 34 was obtained (50 mg, 96%). - To convert it to PF6 - The product was dissolved in a small amount (~1 mL) of acetonitrile and then added Cl - A mixture of ion exchange resin and excess distilled water (25 mL) was prepared and stirred overnight. - The resin is filtered, and the filtrate is collected and the solvent removed using vacuum distillation to obtain the zwitterion Cl. - The product was obtained. ESI-MS (High resolution): Calculated for cation [M] + C 29 H 36 N 10 OOs:732.27 Found:367.1182[M+2] 2+

[0153] 2-18.[Os(TPMA)(L) 1~2 ] n+ (PF6 - ) n Complex(35) [ka]

[0154] The starting material (23) (30 mg, 0.040 mmol) and N-methylimidazole (32 mg, 0.400 mmol) were placed in a glass culture tube, and ethylene glycol (1.5 mL) was added to form a mixed solution. Argon gas was then bubbled through the tube for 10 minutes to create an argon atmosphere, and the mixture was then refluxed at 130 °C for 3 hours. The reaction mixture was slowly added dropwise to a saturated solution of distilled water containing excess ammonium hexafluorophosphate. The precipitate formed in the solution was filtered and washed with distilled water and excess diethyl ether to confirm that the zwitterion was PF6. - The brown solid product 35 was obtained (31 mg, 80%). - To convert it to PF6 - The product was dissolved in a small amount (~1 mL) of acetonitrile and then added Cl - A mixture of ion exchange resin and excess distilled water (25 mL) was prepared and stirred overnight. - The resin is filtered, and the filtrate is collected and the solvent removed using vacuum distillation to obtain the zwitterion Cl. - The product was obtained. ESI-MS (Low resolution): Calculated for cation [M] + C 28 H 34 N8O2Os:706.24 Found:353.2500[M] 2+

[0155] Example 3. Electrochemical characterization of TPMA-based osmium complexes according to the present invention Cyclic voltammetry (CV) was used to analyze the electrochemical properties of the synthesized TPMA-based osmium complexes. A well-cleaned carbon glass electrode with a diameter of 3 mm was used as the working electrode, and measurements were performed at a scan rate of 10 mV / s using an Ag / AgCl electrode as the reference electrode and a Pt electrode as the counter electrode. - The zwitterionic form of the complex was measured in 0.1 M TBAP in acetonitrile at a concentration of 3 mg / mL. - The zwitterionic complex was analyzed using Cl to confirm the position of the redox peaks. -The substances were measured in solution during the anion exchange process, and the concentrations of each substance were not constant. The oxidation / reduction potentials of all the synthesized TPMA-based osmium complexes are shown in Figures 7a to 7c. [Os(TPMA)Cl2] + (Cl - )(18) is the most basic TPMA osmium complex without any substituents, and E 1 / 2 =-0.339 V, and the results are shown in Figure 6. The change in oxidation / reduction potential depending on the type of substituent and coordinated ligand was analyzed with this complex as the center.

[0156] The oxidation / reduction potentials of complexes 19–23, which have the same coordination structure as complex 18 but contain different substituents, were compared. Complexes containing electron-withdrawing groups (EWGs), such as complex 19 (containing two ethyl ester (-COOEt) groups), complex 20 (containing one), and complex 22 (containing an amide (-COONH) group, exhibited more positive oxidation / reduction potentials than complex 18. Conversely, complexes containing electron-donating groups (EDGs), such as complex 21 (containing a methyl (-CH2-) group) and complex 23 (containing two methoxy (-OMe) groups), exhibited more negative oxidation / reduction potentials than complex 18. These results suggest that EWGs decrease the electron density of the central metal, favoring reduction, resulting in a more positive potential shift. EDGs increase electron density, favoring oxidation, resulting in a more negative potential shift. Complexes 20 and 22 shifted the oxidation / reduction potential of complex 18 by approximately 0.102–0.103 V in the positive direction. Both complexes exhibited similar oxidation / reduction potentials, suggesting that the ester and amide groups containing carbonyl groups exert similar electrochemical effects. Complex 19 shifted the oxidation / reduction potential of complex 18 by approximately 0.176 V in the positive direction, confirming that the greater the number of ester groups (EDGs), the greater the electrochemical effect. Complex 21, which contains a hydroxymethyl group, shifted the potential slightly in the negative direction by 0.031 V, while complex 23, which contains two methoxy groups (a better EDG), shifted it by 0.125 V in the negative direction, suggesting that the type and number of EDGs play a larger role. These results suggest that TPMA-based osmium complexes can exhibit potential variations depending on the substituent, thereby confirming their potential applicability and potential improvement as electron transfer mediators with ideal oxidation / reduction potentials.

[0157] Various ligands were coordinated [Os(TPMA)(L) 1~2 ] n+ (PF6 - ) nAll of the complexes (24–30) showed oxidation / reduction potentials that were more positive than those of complex 18. The oxidation / reduction potentials increased significantly in the positive direction in the order of acetylacetone, one pyridine, 4-bromopicolinic acid, picolinic acid, two 1-methylimidazoles, 4-methoxy-2-pyridylcarboxamide, and catechol. Complexes 25 and 28, coordinated with 4-methoxy-2-pyridylcarboxamide and catechol, respectively, exhibited two or more reversible oxidation / reduction peaks and did not exhibit a stable single oxidation / reduction potential, and were therefore deemed unsuitable as electron transfer mediators. However, the TPMA-osmium complex coordinated with picolinic acid exhibited a moderate positive shift in potential, exhibiting an ideal oxidation / reduction potential close to 0 V relative to the Ag / AgCl electrode, and was therefore deemed a suitable TPMA-osmium complex for potential application as an electron transfer mediator. Furthermore, characteristically, [Os(TPMA)Cl2] + (X - Complexes 18 to 23 in the ) series showed similar redox potentials under different solvent conditions due to their zwitterionic forms, but excluding these, [Os(TPMA)(L) 1~2 ] n+ (X - ) n All the complexes are PF6 - Cl is more soluble than when it is in the zwitterionic form. - When in the zwitterionic form, the oxidation / reduction potential appeared in a more negative region, from a minimum of 36 mV to a maximum of 210 mV.

Claims

1. Transition metal complexes useful as electron transfer mediators comprising tetradentate nitrogen donor ligands represented by Formula 1 or 2: [Chemical formula 1] 【Chemistry 1】 [Chemical formula 2] 【Chemistry 2】 In the above Chemical Formula 1 or Chemical Formula 2, M is Os; C ａ , C ｂ and C ｃ are each independently a heterocycle containing one or more nitrogen atoms, preferably linked to the amine group and the methylene group at the 2-position of the ring; L ｍ１ and L ｍ２ are each independently —H, —F, —Cl, —Br, —I, 【Chemistry 2-1】 or 【Chemistry 2-2】 and L ｂ -L ｂ is catechol, acetylacetone, picolinic acid, 4-bromopicolinic acid, 4-methylpicolinic acid, 4-methoxy-2-pyridylcarboxamide, or 2,2′-bithiazole; m is a negative or positive charge representing −1 to −5 or 1 to 5; R １ , R ２ and R ３ are each independently —H; —CN; or —CO ２ H; -CH ２ OH;-CONHCH ２ CH ２ NH ２ or substituted or unsubstituted alkoxycarbonyl, wherein L ｍ１ and L ｍ２ When both are —Cl, R １ , R ２ and R ３ is - not H; X is a counter ion; n means the number of counter ions and is 1 to 5.

2. The transition metal complex of claim 1 , wherein the transition metal complex comprises a tetradentate ligand represented by the following formula 3: [Chemical formula 3] 【Transformation 3】 In the above formula, C ａ , C ｂ and C ｃ are each independently a heterocycle containing one or more nitrogen atoms; R １ , R ２ and R ３ are each independently —H; —CN; or —CO ２ H; -CH ２ OH;-CONHCH ２ CH ２ NH ２ or substituted or unsubstituted alkoxycarbonyl, wherein L ｍ１ and L ｍ２ When both are —Cl, R １ , R ２ and R ３ is not -H.

3. A transition metal complex that is an osmium complex and includes one of the tetradentate ligands having the following structure: 【Chemistry 4】

4. The transition metal complex according to any one of claims 1 to 3, wherein the transition metal complex is one of the following transition metal complexes: Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5

5. a) introducing a ligand represented by Chemical Formula 3 into an ammonium salt of an osmium halide represented by Chemical Formula 5 to synthesize an osmium complex represented by Chemical Formula 6; and b) introducing one or two monodentate or bidentate ligands selected from the group consisting of N-N ligands, N-ligands, N-ligands, N-O ligands, and O-O ligands into the osmium complex of Chemical Formula 6, [Chemical formula 3] 【Transformation 5】 [Chemical formula 5] [(NH ４ ) ２ OsCl ６ ] [Chemical formula 6] 【Transformation 6】 In the above formula, C ａ , C ｂ and C ｃ are each independently a heterocycle containing one or more nitrogen atoms; R １ , R ２ and R ３ are each independently —H; —CN; or —CO ２ H; -CH ２ OH;-CONHCH ２ CH ２ NH ２ or a substituted or unsubstituted alkoxycarbonyl.

6. The method according to claim 5, wherein in step b), one N-N ligand, two N ligands, one N ligand, one N-O ligand, or one O-O ligand is introduced into the osmium complex of Formula 6 to prepare transition metal complexes of the following Formulas 7 to 11: [Chemical formula 7] 【Transformation 7】 [Chemical formula 8] 【Transformation 8】 [Chemical formula 9] 【Chemistry 9】 [Chemical formula 10] 【Chemistry 10】 [Chemical formula 11] 【Chemistry 11】

7. The N ligand is a heterocycle containing one or more nitrogen atoms, the N—N ligand is 2-pyridinecarboxamide or 2,2′-bithiazole; the N—O ligand is 2-picolinic acid; The method of claim 6, wherein the O-O ligand is catechol or acetylacetone.

8. A device comprising the transition metal complex according to any one of claims 1 to 4 as an electron transfer mediator.

9. The device of claim 8 , wherein the device is an electrochemical biosensor.

10. The device of claim 8 , wherein the device is a blood glucose monitoring sensor.

11. an enzyme capable of oxidizing or reducing a liquid biological sample; and A sensing film for an electrochemical biosensor, comprising the transition metal complex according to any one of claims 1 to 4 as an electron transfer mediator.

12. The enzyme is one or more oxidoreductases selected from the group consisting of dehydrogenases, oxidases, and esterases; or 12. The sensing membrane for an electrochemical biosensor according to claim 11, comprising one or more oxidoreductases selected from the group consisting of dehydrogenases, oxidases, and esterases, and one or more cofactors selected from the group consisting of flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), and pyrroloquinoline quinone (PQQ).

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