JPS6187676A - Alkylammonium polycation type 5-alkylphenazinium and its use as an electron carrier - Google Patents

Abstract

NEW MATERIAL:The titled substance wherein at least one of X, Y, and Z of a 5-alkylphenazinium derivative shown by the formula I [R is alkyl, X is side-chain substituent group introduced to 1-position (or 9-position) of phenazinium skeleton; Y and Z are side-chain substituent group or H introduced to either of 2, 3, or 4 position (or 8, 7, or 6-position)] consists of a substituent group containing one or more alkylammoniums [group shown by the formula II (R1-R3 are alkyl, and R3 may be H)]. USE:An electron transferring particle. PREPARATION:A 1-hydroxy-5-alkylphenazinium shown by the formula III is reacted with an alkylaminoalkyl chloride to give a 1-alkylaminoalkoxyphenazine, which is treated with a dialkyl sulfate to give the aimed substance.

Description

Detailed Description of the Invention "Industrial Field of Application" The present invention is a novel substance/Completely new application of cationic 5-alkylzenazinium and an electron carrier using it bound to an anionic carrier. Regarding the field.

"Prior Art" Phenazine mesosulfate (formula ■) is known as a substance that efficiently reacts with NADH or NADPH (hereinafter collectively referred to as NAD(P)H) in a non-enzymatic manner. It has been used as an electron carrier in mediated redox reactions. The principle of its application is as shown in equation [1] below.

Dehydrogenase Here, the string represents the substrate that is oxidized by the action of dehydrogenase and NAD (P), and after oxidation, it changes to NAD (P).
P) becomes NAD(P)H. Detection system B is reduced through the reaction of PMS coexisting with NAD(P)H, and NAD(P)
Return to PMS is very efficient as an electron carrier, and N
This facilitates the reduction of B by AD(P)HK.

A variety of dehydrogenases have traditionally been used. Also, various things are possible according to the formula []]. An example of what has been used is lactate dehydrogenase,
Glutamate dehydrogenase, glycerol dehydrogenase, glucose-6-phosphate dehydrogenase, etc. have become almost routine, especially in the field of clinical chemistry. Formula [1]
Accordingly, these enzymes can be used to directly measure lactic acid, glutamic acid, glycerin, and glucose-6-phosphate in samples, and by using excess amounts of lactic acid, glutamic acid, glycerin, and glucose-6-phosphate as substrates, The activity of the corresponding dehydrogenase itself is measured. For example, the formula for lactate dehydrogenase is as follows.

In addition to lactate dehydrogenase and other connected enzyme systems, various components other than those in the sample on which dehydrogenase acts directly are measured depending on the purpose. - For example, the following two enzyme activities in the blood are important as clinical chemical tests related to liver function, and the amounts are currently measured frequently: L-alanine: 2-
Oxoglutarate aminotransferase (ALT
), L-7 subarucheet:2-oxoglutarate aminotransferase (AST), A
LT produces pyruvic acid and glutamic acid using L-alanine and 2-oxoglutaric acid as substrates, and AST similarly produces oxaloacetic acid and glutamic acid using L-aspartic acid and 2-oxoglutaric acid as substrates.

These two enzymes thus both produce glutamic acid as their product, so ALT or AS
ALT and AST activities in samples such as disintegration are determined by measuring the glutamic acid produced over time by T using the above-mentioned glutamic acid dehydrogenase and the silk combination of NAD or NADP according to the formula [1]. I can do it.

As another example, glucose can be measured directly using glucose dehydrogenase, but due to the enzyme's performance and ease of availability, it is recommended to first treat glucose with hexokinase or glucokinase and ATP. This is often measured using glucose-6-phosphate dehydrogenase and NADP according to the formula []]. In this way, it is already a common technique to connect with other enzyme systems and ultimately guide the reaction to the dehydrogenase system to measure the target component. As described above, the applicability of the measurement system based on the formula [1] using PMS is extremely wide and important.

In addition, dehydrogenase is more NAD (P) shown in formula [1].
) is often used in combination with the formula [
2], the dehydrogenase itself retains the coenzyme or similar cofactor without requiring a combination with these coenzymes, and the dehydrogenase itself can directly oxidize the substrate. There is also an acceptor type dehydrogenase, and when an electron transport system is constituted by the presence of PMS, PMS also efficiently reduces detection system B to achieve measurement.

In both cases of dehydrogenase, NAD(P) is only mediated by PMS.
It can be applied in the measurement of target components in the same way as the dehydrogenases involved.

Next, detection system B will be described. In formulas [1] and [2], two electrons are released per AHI molecule by the action of dehydrogenase, and electron acceptor B of the detection system is reduced via PMS. Therefore, by observing the amount of change in B, an actual detection system is established.

In this method, an electrode is placed on B and the flow of electrons is directly guided to the electrode, and the resulting current is electrically measured. In addition, a substance that changes the light absorption characteristics by accepting BK electrons (i.e., reduction) is placed. There are cases where the amount of change that occurs is measured colorimetrically or spectroscopically. Regarding the former, viewed as the amount of direct electrical change, Yagi et al., J Biochem., 78 (1975), 1347-1352 (J, Bl.
ochem, 78 (1975).

1347-1352) and Japanese Patent Publication No. 53-13990, one electrode (anode) of the battery is used as an electron acceptor, such as platinum or appropriately treated carbon. 2 electrodes (
cathode), such as Amagi, silver/silver chloride, platinum in ferricyanide, etc., and a salt bridge etc. connects the two electrodes. Ichigo produced by the dehydrogenase reaction carried out in the anode-tei is led directly to an electrode such as platinum via PMS, and electrons flow to the cathode via a conductive wire, where they reduce Amagi and generate electric current. Therefore, various dehydrogenase activities or substrate amounts can be determined by measuring this current value. On the other hand, examples of electron acceptors that cause changes in the latter's light absorption properties include 2,4-dichloroindophenol and the divine tetrazolium salt. Currently, it is desirable for these to be accompanied by a color change in the visible region, but it is difficult to analyze components in biological fluids using various dehydrogenases in medical facilities. Absorbance analysis of bulk material is often used, and the electron acceptor VcF
It is common to use a tetrazolium salt commonly known as nitrotetrazolium furyl, and to use formazan 9, which is produced by this reduction reaction, as an indicator.

As mentioned above, PMS is a valuable substance that efficiently mediates the reduction reaction of the detection system in the dehydrogenase reaction, and has been widely used in reaction systems using enzymes.

However, on the other hand, the weaknesses of PMS have been known for a long time, and it has properties that are extremely inconvenient when used. One is instability (inactivation) due to light and the other is corrosivity. Immediately after preparation, the PMS aqueous solution has a pale yellow color, but under light, even with a normal fluorescent knife, the color tone changes rapidly, turning from green to brown. This change can be tracked with the naked eye. At the same time, electron transport activity is lost. Normally, it becomes almost unusable after a few hours or a day or two, and in experiments that require precision, g)1
It has the disadvantage that it can only be used immediately after g. It is also corrosive and causes sores if it comes into contact with the skin. This may be due to the same cause as the instability of PMS to light, but it is thought that PMS that undergoes photo-oxidation produces a toxic substance called biocyanin.

1- solves these weaknesses of PMS all at once.
Methoxy-5-methylzenadinium methyl sulfate (hereinafter abbreviated as 1-methoxy PMS), which has a methoxy group introduced into the 1-position of the 7 enazine skeleton of PMS, as shown in formula [3] It is. This 1-methoxy PMS improves H3 in all respects, both in terms of its original electron transfer performance and ease of use.In practical terms, it is necessary to always consider these aspects, especially from the standpoint of stability against light and corrosion resistance. It is so good that it has become obsolete, and it is now a sufficient substitute for PMS. (Nakamura, Yagi et al., Cl1n, Chim, Acta, +) 101, (1
980), 321-326. and Special Public Service 1973-3919
6) In addition to the above-mentioned application to the measurement system mediated by dehydrogenase, PMS is also used for research on photosynthesis and a light energy conversion system using this. Photosynthesis, carried out by green plants and green algae, is a mechanism that converts solar energy into chemical energy and is the energy source for all life on Earth. This reaction uses light energy to produce the biochemical reducing agent NADPH and the high-energy compound ATP,
It can be divided into a light reaction, which simultaneously releases 02, and a dark reaction, which uses NADPH and ATP to reduce CO□ to carbohydrates (CH20).

Light reactions include non-cyclic phosphorylation, which simultaneously produces NADPH and ATP, and cyclic phosphorylation, which produces only ATP. Avron, Mpaiochim, Biochim, Acta (Avron* M, + Biochim.

Biophya, Ai=ta,,) 40,257~2
72 (1960), Aaada+K, Anal Bio, chem,
υ48.311-315 (1975), BrancL J,
J, , plantphySiol, ) 55.1
87-191 (1975), Homann, P., Ko., Chi., Biochem.
,H,.

Bioahsm, Biophys, Acta) 4
60*1-16 (1977), etc.
Traditionally, JPMS has been used as a useful artificial cofactor in cyclic phosphorylation studies. However, as mentioned above, PMS is sensitive to light, so in these photosynthesis experiments, it is necessary to irradiate it with light, which of course causes changes in the process. In reality, it has been very inconvenient, but photostable 1-methoxy PMS has brought about groundbreaking progress in this aspect as well. Also, as part of these photosynthesis research, the photosynthetic ability of cyanobacteria and chloroplasts is used to convert light energy into electrical energy, and PMS has the function of amplifying this current value, and at this time as well, 1 -Methoxy PMS can be used as a valuable alternative to PMS. Also, synthetic porphyrin can be used instead of chloroplast or the like.

In this way, PMS or 1-methoxy FMS can be used as an efficient electron carrier in detection systems via dehydrogenase reactions, and on the other hand, as an electron carrier in photosynthetic systems. It is widely used as a valuable chemical material.

"Problems to be solved by the invention" This time, the inventors have developed such an efficient electron carrier, P.
He attempted a new field of using MS etc. by bonding it to an anionic carrier, and reached a technical goal of introducing an alkylammonium cation into the side chain of the 7-enazinium skeleton, completing a non-invention. .

The novel 5-alkylzenazinium derivatives having the newly introduced alkylammonium cations are conveniently bonded to an anionic carrier and used. This results in
The efficiency of the 5-alkylzenadinium is enhanced by the proximity of this electron carrier to the Ichigo receptor in the system;
It also enables the system itself to function repeatedly.

For the purpose of bonding with the anionic carrier, this alkylammonium may be located at any position in the heptadium sodium compound as long as it maintains its performance as an electron carrier, and the number is not limited, but in practice, It is placed in the side chain substituents of the zenazinium backbone, either as a side chain substituent itself or included in other suitable substituents. In addition, the above 1
- If the present invention is carried out while retaining the advantages of methoxy PMS, the position of introduction of alkylammonium is - Same as methoxy PMS, the position of introduction of alkylammonium is
The position is appropriate. As a result, a novel electron carrier that meets the purpose of immobilization can be obtained while maintaining the excellent performance of 1-methoxy PMS.

As described above, the introduction of cationic alkylammonium into 5-alkylzenadinium according to the present invention further expands the range of applications of conventional electron carriers, and creates a completely new form of using it after immobilization through ionic bonding. The electron carrier and its reaction mode were made possible.

"Means for Solving the Problems" That is, the present invention has the formula CI] [However, in the formula, RFi alkyl group, X is a side chain substituent introduced at the 1-position (or the contrasting 9-position) of the zenazinium skeleton , Y and 2 represent side chain substituents or hydrogen introduced at different positions of the 2, 3, and 4 positions (or the contrasting positions 8 and 7.6), and x as a substituent
, y, z may be the same or different. ] Only the 5-alkylzenazinium derivatives represented by these may be different, and R3 may be replaced with hydrogen. ) is an alkylammonium polycation type 5-alkylzenazinium composed of substituents containing the following.

Furthermore, the present invention provides a compound of the formula [I] [wherein R is an alkyl group, X is a side chain substituent introduced at the 1-position (or the contrasting 9-position) of the zenazinium skeleton, and Y and 2 are 2.3. 4th place (or 8th place as a control)
, 7.6) indicates a side chain substituent or hydrogen introduced at each different position, and the substituent is x,
y and z may be the same or different. ] Alkylammonium polycation type 5-, which is composed of a substituent containing a 5-alkylzenadinium derivative represented by (, R, R hydrogen can also be replaced). This is a method of using an alkylzenadinium as an electron carrier by bonding it to an anionic carrier.

The new electron carrier according to the present invention, alkylammonium cation type 5-alkyltzenadinium, is a conventional PM.
S% l-Methoxy PMS and other 5-alkylzenadinium type electron carriers can be used by ionically bonding them to a suitable carrier to expand the range of applications, such as hydroxyalkyl,
Alkyl derivatives such as alkyl halides are also possible.

Generally, these alkyl groups are often used as lower alkyl groups having 1 to 3 carbon atoms. Also R1, to, R5
Dialkyl ammoniums in which one of the two is hydrogen can also serve this purpose. That is, the alkylamsonium referred to in the present invention may be any one that maintains stable N and cationic properties. R1 with respect to N,
The remaining conjugates other than R3 are bonded to the zenazinium skeleton or to other substituents attached to the skeleton as side chains. In other words, those in which this alkylammonium is introduced directly as a side chain substituent of the 7-enadinium skeleton or in the form of being included in one or more other side chain substituents are referred to as alkylammonium cation type 5 in the present invention.
- Can be used as an alkylzenadinium in combination with an anionic carrier. The position and number of alkylammonium introduced into zenadinium and the type of side chain substituents to be included are arbitrary, but 5-alkyl zenadinium is usually placed at the 1st position on the zenadinium skeleton where it is stabilized against light. However, if the 1st position is already filled with another substituent, such as an alkoxy group such as methoxy or ethoxy, an acetamido, an amino group or a derivative thereof, or a halodane, to obtain stability against light, cationization is preferable. This alkylammonium or a substituent containing it can be introduced regardless of its position.

However, in accordance with the intent of the present invention, the most efficient way to accomplish this is to place this alkylammonium directly on the 1-position of 5-alkylzenadinium, which is the site that contributes to stabilization to light, or a substituent containing this. is introduced as a side chain. This allows the 5-alkylzenadinium to have both the practical convenience of stability to light and immobilization.

In order to exhibit cationic properties, it is desirable to include one to several alkyl ammonium per zenadinium molecule, and in that sense, an appropriate substituent containing one or more alkylammonium is added to the 1-position of the zenadinium skeleton as a side chain substituent. In this case, the most suitable substituent is an alkoxy group, and an alkoxy group containing one or more alkylammonium in the alkyl moiety is introduced as a side chain substituent to the 1-position. It is better.

This product inherits the superiority of 1-methoxy PMS compared to other 5-alkylzenadiniums, such as electron transfer properties and photostability, and furthermore, it is bonded to an anionic carrier, which is the essence of the present invention. This corresponds to the purpose of causing an electron transfer reaction.

Alkylammonium cation type according to the invention, 5-
By utilizing its ionic bonding properties, alkylzenadiniums have enabled reaction modes and techniques of electron carriers, such as immobilization (PMS), which could not be achieved with conventional 5-alkylzenadiniums. However, its scope of use is not limited to this immobilization, and of course it can also be used in the usual usage forms in which PMS, l-methoxy PM8, etc. have been used. Department,
At this time, unnecessary anionic carriers may not be used. In this sense, the present invention can be understood to further expand the scope and concept of use of electron carriers.

However, the use of an electron carrier, 5-alkylzenadinium, by ionic bonding to an anionic carrier is a new and important point of the present invention, and from the purpose of this invention, a new alkylzenadinium is added to 5-alkylzenadinium. The essence of the present invention is to add and use an ammonium cation, and the range of the individual derivatives is not restricted. Enadinium derivatives are individually novel substances.

Alkylammonium cationic type, 5-alkyl7 enazinium is ionically anionic, i.e. 30 e, -C
OOe, -HPO5θ% noj% Leaving group t Contains tr It is bound to a body, a polymer, a membrane, etc. Practically speaking, 1803e is preferred because of its strong dissociability, but other materials may be used depending on the material, purpose, etc. Immobilization using this ionic bond can be performed very easily. This point also shows the advantages of the present invention. That is, immobilization can be achieved by mixing or simply contacting the alkylammonium cationic, 5-alkylzenadinium according to the invention with a suitable anionic carrier. - For example, 1-r-)limethylamino~n prepared to about 1mM
A polystyrene sulfonic acid membrane immersed in an aqueous solution of pyroxyphenazinium mensulfate at room temperature for about half a day can be used as an electron carrier-immobilized membrane after sufficient water washing and drying.

An important point in the gist of the present invention was that an alkylammonium cation was newly introduced into 5-alkylzenadinium.

To explain this point in more detail, 5-furkyrzenadinium already has cationic properties based on N at position 05 of its zenadinium skeleton, and has an ionic affinity with the anionic carrier. Rimari PMS, ]-Methoxy PMS
etc., it is possible to ionicly bond to an anionic carrier as is. Therefore, the new intention of the present application, which is to immobilize a zenadinium-type Ichigo carrier using ionic bonding, can be achieved to some extent immediately by directing the cation that zenadinium has from its source into an ionic bond with an anionic carrier. It was thought that it would be possible. In fact, this combination is done.

However, to begin with, the conclusion was that although it does stick to the carrier, it eventually comes off even after bonding. It was found that the reaction goes off as soon as the electron transfer reaction begins. The reason for this is inferred as follows.

In the electron transfer reaction, 7-enadinium undergoes reduction and becomes neutral once again, but soon returns to its original state through its own oxidation.
Restores cationic properties. Here, the property of 7enadinium is that once it has lost its e3 charge due to the reaction, it is difficult for it to remain on the anionic carrier even if it quickly returns to itself by releasing πf atoms (oxidation). This can be expressed as follows.

That is, even if 5-alkylzenadinium is simply bonded to an anionic carrier, it has the fate of eventually becoming detached once the actual reaction starts.

In this case, immobilization by ionic bonding is meaningless.

As a result of intensive studies, the inventors have discovered that while taking advantage of the cationic nature of zenadinium, they have also added a new cation for ionic bonding and immobilization of the carrier, in order to achieve the new intention based on the present application. The present invention was achieved by including one or more alkylammonium cations in zenadinium.

The newly introduced 5-alkylzenadinium with an alkylammonium cation supplements the original 7-enadinium cation, allowing the reaction to proceed while being firmly held on the anionic carrier during the reaction. be able to. That is, in order to immobilize the 5-alkylzenazinium type electron carrier by ionic bonding with an anionic carrier and to perform various reaction configurations of the electron carrier by immobilization, it is necessary to introduce a new cation according to the present invention. becomes an indispensable element.

As mentioned above, the efficient introduction position of the alkylammonium cation is the 1st position of the zenazinium skeleton. This position also has the effect of improving the stability of the 5-alkylzenadinium type deuteron carrier against light.

Particularly preferred is 1-alkoxy-5-alkylzenadinium in which an alkyl ammonium is included as an alkoxy group in the alkyl moiety. The synthesis method of this 1-alkoxy type will be described below as an example of the alkylammonium cation type and 5-alkylzenadinium, but the present invention is not essentially limited to these synthesis methods and types of compounds. It is clear that there is no such thing.

The intermediate 1-hydroxy-5-alkylzenadinium (a), which is a common raw material for the above types, was synthesized by organic synthesis by Saray A.R.
s,) col, vol, 3 (1955), P
It is synthesized in good yield by the method described in 753-756.

By using this (a) as a raw material and reacting it with the divine alkylaminoalkyl chloride, various corresponding 1-alkylaminoalkoxyphenazines can be obtained. For example, after reacting dimethylaminoethyl chloride (b) with sodium hydride in dimethyl sulfoxide (a), (b)
1 at 100° C. for 3 hours to obtain ]-]β-trimethylamineethoxyphenazine C). Next, by reacting dialkyl sulfuric acid with the product of the first stage, the desired alkylammonium cation type, 1-alkoxy-5-alkylzenadinium, can be obtained. For example, for (C), dimethyl sulfate (dl) The usage is as follows.

That is, (c) was mixed with 6 times the amount of dimethyl sulfur. (d) Medium, 10
0℃.

After reacting for 10 minutes, add diethyl ether to the reaction mixture! 111-β-Trimethylaminoethoxyphenazinium dimethosulfate (e) is precipitated.

This is purified by dissolving it in acetonitrile and precipitating it again with dimethyl ether.

As mentioned above, if a number of similar alkylaminoalkyl chloride derivatives for (b) are used as raw materials in the first step, various intermediates corresponding to (c) can be obtained. For example, for (b), if γ-diethylaminopropyl chloride or β-dimethylamine butyl chloride is used, the resultant for (C) is 1-γ-
Diethylamine Zorobioxyphenazine or β-
It is dimethylaminotetoxyphenazine.

Similarly, by using an alkyl chloride derivative having a plurality of sialkylamino groups, any alkoxy group containing a plurality of dialkylamine groups can be introduced into the 1-position of the phenazine skeleton. Further, these dialkylamino groups may be monoalkylamino groups in which one of the alkyl groups is replaced with hydrogen.

Furthermore, in the second stage, in addition to dimethyl sulfate (d), any dialkyl sulfate such as diethyl sulfate can be used, and the type of dialkyl sulfate determines the type of alkyl group introduced at the 5-position. ) for any corresponding 5
-Alkylzenadiniums can be obtained. Of course, the type of alkyl group of the dialkyl sulfate selected here is the same as the alkyl group attached to the last N-related alkylamino group contained in the alkoxy group due to the principle of the second step in this synthesis method. be.

On the other hand, carriers, membranes, polymeric substances, etc. that have anionic folding R groups to which they are bonded are available in various materials and forms depending on the purpose. You can choose. In other words, it can be used as a coating part on the electrode surface by binding to an anionic membrane, as a direct ionic bond to an anionic polymer coated on the electrode surface, or as a continuous reaction reactor by binding to a bead-shaped anionic carrier. There are various forms of application. Substances with anionic dissociative groups include polystyrene sulfonic acid, carboxymethyl cellulose, alginic acid, etc. As the dissociative group, sulfonic acid groups are most suitable due to their strong bonding properties, and the synthesis of newly introduced carriers, etc. is also possible.

"Medicine" Hereinafter, the present invention will be explained in detail according to a practical example.

Example 1 Performance of 1-β-trimethylaminoethoxy-5-methylzenadinium (referred to as aminoethoxy PMS) in the Babaon-Phjllpa method a, Method: Linking to nitrotetrazolium via lactate dehydrogenase/NAD system We tested its performance as an electronic carrier.

Reaction conditions, reagents, etc. are provided by JNPL Biochemistry (J, Appl, Blocham, ) 3
(1981).

535-543. (30''C).

l-methoxy PMS (f 1nal concn,
32 μM) was used as a standard, and its reactivity was set as 100.

b.Results Reactivity of Niaminoethoxy PMS Even in the lower concentration range than that of aminoethoxy PMS (e.g. 3.2
μM, 8 μM, etc.) is characterized by the appearance of sufficient lactate dehydronase activity.

The optimal concentration is 3-81M.

Example 2 Immobilization method on S nO2 lightning electrode Sodium polystyrene sulfonate (PS
SNa, Tomoe MM 01 igoZ, W-100
Three molecules (17500° sulfonation rate 73.4%) were previously subjected to dialysis to remove low-molecular impurities. Polyvinyl alcohol (PVA-117, Kurarayvol/F-L, polymerization degree 1700) is used as it is.

First, PYA 3 and F are added to 30 scallions of distilled water and dispersed well at room temperature. Slowly heat this to 80°C, add PSSNa 0.71, completely dissolve without boiling, and then cool slowly to return to room temperature. Bovine serum albumin 15~ is added to this and dissolved.

S nO2 glass'I! The above mixture is applied thinly and uniformly to the electrode, dried under reduced pressure in a P401o desiccator for at least one day, and then dried for at least 15 minutes in a dryer at 190° C. to stabilize.

This electrode can be used in various alkyl ammonium cation type 5-
Alkylzenadinium 1-methoxy PMS or P
After soaking in MS solution (both 0.3 mM) for half a day or more to adsorb it, wash thoroughly with water and air dry.

This is collectively called a PMS electrode.

Example 3 Performance of PM8@pole a. Electrode configuration; the cathode is a calomel electrode.

Place a PMS electrode on the anode.

b. Composition of anolyte: lactate dehydrogenase 0.8r
at, DL-sodium lactate (IM) 0.37!
, NAD" (0, OIM) 0.45 m, ) Lith buffer (pH 8,5, 1M) 0.6 m/, add distilled water to make 2.7-.

C, reaction at the anode; sufficient bone milk j didehydro r
Milk-salt is oxidized by the action of enzyme, and NAD+ is reduced to NADH (!-).NADH reacts with each phenazinium-type constant carrier bound on the electrode by the method of Example 2 to form NAD+. At this time, electrons are given to the electron carrier.The electrons are directly given to the 5n02 electrode from the electron carrier, and reduce the Amagi at the opposite electrode.

An electric current is generated during this process. This @Mt value is proportional to the reactivity of the bound electron carrier if the enzyme zero degree is constant. This time's fruit! f+ is various PMS with constant enzyme concentration.
This is a comparison of the performance of the north pole.

d.Results As shown in Table 2, the SnO□゛ padding without pbrs does not have the ability to convert the oxygen reaction into electric current. In other words, this is a blank reaction. Electrodes containing PMS or l-methoxy PMS have the ability to convert into current only the first time as described above, but cannot be used repeatedly because they elute during use.

However, since the electrode adsorbing 1-β-trimethylaminoethoxy PMS strongly binds PSS anions,
A stable current can be obtained even after the second and subsequent warped uses.

Furthermore, it can be seen that the deuteron transfer performance itself has also increased significantly. Utilizing this, it becomes possible to measure enzyme activity and substrate amount.

Example 4 Alkylammonium cationic PMS Irt m
Preparation of SnO2 electrode coated with PSS-PVA (see Example 2) with 1-β-trimethylaminoethoxy PMS and tetracationic porphyrin (isotopic product. Trade name 'rMPyp) ) After being immersed in a mixed solution of zinc complexes (both concentrations are 0.3mM) for more than half a day to bond, it is washed with water and air-dried.

b. Battery configuration: The cathode is made by soaking Glasseeker 7 in methylbiorodane solution. The battery was constructed as an anode in which 1-β-trimethylaminoethoxy PMS and tetracationic zinc porphyrin were combined. On the anode side, 0.2 M IJ acid buffer was simply added.

C9 result; 500μF only on the anode side, 7m2 II
When irradiated with light, an anode current of about 1 μA is produced, reducing the methylbiorodane on the cathode side (it takes a long time for the reduction to occur to the extent that a blue color is visible). Electrode (anode)
Almost no l-β-trimethylaminoethoxy PMS or porphyrin is eluted in the side solution, and the generation of photoanodic current can be repeated any number of times. Warpage was observed at least 100 times.

From the above, the alkylammonium cation type 5-alkylzenazinium according to the present invention can be bonded to an anionic carrier by the new alkylammonium cation introduced into phenazonium, resulting in a more efficient electron carrier reaction than before. and as a reactor,
It also allows for repeated use. In other words, a new usage pattern for electron carriers has been achieved.

"Effects of the Invention" As described above, the introduction of cationic alkyl ammonium into 5-alkylzenadinium according to the present invention further expands the range of application of the conventional heptatransmitter, and immobilizes it by ionic bonding. We provide a completely new type of electron carrier using this and a method of using the same.

Claims (1)

[Claims] 1) Formula [I] ▲ Numerical formula, chemical formula, table, etc.▼ [I] [However, in the formula, R is an alkyl group, and X is the 1st position of the phenazinium skeleton (or the 9th position as a reference) The side chain substituents, Y and Z introduced into the
, 7, 6)), and the substituent is X,
Y and Z may be the same or different. ] In the 5-alkylphenazinium derivative represented by, at least one of X, Y, and Z is an alkyl ammonium ▲ There are numerical formulas, chemical formulas, tables, etc. ▼ However, R_1, R
_2, R_3 represent an alkyl group, R_1, R_2, R
Each of _3 may be the same or different, and R_3 may be replaced by hydrogen. ) Alkylammonium polycation type 5-alkylphenazinium composed of a substituent containing. 2) The alkylammonium polycation type 5- according to claim 1, wherein X is a side chain substituent containing one or more alkylammonium, and Y and Z are hydrogen.
Alkylphenazinium. 3) The alkylammonium polycation type 5- according to claim 1, wherein X is alkylammonium or an alkoxy group containing one or more alkylammonium in its alkyl moiety, and Y and Z are hydrogen.
Alkylphenazinium. 4) X is a ▲ mathematical formula, chemical formula, table, etc. ▼ (However, m and n are integers from 0 to 5, R_1, R_2, R
_3 represents an alkyl group having 1 to 3 carbon atoms, R_1, R
_2 and R_3 may be the same or different, and R_
3 can be replaced with hydrogen. ), Y and Z are hydrogen, and R is an alkyl group having 1 to 3 carbon atoms, the alkylammonium polycation type 5-alkylphenazinium according to claim 1. 5) Formula [I] ▲There are mathematical formulas, chemical formulas, tables, etc.▼[I] [However, in the formula, R is an alkyl group, and X is a side chain introduced at the 1st position (or the 9th position as a contrast) of the phenazinium skeleton. Substituents, Y and Z, are at the 2, 3, and 4 positions (or the contrasting 8
, 7, 6)), and the substituent is X,
Y and Z may be the same or different. ] In the 5-alkylphenazinium derivative represented by, at least one of X, Y, and Z is an alkyl ammonium (▲There are numerical formulas, chemical formulas, tables, etc.▼, however, R_1,
R_2, R_3 represent an alkyl group, R_1, R_2,
Each R_3 may be the same or different, and R_3 may also be replaced by hydrogen. ) A method for using an alkylammonium polycation type 5-alkylphenazinium constituted by a substituent group as an electron carrier by bonding it to an anionic carrier. 6) The method of use as an electron carrier according to claim 5, wherein X is a side chain substituent containing one or more alkylammonium, and Y and Z are hydrogen. 7) The method of use as an electron carrier according to claim 5, wherein X is alkylammonium or an alkoxy group containing one or more alkylammonium in its alkyl moiety, and Y and Z are hydrogen. 8) X is a ▲ mathematical formula, chemical formula, table, etc. ▼ (However, m and n are integers from 0 to 5, R_1, R_2, R
_3 represents an alkyl group having 1 to 3 carbon atoms, R_1, R
_2 and R_3 may be the same or different, and R_
3 can be replaced with hydrogen. ), Y and Z are hydrogen, and R is an alkyl group having 1 to 3 carbon atoms.