JP2012008084A - Measuring method for active oxygen and measuring apparatus therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply and accurately measure the active oxygen of specimens without performing pretreatment such as fractionation and dilution of a predetermined cell and removal of measurement interfering substance, etc. by using the specimens derived from animals including humans and plants.SOLUTION: A measuring method for active oxygen includes a process for adding a near infrared luminescent compound to a specimen and a process for measuring the emission intensity of the specimen adding the near infrared luminescent compound by using a sensor cooled -5 to 20°C.

Description

  The present invention relates to a method and an apparatus for measuring active oxygen in a specimen.

  Conventionally, active oxygen generated from immune cells in blood is measured by fractionating target cells such as neutrophils and granulocytes from the collected blood, and then reactive oxygen generated by stimulation with drugs, etc. The weak luminescence generated by the reaction between the luminescent compound and the luminescent compound was measured by a luminescence measuring device, or the blood was diluted and generated by the reaction between the active oxygen generated from the cells in the blood by stimulating the drug and the luminescent compound. The weak light emission has been performed by a method of measuring with a light emission measuring device.

  In addition, the measurement of active oxygen generated from animal biological tissues requires removal of blood components, tissue components, and the like that interfere with the generation of active oxygen and the measurement of active oxygen from the collected biological tissues.

  Alternatively, in the measurement of active oxygen in a plant-derived specimen, it was necessary to remove chlorophyll and plant components that interfere with the measurement of active oxygen, particularly pigments.

  In the method for measuring active oxygen in these specimens, Luminol, MCLA, etc. were used as luminescent compounds, but the emission wavelength was 400 to 500 nm, and most of them were absorbed by blood components, biological tissue components, and plant components. Therefore, it is difficult to accurately measure active oxygen. Therefore, pretreatment of the specimen as described above is necessary.

  For example, the operation of fractionating specific cells from blood is very complicated. In addition, since the operation takes time, the biological activity of the cells is reduced. Therefore, the amount of active oxygen generated from the cells is affected by the elapsed time after blood collection. Furthermore, since the operation is complicated, there is a disadvantage that it is not suitable for measuring the active oxygen of a plurality of individuals. Removal of blood components and pigment components from living tissue is complicated, and the active oxygen with a short lifetime disappears during that time, making it impossible to measure the intended active oxygen. In the case of plant-derived specimens, it is necessary to remove plant-derived pigments and plant tissues, and the operation is complicated. During this period, the short-lived active oxygen disappears, and the original intended activity is eliminated. It is impossible to measure oxygen.

  Under such circumstances, the present inventors have succeeded in synthesizing a light-emitting compound that induces near-infrared light emission excellent in light transmittance (Patent Document 1).

  However, in the conventional measuring apparatus and measuring method, even when such a luminescent compound is used, the S / N ratio (Signal to Noise ratio) is small, so that active oxygen cannot be accurately measured.

  JP 2009-215174 A

  The problem to be solved by the present invention is to accurately analyze the activity of a sample from a sample derived from an animal including humans, plants, etc. without pretreatment such as fractionation, dilution, and removal of measurement interfering substances. It is to measure oxygen.

  Under the circumstances as described above, as a result of intensive studies, the present inventors have used a near-infrared light-emitting compound that emits near-infrared light with excellent light transmission as the light-emitting compound, and the reaction between active oxygen and the light-emitting compound. Measure the intensity of near-infrared light generated by the sensor in real time using a sensor cooled to a specific temperature with high time resolution, high sensitivity, high S / N ratio, and close to the living body. Thus, it was found that the target active oxygen can be accurately measured. The present invention is based on such new knowledge.

Accordingly, the present invention provides the following sections:
Item 1. Measurement of active oxygen, including a step of adding a near-infrared luminescent compound to a specimen, and a step of measuring the luminescence intensity of the specimen to which the near-infrared luminescent compound is added using a sensor cooled to −5 to 20 ° C. Method.

Item 2. Item 2. The active oxygen measurement method according to Item 1, wherein the near-infrared light-emitting compound is an indocyanine-linked imidazo [1,2-a] pyrazin-3-one compound represented by the following general formula (I) or a salt thereof: :
A-B-C (I)
[Wherein A represents an imidazo [1,2-a] pyrazin-3-one group represented by the following general formula (II):

(Wherein R ¹ , R ² , R ³ , and R ⁴ are the same or different and are a hydrogen atom, alkyl group, aryl group, halogen atom, alkoxyl group, carboxyl group, formyl group, alkyloxycarbonyl group, aryl. An oxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group or a heterocyclic ring is shown.
However, ^one of R ¹ , R ² , R ³ , and R ⁴ is covalently bonded to the spacer group B. )
Group B represents a spacer group.
The group C represents an indocyanine group represented by the following formula (III):

(In the formula, R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , and R ²⁵ are the same or different and are a hydrogen atom, alkyl group, aryl group, halogen atom, alkoxyl group, carboxyl group, formyl group, sulfonyl group, sulfonic acid A group, an alkyloxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, an aryloxy group, an alkylperoxy group or a heterocyclic ring;
R ¹⁵ and R ¹⁷ may be bonded to each other to form an unsaturated 4- to 10-membered ring.
^{However, R 5, R 6, R} 7, R 8, R 9, R 10, R 11, R 12, R 13, R 14, R 15, R 16, R 17, R 18, R 19, R 20, One of R ²¹ , R ²² , R ²³ , R ²⁴ , and R ²⁵ is covalently bonded to the spacer group B. ]]
Item 3. Item 2. The active oxygen measurement method according to Item 1, wherein the near-infrared light-emitting compound is an indocyanine-linked imidazo [1,2-a] pyrazin-3-one compound represented by the following formula (IV) or (V): .

[Wherein, R ¹⁶ ′ represents an alkyl group, an alkoxyl group or an aryl group.
M represents hydrogen, an alkali metal or an alkaline earth metal.
k shows the integer of 1-7.
l shows the integer of 1-7.
m shows the integer of 1-7.

[In formula, M is the same or different and shows hydrogen, an alkali metal, or an alkaline-earth metal.
k and m are the same as above.
n and p are the same or different and represent an integer of 1 to 7.

  Item 4. Item 2. The active oxygen measurement method according to Item 1, wherein the near-infrared light-emitting compound is an indocyanine-linked imidazo [1,2-a] pyrazin-3-one compound represented by the following formula (VI) or (VII): .

[In formula, M is the same or different and shows hydrogen, an alkali metal, or an alkaline-earth metal. ].

Item 5. A cell for introducing a near-infrared light emitting compound,
Means for measuring the emission intensity, and cooling means for cooling the emission intensity measurement means to -5 to 20 ° C,
A device for measuring active oxygen, comprising:

  Item 6. A kit for measuring active oxygen, comprising a near-infrared light emitting compound and the active oxygen measuring device according to claim 5.

  According to the method of the present invention, active oxygen can be easily and accurately measured from blood collected from animals including humans without pretreatment such as fractionation and dilution of specific cells. Therefore, it becomes possible to evaluate the ability of the cells to generate the active oxygen that is potentially present in a state closer to the living body.

  Further, according to the method of the present invention, the measurement of active oxygen generated from the living tissue of the animal is performed. From the collected biological tissue, the generation of active oxygen and the blood component, tissue component or tissue fixed substance that interferes with the measurement of active oxygen are removed. Active oxygen can be measured easily and accurately without the need for removal.

  Furthermore, according to the method of the present invention, in the measurement of active oxygen in a plant-derived specimen, active oxygen is measured easily and accurately without the need to remove chlorophyll and plant components that interfere with the measurement of active oxygen, particularly pigments. be able to.

  Further, according to the method of the present invention, not only the measurement of active oxygen spontaneously generated from or contained in the sample, but also the measurement of active oxygen after the generation of active oxygen or the addition of active oxygen itself to the sample By performing the above, it is also possible to measure and evaluate the antioxidant ability of the specimen. For example, after adding an active oxygen generation system such as xanthine and xanthine oxidase to a sample such as wine, the antioxidant ability of the sample can be measured and evaluated by measuring the active oxygen over time.

FIG. 1 shows a schematic diagram of an active oxygen measuring device according to one embodiment of the present invention. FIG. 2 shows the change with time of the emission intensity of near-infrared light generated by the reaction between active oxygen generated from a human whole blood sample and a near-infrared light-emitting compound in Example 1. FIG. 3 shows the change with time of the emission intensity of near-infrared light generated by the reaction between active oxygen generated from a bovine blood sample and a near-infrared light-emitting compound in Example 3.

Near-infrared light-emitting compound The near-infrared light-emitting compound used in the present invention may be any compound that reacts with active oxygen and emits light at a near-infrared light emission wavelength.

In a preferred embodiment, the near-infrared light emitting compound may include an indocyanine-linked imidazo [1,2-a] pyrazin-3-one compound represented by the general formula (I) or a salt thereof:
A-B-C (I).
[Wherein A, B and C are as described above].

Here, the spacer group B is not particularly limited as long as it can bond the group A and the group C, and examples thereof include a group represented by the following general formula (VIII):
_{- (CH 2) n - (} C = O) NH- (CH 2) p -NH (C = O) - (CH 2) k -
(VIII)
[Wherein, k, n and p are the same as above. ].

  Moreover, each group shown in the said general formula is specifically as follows.

  Examples of the alkyl group include linear or branched alkyl groups having 1 to 20 carbon atoms. More specifically, for example, methyl, ethyl, propyl, butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosanyl, etc. It is.

  Examples of the aryl group include aromatic hydrocarbons having 6 to 20 carbon atoms. More specifically, for example, phenyl, naphthyl and the like are included.

  Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

  As an alkoxyl group, a C1-C20 linear or branched alkoxyl group can be mentioned. More specifically, for example, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, methoxyethoxy, methoxypropoxy, ethoxyethoxy, ethoxypropoxy, methoxyethoxyethoxy groups and the like are included.

  Examples of the alkyloxycarbonyl group include an alkyloxycarbonyl group in which the alkyl moiety is the alkyl group exemplified above.

  Examples of the arylcarbonyl group include an arylcarbonyl group in which the aryl moiety is the aryl group exemplified above.

  Examples of the heterocyclic group include 5- to 15-membered monocyclic, binary or ternary saturated or unsaturated heterocyclic groups having 1 to 4 nitrogen atoms, oxygen atoms or sulfur atoms. Examples of such heterocyclic groups include pyrrolyl, imidazolyl, pyrazolyl, dihydropyrazolyl, pyridyl, pyridyl N-oxide, dihydropyridyl, pyrimidinyl, tetrahydropyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, triazolyl, tetrazinyl, tetrazolyl, pyrrolidinyl, piperidyl , Piperazinyl, diazepanyl, indolyl, isoindolyl, indolinyl, benzimidazolyl, quinolyl, dihydroquinolyl, isoquinolyl, indazolyl, thienyl, tetrahydrothienyl, furyl, dioxol, tetrahydrofuryl, tetrahydropyranyl and the like.

  Examples of the aryloxy group include an aryloxy group in which the aryl moiety is the aryl group exemplified above.

  Examples of the alkylperoxy group include an alkylperoxy group in which the alkyl moiety is the alkyl group exemplified above.

  Examples of the alkali metal include sodium and potassium.

  Examples of the alkaline earth metal include magnesium and calcium.

Production method of near-infrared light emitting compound The indocyanine-linked imidazo [1,2-a] pyrazin-3-one compound represented by the general formula (I) can be produced by various methods. For example, it is manufactured by the method shown in the following reaction formula-1:
Reaction Formula-1
A-B ₀₁ + B ₀₂ -C → A-B-C
Here, the groups B ₀₁ and B ₀₂ are not particularly limited as long as they react with each other to form the spacer group B.

Examples of the group B ₀₁ include the group — (CH ₂ ) _n — (C═O) NH— (CH ₂ ) _p —NH _2.
[Wherein, n and p are the same as above. ],
Group _{- (CH} 2) n -COOH
[Wherein n is the same as defined above. ]
Etc.

Examples of the group B ₀₂ include a group — (CH ₂ ) _k — (C═O) NH— (CH ₂ ) _p —NH _2.
[Wherein, k and p are the same as above. ],
Based on _{- (CH} 2) k -COOH
[Wherein k is the same as defined above. ]
Etc.

  This reaction is usually a conventional solvent that does not adversely influence the reaction, such as water; alcohol solvents such as methanol, ethanol, isopropanol, n-butanol, trifluoroethanol, ethylene glycol; ketone solvents such as acetone and methyl ethyl ketone. Ether solvents such as tetrahydrofuran, dioxane, diethyl ether and diglyme; ester solvents such as methyl acetate and ethyl acetate; aprotic polar solvents such as acetonitrile, N, N-dimethylformamide and dimethyl sulfoxide; methylene chloride and ethylene chloride In a halogenated hydrocarbon-based solvent; or other organic solvents. Furthermore, this reaction is carried out in a mixed solvent of these conventional solvents.

The proportion of compound _AB01 and compound _B02- C used in the above reaction scheme-1 is usually 1 to 10 moles, preferably 1 to 2 moles per mole of the former.

  The reaction temperature is not particularly limited, and the reaction is usually carried out under cooling, at room temperature, or under heating. The above reaction is preferably carried out for 1 to 30 hours under temperature conditions around room temperature.

  The indocyanine-linked imidazo [1,2-a] pyrazin-3-one compound represented by the aforementioned general formula (IV), which is used in a preferred embodiment of the present invention, is obtained by, for example, the following steps 1 to 5: Can be manufactured.

<Step 1>
A compound represented by general formula (XII) which is a novel indocyanine compound (hereinafter sometimes simply referred to as compound (XII). Similarly, each of compounds represented by general formulas (I) to (XV) is also simply a compound. (I) to (XV) may also be indicated, for example, in the literature Cyaninedye labeling reagents: sulfoindocyanine succinimidyl esters, RB Mujumdar, LA Ernst, SR Mujumdar, CJ Lewis, AS Waggoner, Bioconjugate Chem., 4, 105-111 (1993). The compound can be synthesized from compound (IX), compound (X) and compound (XI). For example, compound (IX), compound (X), and 2- [6-acethlphenylamino] 1,3,5-hexatrienyl] -3,3-dimethyl-5-sulfo-1- (4-sulfobutyl) 3-H- Indolium can be obtained by adding sodium acetate and compound (XI) in methanol at a molar ratio of 1: 1: 1 and reacting at room temperature to 70 ° C. for 1 to 3 hours.

<Process 2>
Compound (XIII), which is a novel indocyanine compound, is obtained, for example, by reacting compound (XII) in methanol with a metal alkoxide such as sodium methoxide or potassium methoxide and reacting at room temperature to 70 ° C. for 1 hour to 50 hours. Obtainable.

<Step 3>
Compound (XIV) is obtained by reacting Compound (XIII) with N, N-disuccimidyl carbonate in a mixture of pyridine and N, N-dimethylformamide at room temperature to 80 ° C. for 30 minutes to 2 hours. Obtainable.
.

<Step 4>
The light-emitting compound represented by the general formula (IV) is obtained by reacting compound (XIV) and compound (XV) in a mixed solution of pyridine and phosphate buffer at 0 ° C. to 50 ° C. for 30 minutes to 24 hours. be able to.

<Step 5>
The luminescent compound represented by the general formula (IV) is prepared by mixing compound (XIII) and compound (XV) in a mixed solution of pyridine and phosphate buffer at 0 ° C. to 50 ° C. for 30 minutes to 24 hours, for example, dicyclohexylcarbodiimide or WSC. It can be obtained by adding a dehydrating condensation agent such as

  The light-emitting compound represented by the general formula (V) can be produced using the method described in JP-A-2009-215174 or according to this method.

  Each target compound obtained by each reaction formula shown above is prepared by cooling the reaction mixture, for example, and then separating the crude reaction product by an isolation operation such as filtration, concentration, extraction, etc., column chromatography, recrystallization The reaction mixture can be isolated and purified by a usual purification operation such as.

Method for Measuring Active Oxygen The present invention measures the step of adding a near-infrared luminescent compound to a specimen, and the luminescence intensity of the specimen to which the near-infrared luminescent compound is added, using a sensor cooled to −5 to 20 ° C. A method for measuring active oxygen is provided.

  Examples of the specimen used in the method of the present invention include those derived from animals including humans, plants and the like. Here, specimens derived from animals including humans, plants, etc. include, for example, animal body fluids, plant extracts, dilutions of these, specific fractions, separated enzymes, and other processed products ( Plant-derived wine, etc.).

  Further, such a specimen may contain an enzyme, a cell, or the like that generates active oxygen, or may not contain this.

  Furthermore, in addition to the blood, biological tissue, and plant-derived specimens as described above, the type of specimen is not particularly limited as long as active oxygen can be measured by near infrared chemiluminescence. For example, processed foods, unprocessed foods, chemical products, cosmetics, pharmaceuticals, agricultural chemicals, plastics, rubbers and the like are listed as other samples.

  The specimen containing cells used in the present invention is not particularly limited as long as the specimen contains cells that generate active oxygen. A preferable sample includes blood of animals including humans. The blood-derived specimen may be obtained by fractionating the target cells such as neutrophils and granulocytes, or by diluting the blood. However, there is no need for pretreatment, and in vivo. In view of the ability to evaluate the ability to generate active oxygen in a state close to the above, whole blood not subjected to these treatments is preferable.

  In the method of the present invention, either spontaneously generated from the specimen, or even if the active oxygen contained in the specimen is measured, the specimen is treated with a stimulant, etc., and the generated reactive oxygen is measured, A substance that generates active oxygen or active oxygen itself may be added to the specimen, and its change with time may be measured.

  Examples of the stimulant include bacteria such as yeast, Staphylococcus aureus, and Escherichia coli, and compounds derived therefrom, such as Zymosan for yeast. Moreover, the thing which opsonized these substances is also mentioned. In addition, phorbol esters as compounds, formyl-methionyl-leucyl-phenylalanin (fMLP), one of leukocyte chemotactic factors, chemokines and interleukin-1 (IL-1) involved in leukocyte migration and inflammation, etc. Is mentioned. Furthermore, the substance which has the stimulating activity discovered by each research or discovered in the future is mention | raise | lifted without being limited to a normal known stimulant.

  When adding a stimulant, it is preferable to add 0.000001 mg to 100 mg of the stimulant as the weight of the active ingredient per 1 ml of the sample. When the molecular weight is a known compound, it can be added in the range of 0.001 iM-100 mM. However, the addition amount is not particularly limited as long as it is an amount suitable for each measurement, and is not limited to use outside the above range.

  When adding a near-infrared luminescent compound to the sample, the amount of the near-infrared luminescent compound is not particularly limited as long as it does not reduce the luminescence measurement and the quality of the sample. For example, as the final concentration after addition, It is preferable to add so that it may become 0.001-1000 micromol.

  In the method of the present invention, the near-infrared light-emitting compound can be added alone or in the form of a light-emitting agent mixed with an excipient, and an active compound containing components useful for active oxygen measurement. You may add in the form of an oxygen measuring agent. In this embodiment, the near-infrared light emitting compound is the main component of the chemiluminescent reaction substrate. The luminescent agent and the active oxygen measuring agent may be in any state of solid, liquid, and gas.

  In addition, components other than the near-infrared luminescent compound, the luminescent agent and the active oxygen measuring agent may be added to the specimen simultaneously or before and after these. For example, after adding an active oxygen generating system, for example, xanthine and xanthine oxidase, to the specimen, the antioxidant capacity of the specimen can be measured and evaluated by measuring the active oxygen over time. Components other than the near-infrared luminescent compound, the luminescent agent, and the active oxygen measuring agent may be in a solid, liquid, or gaseous state. As such other components, not only active oxygen generating substances but also active oxygen itself may be added.

  Then, the emission intensity of near-infrared light emission generated by the reaction between active oxygen and the near-infrared light-emitting compound is measured using a cooled sensor. At this time, a near infrared luminescent compound may be added to the specimen and introduced into a measuring apparatus including the sensor, or a near infrared luminescent compound may be added after the specimen is first introduced into the measuring apparatus. . The cooling temperature is −5 to 20 ° C., preferably −5 to 15 ° C., more preferably 0 to 10 ° C. The emission intensity can be measured with a high S / N ratio by using a near-infrared light-emitting compound as a luminescent reagent and setting the cooling temperature of the sensor within the above range and measuring.

  Here, the specific mode of the measurement of the emission intensity is not particularly limited. For example, the light emitted from the specimen is detected using a cooled sensor, the detected light information is converted into an electric signal, and the electric signal is converted into an electric signal. This can be done by going through a step of processing the converted information.

Active oxygen measuring device Hereinafter, an embodiment of active oxygen measuring apparatus according to the present invention will be described with reference to the drawings. FIG. 1 shows a schematic diagram of an active oxygen measuring device according to one embodiment of the present invention.

  The active oxygen measuring device of the present invention comprises a cell 4 for introducing a near-infrared luminescent compound (sometimes referred to as a sample holder in this specification), a means (sensor) 3 for measuring luminescence intensity, and A cooling means 9 for cooling the emission intensity measuring means to −5 to 20 ° C. is provided.

  In the present invention, the specimen 7 and the near-infrared light emitting compound treated or untreated as described above are introduced into the sample holder 4. A heater 6 may be installed in the vicinity of the sample holder 4 in order to hold the temperature of the sample. Such a heater 6 makes it possible to keep the sample in a condition close to the biological environment.

  In addition, the active oxygen measuring device of the present invention may include an optical unit 8 such as a lens for aggregating near infrared light generated from the measurement sample.

  The means (sensor) 3 for measuring the emission intensity needs to be able to measure the emission intensity in the near infrared region.

System for Measuring Active Oxygen The present invention also provides a system for measuring active oxygen, comprising the above-mentioned near-infrared light emitting compound and the above-mentioned active oxygen measuring device.

  In this invention, you may further provide components other than the near-infrared luminescent compound, the luminescent agent, and the active oxygen measuring agent which were demonstrated in the term of the active oxygen measuring method.

  By using such a system, it is possible to accurately measure active oxygen without requiring special facilities, environments, etc., and even without being a specialist, without requiring special techniques.

  Hereinafter, production examples and examples are described in order to describe the present invention in more detail. These examples embody the present invention and are not intended to limit the scope of the present invention.

Production Example 1: 4- (2-((E) -2-((E) -3-((E) -2- (1- (2-carboxyethyl) -3,3-dimethyl-5-sulfoindolin-2) -ylidene) ethylidene) -2-chlorocyclohex-1-enyl) vinyl) -3,3-dimethyl-5-sulfo-3H-indolium-1-yl) butane-1-sulfonate synthesis (step 1)
4- (2,3,3-trimethyl-5-sulfo-3H-indolium-1-yl) butane-1-sulfonate (1.0 g, in general formula (IX), the corresponding M is hydrogen, and m is 4), 1- (2-carboxyethyl) -2,3,3-trimethyl-3H-indolium-5-sulfonate (0.82 g, in the general formula (X), the corresponding M is hydrogen, k corresponds to a compound of 2), N-[(3- (anilinomethylene) -2-chloro-1-cyclohexen-1-yl) methylene] aniline hydrochloric acid (0.96 g), sodium acetate (0.88 g) , Methanol (24 mL) was stirred at 50 ° C. for 1 hour. Methanol was removed under reduced pressure, and water (50 mL) and chloroform (50 mL) were added to the residue and partitioned.

Further, the aqueous layer was washed with chloroform (50 mL × 5 times), and the aqueous layer was concentrated under reduced pressure. The residue was dissolved in 0.1% TFA aqueous solution, subjected to ODS column chromatography, and eluted with a mixture of water and acetonitrile. The eluate of the target product was concentrated under reduced pressure, and 0.67 g of the target product (in the general formula (XII), the corresponding M was hydrogen, k was 2, 1 was 3, and m was 4) Equivalent) was obtained as a blue-green solid. The instrument analysis data of the target product is shown below.
¹ H NMR (500 MHz, DMSO-d6, 25 ℃, TMS: 0.0 ppm) 1.65 (6H, s), 1.67 (6H, s), 1.75 (2H, quin, J = 7.3 Hz), 1.83 (4H, m ), 2.60 (2H, t, J = 7.3 Hz), 2.70 (6H, m), 4.26 (2H, t, J = 7.3 Hz), 4.38 (2H, t, J = 7.3 Hz), 6.34 (1H, d , J = 14 Hz), 6.45 (1H, d, J = 14 Hz), 7.30 (1H, d, J = 8.5 Hz), 7.47 (1H, d, J = 8.5 Hz), 7.62 (1H, d, d , J = 1.2, 8.5 Hz), 7.67 (1H, d, d, J = 1.2, 8.5 Hz), 7.74 (1H, d, J = 1.2 Hz), 7.81 (1H, d, J = 1.2 Hz), 8.18 (1H, d, J = 14 Hz), 8.29 (1H, d, J = 14 Hz)
ESI-MS m / z calcd for C ₃₇ H ₄₃ N ₂ O ₁₁ S ₃ Cl
822.17, found 823.23 [M + 1]
Production Example 2: 4- (2-((E) -2-((E) -3-((E) -2- (1- (2-carboxyethyl) -3,3-dimethyl-5-sulfoindolin-2) -ylidene) ethylidene) -2-methoxycyclohex-1-enyl) vinyl) -3,3-dimethyl-5-sulfo-3H-indolium-1-yl) butane-1-sulfonate synthesis (step 2)
4- (2-((E) -2-((E) -3-((E) -2- (1- (2-carboxyethyl) -3,3-dimethyl-5-) obtained in Production Example 1 sulfoindolin-2-ylidene) ethylidene) -2-chlorocyclohex-1-enyl) vinyl) -3,3-dimethyl-5-sulfo-3H-indolium-1-yl) butane-1-sulfonate (0.2 g), methanol (12 mL) and potassium butoxide (0.56 g) were stirred at 50 ° C. for 12 hours, and then neutralized by adding water (20 mL) and 1N aqueous HCl (5 mL) under ice cooling. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in 0.1% aqueous TFA solution and subjected to ODS column chromatography. Elution is performed with a mixture of water and acetonitrile, and the eluate is concentrated under reduced pressure. The target product is 0.20 g (in the general formula (XIII), the corresponding M is hydrogen, k is 2, l is 3, corresponding to a compound in which m is 4 and R ¹⁶ ′ is methyl) as a blue-green solid. The instrument analysis data of the target product is shown below.
¹ H NMR (500 MHz, DMSO-d6, 23 ° C, TMS: 0.0 ppm) 1.65 (6H, s),
1.67 (6H, s), 1.70-1.85 (6H, m), 2.55-2.65 (6H, m), 2.70 (2H, t, J = 7.3
Hz), 3.95 (3H, s), 4.21 (2H, br.), 4.35 (2H, br.), 6.20 (1H, d, J = 14.1
Hz), 6.28 (1H, d, J = 14.7 Hz), 7.27 (1H, d, J = 8.0 Hz), 7.42
(1H, d, J = 8.0 Hz), 7.62 (1H, d, J = 8.0 Hz), 7.65 (1H, d, J
= 8.0 Hz), 7.72 (1H, s), 7.79 (1H, s), 7.95 (1H, d, J = 14.1 Hz), 8.06
(1H, d, J = 14.7 Hz)
ESI-MS m / z calcd for C ₃₈ H ₄₆ N ₂ O ₁₂ S ₃
818.22, found 819.19 [M + 1]
Production Example 3: 4- (2-((E) -2-((E) -3-((E) -2- (1- (3- (2,5-dioxopyrrolidin-1-yloxy) -3- oxopropyl) -3,3-dimethyl-5-sulfoindolin-2-ylidene) ethylidene) -2-methoxycyclohex-1-enyl) vinyl) -3,3-dimethyl-5-sulfo-3H-indolium-1-yl) butane Synthesis of -1-sulfonate (process 3)
4- (2-((E) -2-((E) -3-((E) -2- (1- (2-carboxyethyl) -3,3-dimethyl-5-) obtained in Production Example 2 sulfoindolin-2-ylidene) ethylidene) -2-methoxycyclohex-1-enyl) vinyl) -3,3-dimethyl-5-sulfo-3H-indolium-1-yl) butane-1-sulfonate (0.15 g), N , N-disuccimidyl carbonate (0.7 g), pyridine (3 mL), and N, N-dimethylformamide (3 mL) were stirred at 50 ° C. for 40 minutes. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in a mixed solution of methylene chloride and methanol and subjected to silica gel column chromatography. Elution is performed with a mixture of methylene chloride and methanol, and the eluate containing the target product is concentrated under reduced pressure. The target product is 0.143 g (in formula (XIV), the corresponding M is hydrogen, k is 2, Was 3, and m was 4, corresponding to a compound in which R ¹⁶ ′ was methyl). The instrument analysis data of the target product is shown below.
¹ H NMR (500 MHz, DMSO-d6, 23 ° C, TMS: 0.0 ppm) 1.63 (6H, s), 1.66 (6H, s), 1.70-1.85 (6H, m), 2.55 (2H, t, J = 7.3 Hz), 2.60 (4H, Br.), 2.78 (4H, s), 3.25 (2H, t, J = 7.3 Hz), 3.94 (3H, s), 4.23 (2H, br.), 4.43 (2H, br.), 6.08 (1H, d, J = 13.3 Hz), 6.35 (1H, d, J = 14.6 Hz), 7.23 (1H, d, J = 8.5 Hz), 7.45 (1H, d, J = 8.5 Hz) ), 7.56 (1H, d, d, J = 1.2, 8.5 Hz), 7.66 (1H, d, J = 8.5 Hz), 7.67 (1H, s), 7.80 (1H, s), 7.88 (1H, d, J = 13.3 Hz), 8.10 (1H, d, J = 14.6 Hz)
ESI-MS m / z calcd for C ₄₂ H ₄₉ N ₃ O ₁₄ S ₃
915.24, found 916.11 [M + 1]
Production Example 4: 4- (2-((E) -2-((E) -2-methoxy-3-((E) -2- (1- (3- (2- (3- (6- ( 4-methoxyphenyl) -3-oxo-3,7-dihydroimidazo [1,2-a] pyrazin-2-yl) propanamido) ethylamino) -3-oxopropyl) -3,3-dimethyl-5-sulfoindolin-2-ylidene Synthesis of) ethylidene) cyclohex-1-enyl) vinyl) -3,3-dimethyl-5-sulfo-3H-indolium-1-yl) butane-1-sulfonate (step 4)
4- (2-((E) -2-((E) -3-((E) -2- (1- (3- (2,5-dioxopyrrolidin-1-yloxy)) obtained in Production Example 3 -3-oxopropyl) -3,3-dimethyl-5-sulfoindolin-2-ylidene) ethylidene) -2-methoxycyclohex-1-enyl) vinyl) -3,3-dimethyl-5-sulfo-3H-indolium-1- yl) butane-1-sulfonate (0.05 g) and N- (2-aminoethyl) -3- (6- (4-methoxyphenyl) -3-oxo-3,7-dihydroimidazo [ 1,2-a] pyrazin-2-yl) propanamide (0.0087 g) was purged with hydrogen in a mixture of pyridine (0.6 mL) and 0.1 M phosphate buffer (pH 7.4, 0.25 mL) at room temperature. For 4 hours. The reaction solution was concentrated under reduced pressure, the residue was dissolved with 0.1% TFA aqueous solution, and this solution was subjected to ODS column chromatography. Elution was performed with a mixture of water and acetonitrile, and the eluate of the target product was concentrated under reduced pressure, and 0.009 g of the target product (in formula (IV), the corresponding M was hydrogen, k was 2, and l was 3 Corresponding to a compound in which m is 4 and R ¹⁶ ′ is methyl) as a yellow-green solid. The instrument analysis data of the target product is shown below.
¹ H NMR (500 MHz, D2O, 22 ° C, Aceton: 2.07 ppm) 1.21 (6H, s), 1.38 (6H, s), 1.52 (2H, br.), 1.62 (2H, br.), 2.37 (2H , t, J = 7.3 Hz), 2.45 (2H, br.), 2.72 (4H, m), 3.00 (4H, m), 3.42 (3H, s), 3.46 (3H, s), 3.62 (2H, br .), 4.00 (2H, br.), 5.25 (1H, br.), 5.55 (1H, br.), 6.48 (2H, d, J = 7.9 Hz), 6.78 (1H, br.), 6.93 (1H , br.), 7.10 (2H, d, J = 7.9 Hz), 7.35 (1H, s), 7.40 (1H, d, J = 7.4 Hz), 7.48 (1H, br.), 7.55 (3H, m) , 7.63 (1H, s), 7.83 (1H, s)
ESI-MS m / z calcd for C ₅₆ H ₆₅ N ₇ O ₁₄ S ₃
1155.38, found 1156.33 [M + 1]
Production Example 5: 4- (2-((E) -2-((E) -2-methoxy-3-((E) -2- (1- (3- (2- (3- (6- ( 4-methoxyphenyl) -3-oxo-3,7-dihydroimidazo [1,2-a] pyrazin-2-yl) propanamido) ethylamino) -3-oxopropyl) -3,3-dimethyl-5-sulfoindolin-2-ylidene Synthesis of) ethylidene) cyclohex-1-enyl) vinyl) -3,3-dimethyl-5-sulfo-3H-indolium-1-yl) butane-1-sulfonate (Step 5)
4- (2-((E) -2-((E) -3-((E) -2- (1- (2-carboxyethyl) -3,3-dimethyl-5-) obtained in Production Example 2 sulfoindolin-2-ylidene) ethylidene) -2-methoxycyclohex-1-enyl) vinyl) -3,3-dimethyl-5-sulfo-3H-indolium-1-yl) butane-1-sulfonate (0.03g), general N- (2-aminoethyl) -3- (6- (4-methoxyphenyl) -3-oxo-3,7-dihydroimidazo [1,2-a] pyrazin-2-yl) propanamide represented by the formula (XV) 0.017 g), WSC HCl (0.22 g), pyridine (0.6 mL) and 0.1 M phosphate buffer (pH 7.4, 0.05 mL) were purged with hydrogen and stirred at room temperature for 2 hours. Acetone was added to the reaction solution to separate the produced solid from the supernatant, and the solid was dissolved in 0.1% TFA aqueous solution. The solution was subjected to ODS column chromatography. Elution was performed with a mixture of water and acetonitrile, and the eluate of the target product was concentrated under reduced pressure, and 0.007 g of the target product (in formula (IV), the corresponding M was hydrogen, k was 2, and l was 3 Corresponding to a compound in which m is 4 and R ¹⁶ ′ is methyl) as a yellow-green solid.

Example 1 Measurement of active oxygen generated from human whole blood Human blood was used as a measurement sample. As the measurement sample, blood collected with a heparin-treated blood collection tube and kept at 37 ° C. after blood collection was used.

As the luminescent reagent, 4- (2-((E) -2-((E) -2-methoxy-3-((E) -2- (1- (3- (2- (3- (6- (4-methoxyphenyl) -3-oxo-3,7-dihydroimidazo [1,2-a] pyrazin-2-yl) propanamido) ethylamino) -3-oxopropyl) -3,3-dimethyl-5-sulfoindolin-2- ylidene) ethylidene) cyclohex-1-enyl) vinyl) -3,3-dimethyl-5-sulfo-3H-indolium-1-yl) butane-1-sulfonate (in formula (IV), the corresponding M is hydrogen) Equivalent to a compound in which k is 2, l is 3, m is 4, and R ¹⁶ ′ is methyl. Hereinafter, in this example, it may be simply referred to as NIR-CLA) A 0.1 mM aqueous solution was used. A Zymosan solution (25 mg / mL PBS (−)) was used as a stimulant.

  A near infrared measurement device was used for the measurement. 1.0 mL of blood and 10 μL of luminescent reagent were added to the measurement container, and measurement was started. The measurement was performed in a cycle of [30 seconds measurement + 30 seconds rest]. The temperature of the sensor part was set to 5 ° C. The emission intensity was measured with the sample temperature maintained at 37 ° C. After 5.5 minutes of measurement, 50 μL of Zymosan solution was added, and the same measurement was performed again with a near-infrared emission measuring device.

  The measurement results are shown in FIG. As shown in FIG. 2, active oxygen generated from cells in human whole blood could be measured without any treatment such as neutrophil fractionation.

Example 2 A near-infrared light emission measurement device was used as a comparative measurement device for measurement accuracy according to sensor temperature conditions , and the light emission intensity was measured while maintaining the temperature of the sensor portion at 25 ° C. or 5 ° C.

  As the luminescent reagent, 1 mg of the aforementioned NIR-CLA dissolved in 866 uL of milli-Q water was used. The solution was dispensed in 100 μL aliquots and stored at −80 ° C. until testing.

  During the test, the luminescent reagent solution was further diluted with milliQ water. Specifically, a 0.3 μM solution (30 μL / 100 μL) was first prepared, and a 0.03 μM solution was further prepared from this solution. A 2-fold dilution series was prepared from this solution to make 0.03 and 0.015 μM solutions.

The XOD-Xanthine system was used as the active oxygen generation system. Specifically, the following ATTO AB2970 CLETA-S enzyme / substrate was used:
0.125unit / mL XOD 1.2M (NH ₄ ) ₂ SO ₄ -0.25M HEPES 0.05mM EDTA pH7.5
0.1mM Xanthine 0.25M HEPES 0.05mM EDTA pH7.5
4. Measurement The sample was mixed in the following volume, set in the apparatus, the luminescence intensity was measured for 10 seconds, and the measured values were integrated.

Luminescent reagent solution: 10 μL (final conc. 0.001 and 0.005 μM)
XOD (xanthine oxidase): 80μL (final conc. 0.01unit / tube)
Xanthine 200μL
The measurement was performed three times for each test section, and the average value was calculated.

  As measurement results, the emission intensity of each test section is shown in Table 1, and the S / N ratio is shown in Table 2.

  As shown in Table 2, when the sensor temperature was 25 ° C., the S / N ratio remained at a low value of 2.3 or less. On the other hand, when the sensor temperature is 5 ° C., the S / N ratio is very high as 9.0 or more, and it can be seen that the active oxygen concentration can be measured with high accuracy.

Example 3 Measurement of active oxygen generated from a bovine blood sample Blood (Holstein) of a 27-day-old bull was used as a measurement sample. As the measurement sample, blood collected with a heparin-treated blood collection tube and kept at 37 ° C. after blood collection was used.

  As the luminescence reagent, the aforementioned NIR-CLA aqueous solution of 0.0125 mM was used. A Zymosan solution (25 mg / mL PBS (−)) was used as a stimulant.

  A near infrared measurement device was used for the measurement. Measurement was started by adding 50 μL of blood, 200 μL of PBS (−), and 10 μL of luminescent reagent to the measurement container. The measurement was performed in a cycle of [10 seconds measurement + 50 seconds rest]. The temperature of the sensor part was set to 5 ° C. The emission intensity was measured with the temperature of the sample held at 37 ° C. Ten minutes after the measurement, 10 μL of the Zymosan solution was added, and the same measurement was performed again with the near-infrared emission measuring device.

  The measurement results are shown in FIG. As shown in FIG. 3, the active oxygen generated from cells in bovine blood could be measured without fractionation of neutrophils and the like.

1. Housing 2. 2. Dark box Sensor 4. Sample holder 5. 5. Measurement sample container Heater (for holding sample temperature)
7). Measurement sample 8. Optical unit 9. Cooling unit (for holding sensor temperature)

Claims (6)

Measurement of active oxygen, including a step of adding a near-infrared luminescent compound to a specimen, and a step of measuring the luminescence intensity of the specimen to which the near-infrared luminescent compound is added using a sensor cooled to −5 to 20 ° C. Method. The active oxygen measurement according to claim 1, wherein the near-infrared light-emitting compound is an indocyanine-linked imidazo [1,2-a] pyrazin-3-one compound represented by the following general formula (I) or a salt thereof: Method:
A-B-C (I)
[Wherein A represents an imidazo [1,2-a] pyrazin-3-one group represented by the following general formula (II):

(Wherein R ¹ , R ² , R ³ , and R ⁴ are the same or different and are a hydrogen atom, alkyl group, aryl group, halogen atom, alkoxyl group, carboxyl group, formyl group, alkyloxycarbonyl group, aryl. An oxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group or a heterocyclic ring is shown.
However, ^one of R ¹ , R ² , R ³ , and R ⁴ is covalently bonded to the spacer group B. )
Group B represents a spacer group.
The group C represents an indocyanine group represented by the following formula (III):

(In the formula, R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , and R ²⁵ are the same or different and are a hydrogen atom, alkyl group, aryl group, halogen atom, alkoxyl group, carboxyl group, formyl group, sulfonyl group, sulfonic acid A group, an alkyloxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, an aryloxy group, an alkylperoxy group or a heterocyclic ring;
R ¹⁵ and R ¹⁷ may be bonded to each other to form an unsaturated 4- to 10-membered ring.
^{However, R 5, R 6, R} 7, R 8, R 9, R 10, R 11, R 12, R 13, R 14, R 15, R 16, R 17, R 18, R 19, R 20, One of R ²¹ , R ²² , R ²³ , R ²⁴ , and R ²⁵ is covalently bonded to the spacer group B. ]] The active oxygen measurement according to claim 1, wherein the near-infrared light-emitting compound is an indocyanine-linked imidazo [1,2-a] pyrazin-3-one compound represented by the following formula (IV) or (V): Method.

[Wherein, R ¹⁶ ′ represents an alkyl group, an alkoxyl group or an aryl group.
M represents hydrogen, an alkali metal or an alkaline earth metal.
k, l, and m are the same or different and represent an integer of 1 to 7. ]

[In formula, M is the same or different and shows hydrogen, an alkali metal, or an alkaline-earth metal.
k and m are the same as above.
n and p are the same or different and represent an integer of 1 to 7. ]. The active oxygen measurement according to claim 1, wherein the near-infrared light emitting compound is an indocyanine-linked imidazo [1,2-a] pyrazin-3-one compound represented by the following formula (VI) or (VII): Method.

[In formula, M is the same or different and shows hydrogen, an alkali metal, or an alkaline-earth metal. ].

A cell for introducing a near-infrared light emitting compound,
Means for measuring the emission intensity, and cooling means for cooling the emission intensity measurement means to -5 to 20 ° C,
A device for measuring active oxygen, comprising:   A kit for measuring active oxygen, comprising a near-infrared light emitting compound and the active oxygen measuring device according to claim 5.

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