JPH0892211A - Labeling dye, its precursor, their synthesis, use of labeling dye and apparatus for detecting cocaine or methamphetamine using the labeling dye - Google Patents

Abstract

PURPOSE: To provide a labeling dye capable of detecting cocaine and methamphetamine by immunoassay in high sensitivity, a precursor for the dye and a process for synthesizing the dye. CONSTITUTION: The labeling dye for the detection of cocaine is composed of a merocyanine derivative of formula I and the labeling dye for the detection of methamphetamine is composed of a methamphetamine derivative of formula II (X is an anion). The compound of formula I can be produced by dissolving a halogenated propyl derivative of formula III (X1 and X2 are each a halogen) (a precursor of the compound of formula I) in an organic solvent, adding norcocaine of formula IV to the solution and reacting under heating. The dye of formula I is reacted with an anticocaine antibody, the intensity of fluorescence is measured at an excitation wavelength of 530-590nm, the fluorescent intensity is measured again after adding a determination specimen liquid and cocaine is detected by the variation of the intensity. Methamphetamine can be detected by a similar method using the dye of formula II and an antimethamphetamine antibody.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a merocyanine-based or methamphetamine derivative-labeled dye useful for immunoassays and other biochemical experiments, a precursor compound thereof, a method for synthesizing them, and a method for using the dye. The present invention relates to a detection device for detecting cocaine and methamphetamine by an immunoassay.

[0002]

2. Description of the Related Art First, a case where methamphetamine (MA) is a detection target will be described as an example of the prior art. As a labeling dye in which MA and a fluorescent substance are chemically bonded, a compound in which a dansyl group is bonded to MA has been proposed, as described in Japanese Patent Application Laid-Open No. 3-223673. The dansylated MA (hereinafter DNS-MA) is 330n
It is known that when excited by m, it emits fluorescence of 525 nm and binds to an anti-MA antibody to change its fluorescence intensity. When DNS-MA is bound to the anti-MA antibody in advance and MA is added thereto, DNS-MA is desorbed from the antibody as MA binds to the antibody, and the fluorescence intensity at 525 nm changes. By measuring the intensity change, MA can be detected with high sensitivity.

[0003]

However, there is no precedent for detecting cocaine (CC) as a detection target, and CC cannot be detected by the conventional technique. Therefore, C
In order to detect C, development of a labeling dye that can be detected by an immunological method has been required. In addition, since many substances having fluorescence around 525 nm are naturally present, when these impurities are mixed, the measurement method using DNS-MA described above is used.
Was difficult to detect.

In order to solve the above problems, the present invention provides a labeled dye capable of detecting CC or methamphetamine contained in a substance to be measured, a precursor compound thereof, a method for synthesizing them, and a method for using the dye. With the goal. Furthermore, it aims at providing the detection apparatus of cocaine methamphetamine using a labeling dye.

[0005]

To achieve the above object, the labeling dye of the present invention comprises a merocyanine derivative represented by the above formula (Formula 1).

Next, the propyl halide derivative of the present invention is a precursor of a labeling dye composed of the merocyanine derivative represented by the above formula (Formula 1) and is represented by the above formula (Formula 2). Next, the first method for producing a labeled dye of the present invention is to dissolve the halogenated propyl derivative represented by the formula (Formula 2) in an organic solvent and add norcocaine represented by the formula (Formula 3) to the solution. It has a configuration of heating.

Next, in the method for producing a dye precursor of the present invention, a trimethylindorhenium salt represented by the formula (Formula 4) and a dimethylaminobenzaldehyde represented by the formula (Formula 5) are dissolved in an organic solvent, It has a constitution in which the halogenated propyl derivative represented by the above formula (Formula 2) is synthesized by heating.

The second labeling dye of the present invention comprises a methamphetamine derivative represented by the above formula (Formula 6).
Next, the methanesulfonic acid derivative of the present invention is represented by the above formula (Formula 7) and is a precursor of the second labeling dye of the present invention.

Next, the hydroxy derivative of the present invention is represented by the above formula (Formula 8) and is a precursor of the second labeling dye of the present invention. Next, the acetyl derivative of the present invention is represented by the above formula (Formula 9) and is a precursor of the second labeling dye of the present invention.

Next, the second method for producing a labeled dye of the present invention has a constitution in which a methanesulfonic acid derivative represented by the above formula (Formula 7) and methamphetamine are subjected to a substitution reaction in an acidic solvent.

Next, the method for using the dye of the present invention is as follows. The fluorescence intensity of the dye-derived fluorescence emitted from the buffer solution containing the labeling dye consisting of the merocyanine derivative represented by the above formula (Formula 1) and the anti-cocaine antibody, or Intensity change between the fluorescence intensity of the fluorescence derived from the dye emitted from the buffer solution containing the labeling dye represented by the formula (Formula 6) and the anti-methamphetamine antibody, and the fluorescence intensity when the substance to be measured is added to the buffer solution. Is measured, and cocaine or methamphetamine contained in the substance to be measured is detected.

Next, in the cocaine / methamphetamine detection apparatus of the present invention, a sample solution prepared by dissolving the sample in a dye solution containing an antigen-labeling dye is reacted with an anti-methamphetamine antibody or an anti-cocaine antibody to change the fluorescence intensity of the sample solution. A method of detecting methamphetamine, cocaine or a derivative thereof for measuring the presence or absence of methamphetamine, cocaine or a derivative thereof in a sample solution, wherein the labeling dye is represented by the formula (Formula 1) or (Formula 6).
A container for storing each of the dye solution and the antibody solution containing the antibody, and an elution device for eluting the sample, and the sample solution. And a reaction container for reacting the antibody liquid, a supply means for supplying the antibody liquid and the sample liquid to the reaction container, and a fluorescence detector for detecting the fluorescence intensity of the liquid to be measured in the reaction container. And at least.

In the above arrangement, the supply means is preferably a syringe type pump and a valve. Further, in the above structure, it is preferable that the reaction container is a rectangular cell having an inlet and an air vent hole in the upper portion and an ejection nozzle in the lower portion.

Further, in the above constitution, in the reaction vessel,
It is preferable to provide a stirring means driven by a magnetic force for stirring the sample solution, the dye solution and the antibody solution. Moreover, in the said structure, it is preferable to equip the reaction container with the thermostat which used the Peltier element.

Further, in the above structure, it is preferable that at least the storage device and the supply means are provided inside the air conditioning constant temperature device.

[0016]

The labeling dye represented by the above formula (Formula 1) of the present invention (hereinafter abbreviated as CCIMC) has a long wavelength region (59).
It has fluorescence around 0 nm). And CCIMC is anti-CC
It has an affinity for the antibody, but its value is CC
Not as expensive as Therefore, in the absence of CC, CCIMC binds to the anti-CC antibody to change the fluorescence intensity, but when CC is added thereto, CCIMC bound to the antibody replaces CC with high affinity and The fluorescence intensity approaches the initial fluorescence intensity according to the amount.
CC can be detected by measuring this change in fluorescence intensity. Therefore, the CCIMC dye of the present invention is
It enables to detect CC with high sensitivity.

Further, since the halogenated propyl derivative represented by the above chemical formula (Formula 2) of the present invention has a halogen which is excellent as a leaving group, CCIMC which is the first dye of the present invention is synthesized. It is possible to provide a precursor (intermediate) useful for

Further, according to the first method for producing a labeled dye of the present invention, the halogenated propyl derivative represented by the formula (Formula 2) is dissolved in an organic solvent, and the compound is represented by the formula (Formula 3). It is possible to provide a novel method for synthesizing CCIMC, which can be synthesized by adding norcocaine to the mixture and heating.

According to the method for producing a dye precursor of the present invention, the trimethylindorhenium salt represented by the above formula (Formula 4) and the dimethylaminobenzaldehyde represented by the above formula (Formula 5) are dissolved in an organic solvent. Then, the propyl halide derivative represented by the above formula (Formula 2) can be synthesized by heating, and a novel method for synthesizing the halogenated propyl derivative can be provided.

The labeling dye represented by the above formula (Formula 6) of the present invention (hereinafter abbreviated as MAIMC) has fluorescence in a long wavelength region (near 590 nm). And although MAIMC has an affinity for anti-MA antibodies, its value is not as high as for MA. Therefore, in the absence of MA, MAIMC binds to the anti-MA antibody and the fluorescence intensity changes, but when MA is added thereto, MAIMC bound to the antibody replaces MA with high affinity and The fluorescence intensity approaches the initial fluorescence intensity according to the amount. MA can be detected by measuring this change in fluorescence intensity, but the dye consisting of MAIMC of the present invention is 55
It absorbs light centered at a wavelength of 0 nm,
Since it is a dye with fluorescence in m), it is less susceptible to the effects of impurities having absorption in the ultraviolet region and the influence of light scattering due to a significant insoluble component in the ultraviolet region, making it easy to detect MA with high sensitivity. A dye can be provided.

Further, since the methanesulfonyl derivative represented by the above formula (Formula 7) has a methanesulfonyl group which is excellent as a leaving group, M which is the second dye of the present invention.
A precursor (intermediate) useful for synthesizing AIMC can be provided.

Since the hydroxy derivative represented by the above formula (Formula 8) has a hydroxyl group, it is highly reactive with an acid chloride. Therefore, the methanesulfonyl derivative can be synthesized by rapidly reacting with methanesulfonic anhydride.

Further, since the acetyl derivative represented by the above formula (Formula 9) removes the reaction inhibiting effect of the hydroxyl group, the dye skeleton can be synthesized in a high yield. That is, in order to synthesize the hydroxy derivative, the reaction of N-hydroxypropyl-2,3,3-trimethylindolenine and pN, N-dimethylbenzaldehyde seems to be the shortest route, but this reaction is N-hydroxypropyl
It is inhibited by the hydroxyl group of -2,3,3-trimethylindolenine and practically does not proceed. Therefore, a dye skeleton, that is, an acetyl derivative represented by the above formula (Formula 9) is synthesized with the hydroxyl group on trimethylindolenine protected by an acetyl group, and then the acetyl group is removed by hydrolysis to enhance the hydroxy derivative. It can be synthesized in yield.

According to the second method for producing a labeled dye of the present invention, it can be synthesized by subjecting the methanesulfonic acid derivative represented by the above formula (Formula 7) and methamphetamine to a substitution reaction in an acidic solvent. , MAIMC can be provided.

The method of using the labeling dye of the present invention is C
Buffer solution containing CIMC and anti-CC antibody, or MAIM
Cocaine or methamphetamine can be detected with high sensitivity by measuring the intensity change between the fluorescence intensity of the buffer solution containing C and the anti-methamphetamine antibody and the fluorescence intensity when the substance to be measured is added to the buffer solution. It will be possible.
That is, since the labeled dye of the present invention is a labeled dye having fluorescence in a long wavelength region, it is possible to provide a detection method which is not easily affected by impurities and which can detect CC with high sensitivity.
When CCIMC and MAIMC are used as a mixture, the drug contained in the liquid to be measured can be detected, and at the same time, it can be identified whether the detected drug is CC or MA.

Next, the apparatus for detecting MA, CC or a derivative thereof according to the present invention uses an anti-MA antibody or anti-CC in a sample solution prepared by dissolving the sample in a dye solution containing an antigen labeling dye.
A detection device for MA, CC or their derivatives, which comprises reacting an antibody and measuring the presence or absence or the concentration of MA, CC or their derivatives in the sample liquid from the change in the fluorescence intensity of the sample liquid, wherein the labeling dye is At least one selected from the labeling dyes represented by the formulas (Formula 1) and (Formula 6)
And a container for respectively storing the antibody solution containing two dyes and the antibody, an elution device for eluting the sample, and a reaction container for reacting the sample solution and the antibody solution And a supply means for supplying the antibody solution and the sample solution to the reaction container,
By providing at least a fluorescence detector for detecting the fluorescence intensity of the liquid to be measured in the reaction container, a detection device capable of quickly detecting MA, CC or their derivatives in the sample liquid is achieved. it can.

Further, according to a preferable example of the present invention in which the supply means is a syringe type pump and a valve, the liquid can be easily supplied from the storage container to the reaction container and can be easily detected.

Further, according to a preferable example of the present invention in which the reaction container is a rectangular cell having an inlet and an air vent hole in the upper portion and a discharge nozzle in the lower portion, the supply means and the discharge nozzle are provided. By controlling the opening and closing, continuously different samples can be easily measured.

Further, according to a preferred embodiment of the present invention in which a stirring means driven by a magnetic force for stirring the sample solution, the dye solution and the antibody solution is provided in the reaction container, the sample solution is allowed to react quickly. The processing time is shortened.

Further, according to the preferable example of the present invention in which the reaction vessel is provided with a thermostatic device using a Peltier element, detection can be performed under a constant temperature environment. Further, according to the preferable example of the present invention in which the storage device and the supply means are provided at least inside the air-conditioning type constant temperature device, it becomes possible to detect CC or the like under any temperature environment.

[0031]

Embodiments of the present invention will be described below with reference to the drawings. The trimethylindorhenium salt represented by the chemical formula (Formula 4) is, for example, chloroiodopropane and
Mix with 0.5 to 2 molar ratio of trimethylindolenine,
It can be obtained by heating at about 60-100 ° C for about 1-5 hours.

The propyl halide derivative represented by the chemical formula (Formula 2) is, for example, a solution of a trimethylindorhenium salt represented by the chemical formula (Formula 4) in an organic solvent,
For example, an equimolar amount of dimethylaminobenzaldehyde represented by the above formula (Formula 3) is added to an ethanol solution,
By heating to 60 to 100 ° C., dimethylaminobenzaldehyde serves as a base, and it can be obtained in a short time without adding pyridine which was conventionally required as a base.

Further, CC represented by the above chemical formula (Formula 1)
The IMC is, for example, about 2 to 10 times mol of norcocaine added to an organic solvent solution of the iodopropyl derivative represented by the chemical formula (Formula 2), for example, a chloroform solution, to obtain about 60
It can be obtained by heating at -100 ° C for about 5-12 hours.

The acetyl derivative represented by the above formula (Formula 9) is equimolar to a solution of the acetyl-substituted trimethylindorhenium salt represented by the above Formula (Formula 4) in a basic organic solvent such as a pyridine solution. It can be obtained in a short time by adding dimethylaminobenzaldehyde (1) and heating to about 110 to 150 ° C.

The hydroxy derivative represented by the above formula (Formula 8) is obtained by dissolving the acetyl derivative represented by the above formula (Formula 9) in an organic solvent such as methanol, and adding a several-fold amount of alkali such as sodium hydroxide. The reaction is terminated by mixing the aqueous solution of (1) and stirring at room temperature and atmospheric pressure for 1-2 hours, and the solvent is removed after pH adjustment.

The methanesulfonic acid derivative represented by the formula (Formula 7) is added to a solution of the hydroxy derivative represented by the formula (Formula 8) in an organic solvent such as a chloroform solution at a molar ratio of 2 to 20. It can be obtained by adding an acid anhydride and stirring at room temperature or a low temperature of about 4 ° C. and normal pressure for 1 to 10 days.

The dye represented by the general formula (Chemical formula 6) is obtained by adding MA in a molar ratio of 2 to 20 to an acetic acid solution of the methanesulfonic acid derivative represented by the general formula (Chemical formula 7) to obtain about 50-
It can be obtained by heating at 80 ° C for several hours.

FIG. 1 is a block diagram showing the structure of the detection apparatus of the present invention. In FIG. 1, all the constituent parts are placed in an air-conditioned constant temperature room 17 provided with an air-conditioned constant temperature device 18,
It is kept at ℃. An eluate, an antibody solution and a dye solution are stored in the containers 1, 2 and 3, respectively. A valve 4 and a pump 7 are connected to the container 1, a valve 5 and a pump 8 are connected to the container 2, and a valve 6 and a pump 9 are connected to the container 3. The container 1 is connected to a sample elution device 21 via a valve 4 and a valve 22. A liquid reservoir 23 is provided below the sample elution device 21. The liquid reservoir 23 is connected to the reaction container 11 in the fluorescence detector 14 via the switching valve 10.
It is connected to. A magnetic stirrer bar 12 and a magnetic stirrer 13 are provided as stirring means in the reaction vessel 11. The reaction vessel 11 is kept at 25 ° C. by the Peltier device 19. The reaction device 11, the sample elution device 21, and the liquid reservoir 23 are connected to the waste liquid reservoir 16 via a waste liquid valve 24 and a waste liquid pump 15, respectively.

The reaction vessel 11 is a so-called rectangular cell having a rectangular cross section as shown in FIG. 2, and a switching valve 10 and a connecting pipe 11c from the valve 5 and an air vent hole 11d are provided in the upper portion 11a. There is. In general, the shape of the reaction container is not limited to the one having a rectangular cross section, and the one having a shape other than the rectangular cross section such as a circular cross section can be used. However, when a shape other than a rectangular cross section is used, a light scattering phenomenon occurs when measuring the fluorescence intensity, which is not very suitable. In addition, when injecting a liquid such as a sample liquid, it is preferable to provide an air vent hole inside the reaction container 11 so as not to apply a back pressure, in order to accurately control the injection amount.

FIG. 3 (a) is a front view showing a cell holder to which the Peltier element 19 of the fluorescence detector 14 of the detection device of this embodiment is attached, and FIG. 3 (b) is a side view thereof. Figure 3
In (a), 11c is a liquid injection port, 11b is a waste liquid port,
32 is an aluminum fin. In FIG. 3B, the rectangular reactor 11 is fixed to one surface of an aluminum block 31, and a thermostatic device 33 having a Peltier element 19 is provided near it. The Peltier element 19 is fixed to the other surface of the block 31. further,
An aluminum fin 3 is provided on the surface opposite to the Peltier element 19.
2 is attached. As described above, the Peltier element 19 is connected to the reactor 11 via the block 31 having a large heat capacity.
By mounting, the rapid temperature change when the sample liquid or the like is injected into the reaction container 11 or when the Peltier element 19 generates heat is relieved. Further, since the entire apparatus is provided inside the air-conditioned constant temperature room 17, stable detection can be performed regardless of temperature conditions such as cold regions and warm regions.

The sample elution device 21 is, as shown in FIG.
Piston 41, O-ring 42, eluent injection / discharge port 4
3 and a filter mounting portion 44. Further, the computer 20 causes the pumps 7, 8, 9 and the valves 4, 5,
6, 10, 22, 24 and magnetic stirrer 1
3 is controlled and the data from the fluorescence detector 14 is acquired and the data is processed. Therefore, a person who is unfamiliar with the handling of the device can easily perform the detection work without having any special knowledge.

Since the method for detecting CC or MA using the labeling dye of the present invention is to measure the change in the fluorescence intensity of CCIMC or MAIMC, the wavelength of the fluorescence to be measured should be 550 to 640 nm, which has a high intensity. Preferably 590n
The fluorescence of m is the strongest and is therefore preferred. In addition, the measurement using such a long wavelength has a small effect even if impurities are mixed, as described above, and the CC sensitivity is high.
Alternatively, MA can be detected, which is preferable. Also, the excitation light
It is optimal to use 550 nm in terms of fluorescence intensity, but the excitation wavelength separated from the wavelength of half-face fluorescence is superior in terms of stray light. Therefore, it is practically preferable to excite near 530 nm.

The present invention will be described in more detail with reference to specific examples. In the following experiments, all liquid chromatography (hereinafter, HPLC) measurements were performed under the following conditions.

Device: Japan Millipore Limited, 600 system Detector: Otsuka Electronics, MCPD3600 Column: Japan Millipore Limited, C18 (15μ, 100
Solvent: Methanol / hydrochloric acid (0.01N) = 3/2 Flow rate: 2.0 ml / min Quantitative: Quantitative by area of absorption peak at wavelength of 550 nm (Example 1) (Synthesis of trimethylindorhenium salt) ) Trimethylindolenine 10 g (MW = 159.2, 0.063 mol) and chloroiodopropane 50 g (MW = 204.4, 0.24 mol) were mixed using 30 ml of benzene, and heated under a nitrogen stream at 80 ° C. for 6 hours. Benzene evaporated during the reaction time. The reaction was cooled and washed repeatedly with diethyl ether to give a pale red powder. The product was identified by NMR as 1- (3-iodopropyl) -2,3,3-trimethylindolenium chloride (MW = 363.5). The yield was 20 g (87%).

Table 1 shows the chemical shifts of ¹ H-NMR in deuterated chloroform and the assignment of each peak.

[0046]

[Table 1]

(Example 2) (Synthesis of iodopropyl derivative) 4.4 g (12.10 mmol) of trimethylindolenium salt was dissolved in 200 ml of ethanol, and 1.81 g (12.10 mmol) of dimethylaminobenzaldehyde was added to the solution. Refluxed for 2 hours. The reaction product was cooled to room temperature, and the precipitated solid was filtered out and washed with ether. Recrystallized from ethanol to give 6.3g (MW = 49
4.7, 100%) iodopropyl derivative was obtained.

Table 2 shows the chemical shifts of ¹ H-NMR in deuterated chloroform and the assignment of each peak.

[0049]

[Table 2]

(Example 3) (Synthesis of norcocaine) 1 g (3.29 mmol) of cocaine was dissolved in a mixed solvent of 55 ml of acetonitrile and 110 ml of water, and 1 ml of acetic acid was added thereto to prepare a weakly acidic solution. .
To this solution, 42 ml of 1.06 g (7.55 mmol) potassium permanganate aqueous solution was added dropwise over 8 hours. After the dropping was completed, the mixture was stirred at room temperature overnight. The precipitate formed is filtered off,
Potassium carbonate was added to the filtrate to make a weakly basic solution. This solution was extracted with diethyl ether three times, and anhydrous sodium sulfate was added to the organic layer for drying. After the ether was distilled off under reduced pressure, silica gel thin layer chromatography (developing solvent:
Ammonia saturated chloroform), 536mg
We obtained Norcocaine (MW = 289.4, 56%).

Table 3 shows the chemical shifts of ¹ H-NMR in deuterated chloroform and the assignment of each peak.

[0052]

[Table 3]

Example 4 (Synthesis of CCIMC) 100 mg (0.20 mmol) of iodopropyl derivative and 292 mg (1.01 mmol) of norcocaine were added to 30 m.
It was dissolved in 1 l of chloroform and refluxed under a nitrogen stream for 8 hours. The reaction solution was cooled to room temperature and then added to about 1000 ml of ether with stirring. The solid formed was filtered and washed with ether to give 125 mg (MW = 655.5, 94%) of C.
I got CIMC.

Table 4 shows the chemical shifts of ¹ H-NMR in deuterated chloroform and the assignment of each peak.

[0055]

[Table 4]

(Example 5) (Measurement of change in fluorescence intensity of CCIMC) CCIMC (final concentration 7.09 × 10 ^-6 M) in PBS (phosphate buffer) solution 1487.
After taking 5 μl and stirring at 25 ° C for 1 minute, the excitation wavelength is 530 nm.
The fluorescence intensity at 590 nm was measured by. Its fluorescence intensity was 110.4. 114.3 μl of anti-CC antibody adjusted to a final concentration of 1 × 10 ⁻⁶ M was added to this solution, and the mixture was stirred at 25 ° C. for 1 minute, and then the fluorescence intensity was measured under the same conditions as above, and it was enhanced to 165.5.

Next, 150 μl of CC aqueous solution having a final concentration of 1.9 × 10 ⁻⁴ M was added to this solution, and the excitation wavelength was 530 nm.
The fluorescence intensity at 590 nm was measured by. The fluorescence intensity was 113.9. That is, it can be seen that the fluorescence enhancement due to the binding between the antibody and CCIMC was almost completely inhibited by the addition of CC.

Final concentration of CC was 7.71 × 10 ^-5 M, 1.94 × 10
^-5 M, 7.84 x 10 ^-6 M, 1.97 x 10 ^-6 M, 7.96 x 10 ^-7 M, 1.99 x
For 10 ⁻⁷ M, 7.93 × 10 ⁻⁸ M, 1.84 × 10 ⁻⁸ M, and 6.21 × 10 ⁻⁹ M, the fluorescence intensity was measured by the same method as above. Each fluorescence intensity is 116.6 (7.71 × 10 ^-5 M), 12
5.1 (1.94 x 10 ^-5 M), 135.2 (7.84 x 10 ^-6 M), 149.8 (1.97 x
10 ^-6 M), 156.5 (7.96 × 10 ^-7 M), 161.1 (1.99 × 10 ^-7 M),
162.8 (7.93 x 10 ^-8 M), 163.7 (1.84 x 10 ^-8 M), 165.1
(6.21 × 10 ^-9 M).

FIG. 5 is a CC detection curve obtained in one embodiment of the present invention. Also in this embodiment, such a characteristic curve could be obtained. (Example 6) (Synthesis of acetyl derivative) Synthesis of propyl 3-bromoacetate (3-Bromopropylacetate, BPA) Bromopropanol (BPO) 102.520 g (MW = 139.00, 0.737)
Acetic anhydride (Acetic unhydrid
e, AcU) 91.364g (MW = 102.99, 0.8871mole) benzene 5
The 0 ml solution was gradually added to avoid heat generation, and the mixture was stirred overnight at room temperature. Gas-liquid chromatography (GLC) confirmed that all bromopropanol had reacted. By washing the reaction solution with pure water five times, unreacted acetic anhydride was hydrolyzed and most of acetic acid was removed.
After drying the benzene layer for one day with magnesium sulfate,
The benzene was evaporated. Distilled, 66mmHg, 11
Fractions of 3 to 115 ° C were collected. Alkylation of 2,3,3-trimethylindolenine (TMI) TMI 43.9g (MW = 159.23, 0.2758mole) and BPA 50g (MW = 181.
3, 0.2758 mole) was mixed with 30 ml of benzene and heated under a nitrogen stream at 130 ° C. for 6 hours. Benzene evaporated during the reaction time. The reaction was tar-like upon cooling. By repeatedly washing the reaction product with diethyl ether, a pale red powder was obtained. The product is 1-
It was identified as (3-acetylpropyl) -2,3,3-trimethylindolenium bromide (APTMI, MW = 340.26). The yield was 81 g (0.2380 mole, 86.3%). Here, APTMI seems to have a strong bond with unreacted TMI, and the tar-like reaction product is difficult to purify. When a large amount is handled, each is transferred to a mortar and pulverized in ether for purification. Further, when the solidification is completed after the rough purification, it is dispersed in ether in a flask and subjected to ultrasonic waves for 0.5 to 2 hours for purification. The purity at this point is sufficient for synthesis. In the case of further purification for analytical purposes, etc., it was dissolved in a small amount of acetone and diluted with benzene. Synthesis of Acetyl Derivative APTMI of 9.9906 g (MW = 340.263, 0.0294 mole) was dissolved in 75 ml of ethanol with gentle heating to obtain a solution 1. 4.5994 g of p-dimethylaminobenzaldehyde (MW = 14
9.19, 0.0308 mole) was dissolved in 50 ml of ethanol with gentle heating to give a solution 2. When solutions 1 and 2 were mixed, reddish purple coloring was immediately observed at room temperature. After refluxing for 2 hours and then cooling overnight at room temperature, a purple solid precipitate was obtained. It was filtered and washed with benzene. The solid component was 9.7609 g. The product obtained from the NMR results could be identified as the acetyl derivative of the above formula (Formula 9). When the solvent was distilled off from the filtrate, it became a brown candy-like product. At this time, caution is required because bumping easily occurs due to high viscosity. It is presumed that the cause of such a candy-like substance that should originally be a solid is water, and it was solidified by vacuum drying at 60 ° C. in the presence of diphosphorus pentoxide. Then, it was dissolved in 50 ml of hot ethanol and left overnight at -20 ° C, and purple crystals appeared. After filtration in the same manner as above, it was washed with benzene. It was found that the present conditions could easily increase the purity of the product. The yield obtained by this operation was 4.16 g. The molecular weight of the acetyl derivative was 471.44, and the mass obtained by the two operations was 13.92 g, that is, 0.0295 mole, and the yield was 100%.

Table 5 shows ¹ H-NM in heavy chloroform.
The chemical shift of R and the attribution of each peak are shown.

[0061]

[Table 5]

(Example 7) (Synthesis of hydroxy derivative) acetyl derivative 7.6582 g (1
6.24 mmole) was dissolved in 500 ml of methanol. While stirring the solution, 3 ml (30 mmole) of 10 N sodium hydroxide was added, and the mixture was left at room temperature. The solution, which was initially dark purple, was mostly decolorized after a few minutes. When checked by liquid chromatography, it was confirmed that about 25% of the acetyl derivatives were unreacted after 0.5 hours, but all the acetyl derivatives were reacted after 1 hour. Just in case, total 2
After stirring for 5 hours, 5.1019 g (84.96 mmole) of acetic acid was added. The decolorized solution immediately returned to the original color.
Methanol and acetic acid were removed by evaporation. Chloroform was added to the near dry product and undissolved sodium acetate was filtered. The filtrate was evaporated again and the remaining deep purple paste-like product was ultrasonically washed with diethyl ether to give a purple solid. The final product was vacuum dried (60 ° C.) in the presence of diphosphorus pentaoxide. The yield is 5.
It was 7413g (MW = 429.5, 13.37mmole, 82.3%).

When the infrared absorption spectra (KBr tablet method) of the acetyl derivative and the hydroxy derivative, which are the raw materials in this reaction, were compared, the peak derived from the acetyl group (1700, 1240 cm ⁻¹ ) found in the acetyl derivative was found to be complete. Had disappeared. As a result, it was determined that this reaction product was the target HPIMC.

(Example 8) (Synthesis of methanesulfonic acid derivative) Hydroxy derivative
1.000 g (MW = 429.5, 2.33 mmole) and methanesulfonic anhydride 2.5377 g, (MW = 174.2, 14.57 mmole) were dissolved in 15 ml of chloroform and stirred at room temperature for 13 days. 3% with 3% saline
After repeated washing, the chloroform layer was dried over magnesium sulfate. After 20 days, magnesium sulfate was filtered, the solvent was distilled off, and the residue was washed with diethyl ether. Yield 0.6853g (MW = 507.4
9, 1.35mmole, 58.0%). When the raw hydroxy derivative was compared with the product of this reaction by HPLC, the retention time of HPLC was about 11 minutes and the retention time of this reaction product was about 17 minutes. It was also found at the same time that the reaction product did not contain any raw materials.

(Example 9) (Synthesis of MAIMC) Methanesulfonic acid derivative 55.1 mg
(MW = 507.49, 0.109mmole) and methamphetamine 133.8mg
(MW = 149.24, 0.897mmole) was dissolved in acetic acid 235.7mg, HPLC
The mixture was stirred at 80 ° C while monitoring the progress of the reaction. After the reaction for 2 hours, the retention time of the methanesulfonic acid derivative, which is the raw material, was about 17 minutes, but the reaction time was 10 minutes at 11 minutes.
% Reaction product was found. After the reaction for 17 hours, it was confirmed that all the methanesulfonic acid derivatives were converted into the product at the position of 11 minutes almost quantitatively. From the structures of the compounds of Examples 1 to 3 and the fact that this reaction proceeded with almost no side reaction, and the general reaction route of methanesulfonic acid ester and amine, this reaction product had the structure shown in Chemical formula 1. It was judged to be MAIMC. The product is under ultrasound,
After washing three times with diethyl ether, it was confirmed to be pure by HPLC. From the HPLC monitoring results, it can be said that the reaction yield was almost 100%.

(Example 10) (Measurement of change in fluorescence intensity of MAIMC) MAIMC (8.6
1600 μl of a PBS (phosphate buffer) solution (× 10 ⁻⁶ M) was taken and stirred at 25 ° C. for 1 minute, and then the fluorescence intensity at 590 nm was measured at an excitation wavelength of 530 nm. Its fluorescence intensity was 32.0. To this solution, 100 μl of anti-MA antibody adjusted to a final concentration of 6.1 × 10 ⁻⁶ M was added, and the mixture was stirred at 25 ° C. for 1 minute, and the fluorescence intensity was measured under the same conditions as above, and it was enhanced to 54.8. Then, ^add MA to this solution to a final concentration of 2.3 x 10 ^-5 M.
100 μl of the / PBS solution was added, and the fluorescence intensity at 660 nm was measured at an excitation wavelength of 600 nm. The fluorescence intensity was 31.0. That is, it is understood that the fluorescence enhancement due to the binding between the antibody and MAIMC was almost completely inhibited by the addition of MA.
Further, when the same experiment was performed and the same amount of PBS was added instead of the MA / PBS solution, the fluorescence intensity was 53.0 and no inhibition of fluorescence enhancement was observed. That is, it can be said that MA was detected by the labeling dye MAIMC of the present invention by an immunological method.

(Embodiment 11) In this embodiment, a mixed dye of a labeling dye represented by the above formula (Formula 1) and a labeling dye represented by the above formula (Formula 6) is used by using the detection apparatus shown in FIG. The change in fluorescence intensity due to the binding of the solution with the anti-MA antibody and the anti-CC antibody was examined. First, 1.25 ml of the dye solution was injected from the eluate dye container 1 through the eluate valve 4 into the eluent syringe pump 7. This dye solution contains a 6 × 10 ⁻⁷ M (mol / l) aqueous solution of the dye represented by the formula (Formula 1) and a 6 × 10 ⁻⁷ M aqueous solution of the dye represented by the formula (Formula 6). A mixture prepared by mixing each volume was used. After being left for several seconds, the valve 22 for the sample elution device was switched to store the eluate in the liquid reservoir 23. At this time, the reason that the eluate is once stored in the liquid reservoir 23 is to remove bubbles contained in the eluate. Then, 1.25 ml of the eluate was sucked into the dye solution syringe pump 9 through the switching valve 10 and the dye solution valve 6. After that, the switching valve 10 is switched, and the square cell 11 of the fluorescence detector 14 (JASCO, FP-821) is driven by the dye liquid syringe pump 9.
The eluate (1.10 ml) was injected into. Since the upper portion of the angular cell 11 is an open system and no back pressure is applied, quantitative injection could be performed. While stirring the solution by rotating the magnetic stirrer bar 12, the excitation wavelength is 600 nm and the fluorescence wavelength is 6 nm.
The fluorescence intensity of the eluate was measured at 60 nm, and the intensity was 6.
It was 44. After that, from the antibody solution container 2 through the antibody solution valve 5, 1 × into the antibody solution syringe type pump 8.
0.08 ml of 10 ⁻⁵ M anti-MA antibody solution was aspirated. The valve 5 for antibody solution is switched, and 0.08 ml of 1 × 10 ⁻⁵ M anti-MA antibody solution is injected into the square cell 11 of the fluorescence detector 14 (FP-821, manufactured by JASCO Corporation) from the syringe type pump 8 for antibody solution. did. Then, when the fluorescence intensity was measured under the same conditions as above, it was enhanced to 6.55. The same measurement was performed several times while increasing the amount of injected antibody.

After the completion of each measurement, the following washing operation was performed. The rotation of the magnetic stirrer bar 12 was stopped, the waste liquid pump 15 was operated, and the measurement liquid present in the square cell 11 was discarded into the waste liquid container 16 through the waste liquid port 11b shown in FIG. From the dye liquid container 3 through the dye liquid valve 6 into the dye liquid syringe type pump 9,
Aspirated ml. The dye liquid valve 6 is switched, and the square cell 11 of the fluorescence detector 14 is driven by the dye liquid syringe pump 9.
Then, 1.5 ml of the dye solution was injected, the waste liquid pump 15 was operated again, and the dye liquid existing in the square cell 11 was discarded into the waste liquid container 16. The inside of the square cell 11 and the inside of the tube were washed by repeating this operation a few times.

Next, the same measurement as above was carried out using a 1 × 10 ^-5 M anti-CC antibody solution, with an excitation wavelength of 530 nm and a fluorescence wavelength of 5.
The measurement was performed at 90 nm, and changes in fluorescence intensity due to the binding of the dye solution and the anti-CC antibody were measured. After the completion of each measurement, the same washing as above was performed.

Next, by the same operation as above, the anti-MA antibody and the anti-CC antibody were injected into the mixed dye solution, and the excitation wavelength was set to 600.
nm, and the fluorescence intensity of the eluate was measured at a fluorescence wavelength of 660 nm, and then a MA solution having a known concentration was temporarily injected to measure the fluorescence intensity change, and a characteristic curve similar to that of FIG. 5 was obtained.
After the completion of each measurement, the same washing as above was performed.

Further, the same measurement as above is performed by using the excitation wavelength 53.
After performing 0 nm and fluorescence wavelength 590 nm, CC solution of known concentration was temporarily injected into this, and the fluorescence intensity change was measured,
The characteristic curve was obtained. After the completion of each measurement, the same washing as above was performed.

In the actual work of detecting methamphetamine and the like, the filter after collection is set in the filter mounting portion 44 of the elution device 21, and the sample is eluted from the filter,
The presence and concentration of methamphetamine and the like in the eluate can be determined from the fluorescence intensity of the eluate with reference to the characteristic curve obtained in this example.

Further, when the excitation light of a plurality of wavelengths is irradiated, the emitted fluorescence is dispersed by the spectroscopic device built in the fluorescence detector, and then the fluorescence intensity is measured at the same time.

[0074]

As described above, the halogenated propyl derivative represented by the chemical formula (Formula 2) of the present invention easily reacts with dimethylaminobenzaldehyde to form a basic skeleton of a dye. Therefore, it becomes a useful precursor when synthesizing CCIMC represented by the chemical formula (Formula 1). Further, CCIMC composed of the merocyanine derivative represented by the chemical formula (Formula 1) can provide a labeled dye capable of detecting CC with high sensitivity. Further, the halogenated propyl derivative of the present invention and the novel method of synthesizing CCIMC have enabled the production of CCIMC as a labeling dye.

Further, the acetyl derivative represented by the above formula (Formula 9) of the present invention can be easily deprotected in an alkaline aqueous solution, and is a useful precursor in synthesizing the hydroxy derivative of the formula (Formula 8). Become a body. The hydroxy derivative represented by the above formula (Formula 8) can easily react with an acid anhydride to introduce an excellent leaving group in high yield. Therefore, it becomes a useful precursor when synthesizing the methanesulfonic acid derivative represented by the formula (Formula 7). Since the methanesulfonic acid derivative represented by the above formula (Formula 7) has an excellent leaving group, a methanesulfonyl group, it is a useful precursor for synthesizing the MAIMC represented by the above formula (Formula 6). Become a body.

Further, the MAIMC represented by the above formula (Formula 6) of the present invention is not easily affected by impurities and has high sensitivity and M
A labeled dye capable of detecting A can be provided. In addition, the novel method for synthesizing MAIMC of the present invention made it possible to produce MAIMC, which is a labeling dye. Further, according to the method of using the labeled dye of the present invention, since CCIMC or MAIMC is used as the immunization method, CC or MA can be detected with high sensitivity, being less affected by impurities.

Next, the apparatus for detecting MA, CC or a derivative thereof according to the present invention uses a sample solution prepared by dissolving a sample in a dye solution containing an antigen-labeling dye, and then adding anti-MA antibody or anti-CC to the sample solution.
A detection device for MA, CC or their derivatives, which comprises reacting an antibody and measuring the presence or absence or the concentration of MA, CC or their derivatives in the sample liquid from the change in the fluorescence intensity of the sample liquid, wherein the labeling dye is At least one selected from the labeling dyes represented by the formulas (Formula 1) and (Formula 6)
And a container for respectively storing the antibody solution containing two dyes and the antibody, an elution device for eluting the sample, and a reaction container for reacting the sample solution and the antibody solution And a supply means for supplying the antibody solution and the sample solution to the reaction container,
By providing at least a fluorescence detector for detecting the fluorescence intensity of the liquid to be measured in the reaction container, a detection device capable of quickly detecting MA, CC or their derivatives in the sample liquid is achieved. it can. Further, when the supply means is a syringe type pump and a valve, the liquid can be easily supplied from the storage container to the reaction container and can be easily detected. Further, when the reaction container is a rectangular cell having an inlet and an air vent hole in the upper part and an exhaust nozzle in the lower part, it is continuously different by controlling the opening / closing of the supply means and the exhaust nozzle. The sample can be easily measured. Further, when the reaction container is provided with a stirring means driven by magnetic force for stirring the sample solution, the dye solution and the antibody solution, the sample solution can be reacted quickly and the time required for the treatment can be shortened. Further, when the reaction container is equipped with a thermostatic device using a Peltier device, detection can be performed under a constant temperature environment. Further, if at least the storage device and the supply means are provided inside the air-conditioning type constant temperature device, it becomes possible to detect CC or the like under any temperature environment.

[Brief description of drawings]

FIG. 1 is a block diagram showing a cocaine / methamphetamine detection apparatus according to an embodiment of the present invention.

FIG. 2 is a perspective view of a reaction device mounted on a fluorescence detector of a detection device according to an embodiment of the present invention.

FIG. 3 (a) is a front view showing the configuration of a reaction device equipped with a Peltier element of a fluorescence detector of a detection device in one embodiment of the present invention, and FIG. 3 (b) is a side view thereof.

FIG. 4 is a perspective view of a sample elution device of a detection device according to an embodiment of the present invention.

FIG. 5 is a CC detection curve diagram obtained by using the detection apparatus according to the embodiment of the present invention.

[Explanation of symbols]

 1, 2, 3: Storage container 4, 5, 6: Valve 7, 8, 9: Pump 10: Switching valve 11: Reaction container 11a: Upper part of reactor 11b: Inlet port 11c: Waste liquid port 12: Magnetic stirrer bar 13: Magnetic stirrer 14: Fluorescence detector 15: Waste liquid pump 16: Waste liquid reservoir 17: Air conditioning constant temperature chamber 18: Air conditioning constant temperature device 19: Peltier element 20: Computer 21: Sample elution device 22: Sample elution device valve 23 : Liquid reservoir 24: Waste liquid valve 31: Aluminum block 32: Aluminum fin 33: Constant temperature device 41: Piston 42: O-ring 43: Injection / discharge port 44: Filter mounting part

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tadayasu Kobata 1006 Kadoma, Kadoma City, Osaka Prefecture Matsushita Electric Industrial Co., Ltd.

Claims (16)

[Claims] 1. A labeling dye comprising a merocyanine derivative represented by the following formula (Formula 1). [Chemical 1] 2. A halogenated propyl derivative represented by the following formula (formula 2), which is a precursor of a labeling dye comprising the merocyanine derivative represented by the formula (formula 1). [Chemical 2] 3. A propyl halide derivative represented by the above formula (Formula 2) is dissolved in an organic solvent, and norcocaine represented by the following formula (Formula 3) is added thereto and heated to obtain the above formula (Formula 1). A method for producing a labeled dye for synthesizing the indicated merocyanine derivative. [Chemical 3] 4. A trimethylindolenium salt represented by the following formula (Formula 4) and a dimethylaminobenzaldehyde represented by the following formula (Formula 5) are dissolved in an organic solvent and heated to produce the above formula (Formula 2). A method for producing a dye precursor for synthesizing the indicated propyl halide derivative. [Chemical 4] [Chemical 5] 5. A labeling dye comprising a methamphetamine derivative represented by the following formula (Formula 6). [Chemical 6] 6. A methanesulfonic acid derivative represented by the following formula (Formula 7). [Chemical 7] 7. A hydroxy derivative represented by the following formula (Formula 8). Embedded image 8. An acetyl derivative represented by the following formula (Formula 9). [Chemical 9] 9. A method for synthesizing a methamphetamine derivative of a dye, which comprises subjecting a methanesulfonic acid derivative represented by the formula (Formula 7) and methamphetamine to a substitution reaction in an acidic solvent. 10. The fluorescence intensity of fluorescence derived from a dye emitted from a buffer solution containing the labeling dye represented by the formula (Formula 1) and an anti-cocaine antibody, or the labeling dye represented by the formula (Formula 6) and Fluorescence intensity of fluorescence derived from a dye emitted from a buffer solution containing a methamphetamine antibody, and measuring the intensity change of the fluorescence intensity when the measurement target substance is added to the buffer solution, cocaine contained in the measurement target substance or A method of using a dye for detecting methamphetamine. 11. A sample solution prepared by dissolving a sample in a dye solution containing an antigen-labeling dye is reacted with an anti-methamphetamine antibody or an anti-cocaine antibody, and methamphetamine, cocaine or the like in the sample solution is determined from the change in fluorescence intensity of the sample solution. Is a detector for methamphetamine, cocaine, or a derivative thereof for measuring the presence or absence of the derivative or the concentration thereof, wherein the labeling dye is at least one selected from the labeling dyes represented by the formulas (1) and (6). A container for storing an antibody solution which is a dye and contains the dye solution and the antibody, an elution device for eluting the sample, and a reaction container for reacting the sample solution with the antibody solution. A supply means for supplying the antibody solution and the sample solution to the reaction vessel, and the measurement target in the reaction vessel A detection device for methamphetamine, cocaine, or a derivative thereof, comprising at least a fluorescence detector for detecting the fluorescence intensity of a liquid. 12. The detection device according to claim 12, wherein the supply means is a syringe type pump and a valve. 13. The detection device according to claim 12, wherein the reaction container is a prismatic cell having an inlet and an air vent hole in the upper portion and a discharge nozzle in the lower portion. 14. The detection device according to claim 12, further comprising stirring means driven by magnetic force for stirring the sample solution, the dye solution and the antibody solution in the reaction container. 15. The detection device according to claim 12, further comprising a thermostat using a Peltier device in the reaction container. 16. The detection device according to claim 12, wherein the storage device and the supply means are provided at least inside the air conditioning constant temperature device.