JP2009143821A - Reagent for delivatizing fluorescensce and method for derivatizing fluorescensce - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reagent and a method for derivatizing many kinds of amine- and amino acid-associated substances existing in a sample. <P>SOLUTION: The reagent and method for derivatizing the fluorescensce can derivatize various kinds of amine- and amino acid-associated substances in the sample by using the fluorescensce derivatizing reagent expressed by chemical formulae [I] or [II], and at the same time, adsorbs and removes the un-reacted reagent released from a fluorous tag. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a fluorescent derivatization reagent and a fluorescent derivatization method, and more specifically, a fluorescent derivatization reagent having a fluorous tag, a fluorescent derivatization method using the same, and separation measurement of amines or amino acid-related substances in a sample The present invention relates to a method and a method for removing residual unreacted reagents.

  A biological sample contains many biological components including amines and amino acid-related substances. In the analysis of a biological component contained in a very small amount in a biological sample, it is required to analyze with high sensitivity and high accuracy using a very small amount of sample. Therefore, various analytical instruments and analytical methods have been adopted. Among them, high-performance liquid chromatography (HPLC) is one of the useful separation / analysis methods that can measure multiple components simultaneously. Widely used in various fields such as medicine, food and environment. The detection by HPLC is based on the physical and chemical properties of the substance to be analyzed, and detectors such as absorbance, differential refractive index, electrochemistry, mass spectrometry, fluorescence, and chemiluminescence are used. These detection methods are suitably selected according to the type and state of the sample to be measured, the chemical properties and concentration of the analysis target substance, and the like. However, these detection methods can be used when the substance to be measured shows a strong response to each measuring device, but it can be measured, but it does not show a sufficient response for the measurement or does not show any response at all. Cannot be measured as is.

  If the response of the analyte is insufficient or does not respond to these detection methods, a substance that shows a strong response to the detector by a chemical reaction or the like so that the analyte can be measured. It is necessary to convert it into, for example, a fluorescent derivative so as to emit fluorescence. Such conversion to a fluorescent derivative is called so-called fluorescent derivatization (labeling or labeling), and a reagent used for the conversion is called a derivatization (labeling) reagent. Such derivatization is a particularly useful technique for analyzing a substance to be analyzed which is a trace amount of a target component present in a sample composed of a complex matrix.

  In recent years, development of this derivatization method and derivatization reagent has progressed, and some are being used on a daily basis. Among these, fluorescent derivatization is widely used because of its high sensitivity and high selectivity, and the fluorescence method has become the mainstream of derivatization. This fluorescence detection method is a method for detecting fluorescence emitted from a target component that has been irradiated with light of a specific wavelength (excitation light) and absorbed. Since only a small part of the fluorescent material emits the light energy as fluorescence, this fluorescence detection method has higher selectivity and higher sensitivity than other detection methods.

  Further, in most of such fluorescent derivatization reagents, a reaction site for labeling a target substance and a fluorescent site involved in fluorescence generation coexist in the same molecule. Many fluorophores constituting such fluorescent sites have been developed (Non-Patent Documents 1, 2, and 3). However, conventional fluorescent derivatization methods and fluorescent derivatization reagents have their merits and demerits, and further development of highly sensitive and highly selective fluorescent derivatization methods and fluorescent derivatization reagents is required.

Therefore, in order to solve these problems, development of a new fluorination derivatization method using a non-fluorescent reagent and special fluorescence phenomena such as fluorescence polarization, time-resolved fluorescence, fluorescence resonance energy transfer, excimer fluorescence Attempts have been made to develop a derivatization method incorporating the above. Among them, the excimer fluorescence derivatization method is an excitation dimer (excimer) formed by bringing together polycyclic aromatic fluorescent molecules such as pyrene in close proximity and absorbing excited light to form excited states. ) Is a fluorescence derivatization method in which the excimer fluorescence phenomenon in which light is emitted is introduced into derivatization. In this excimer fluorescence derivatization method, it is possible to spectrally distinguish between a derivative into which one luminophore is introduced and a plurality of derivatives into which one luminophore is introduced per molecule of the target substance, which has been difficult with the conventional derivatization method. (Non-Patent Document 4).

However, most of the fluorescent derivatization reagents reported so far have a problem in that detection of the target component is hindered because fluorescence is emitted also from the unreacted reagent itself remaining in excess. Moreover, although the reagent itself is non-fluorescent and a fluorescent derivatization reagent in which only the amine derivative emits fluorescence is commercially available, the type of fluorophore that can be used is limited because it uses the special luminescence mechanism of the fluorophore. Yes, it was not always satisfactory for practical use.
Ohkura, Y., et al., J. Chromatogr. B, 659,85-107 (1994) Yamaguchi, M., et al., "Reagentsfor FluorescenceDetection", ed. By Toyooka, T /, John Wiley and Sons Ltd., New York, 1999, pp. 99-166 Fukushima, K., et al., J. Pharm. Biomed. Anal., 30, 1655-1687 (2003) Hideyuki Yoshida, YAKUGAKU ZASSHI 123 (8) 691-696 (2003)

Therefore, the present inventors have conducted intensive research on fluorescent derivatization reagents and fluorescent derivatization methods in which amines and amino acid-related substances can be specifically analyzed and unreacted reagent peaks do not appear on the chromatogram. F-trap with no reagent peak detected on gram
The present invention was completed by finding a fluorescent derivatization method based on the new concept.
Furthermore, the present inventors synthesized F-trap derivatives as derivatization reagents for amines based on the new concept of F-trap, and confirmed that the F-trap derivatives do not interfere with HPLC.

Accordingly, an object of the present invention is to provide a fluorescent derivatization reagent having a fluorescent moiety, an amine reaction site, and a fluoro moiety called a fluorous tag.
As used herein, the term “fluorous” is a coined word meaning “parent fluorocarbon”, and “fluorous tag” is a labeled moiety having a region rich in fluorine bonds in the molecule. Means.

  A preferred embodiment of the present invention is represented by the general formula [I]:

Wherein Rf represents a fluoroalkyl group completely or partially substituted with a linear or branched fluorine atom having 4 to 10 carbon atoms, and n is 0 or 1 to 6 Means an integer)
Or general formula [II]:

（式中、Ｒ_１は炭素原子数１ないし１０個の直鎖状もしくか分岐状のアルキル基またはアルコキシ基を意味し、Ｒｆおよびｎは前記と同じ意味を有する）
で表される多環化合物である蛍光誘導体化試薬を提供することを目的としている。

(Wherein R ₁ represents a linear or branched alkyl group or alkoxy group having 1 to 10 carbon atoms, and Rf and n have the same meaning as described above)
It aims at providing the fluorescence derivatization reagent which is a polycyclic compound represented by these.

  In a further preferred embodiment of the present invention, the chemical structural formula [Ia]:

で表されるF-trap ピレンまたは化学構造式［IIａ］：

F-trap pyrene represented by the chemical structural formula [IIa]:

で表されるF-trap クマリンであることからなる蛍光誘導体化試薬を提供することを目的としている。

It aims at providing the fluorescence derivatization reagent which consists of F-trap coumarin represented by these.

  In another aspect, the present invention provides a method for derivatizing amines or amino acid-related substances using the above fluorescent derivatization reagents [I], [II], [Ia] or [IIa], or amines Alternatively, an object of the present invention is to provide a method for measuring amines or amino acid-related substances, comprising separating and measuring amino acid-related substances.

  Furthermore, the present invention selectively removes excess unreacted reagent with a fluorinated adsorbent when amines and amino acid-related substances in a sample are derivatized using a fluorescent derivatization reagent or derivatization method. It is an object of the present invention to provide a removal method comprising:

  In order to achieve the above object, the present invention provides a fluorescent derivatization reagent having a fluorescent moiety, an amine reaction site, and a fluoro moiety called a fluorous tag.

  As a preferred embodiment of the present invention, the general formula [I]:

（式中、Ｒｆは炭素原子数４ないし１０個の直鎖状もしくか分岐状のフッ素原子で完全にもしくは部分的に置換されたフルオラスアルキル基を意味し、ｎは０または１ないし６の整数を意味する）
または一般式［II］：

Wherein Rf represents a fluoroalkyl group completely or partially substituted with a linear or branched fluorine atom having 4 to 10 carbon atoms, and n is 0 or 1 to 6 Means an integer)
Or general formula [II]:

（式中、Ｒ_１は炭素原子数１ないし１０個の直鎖状もしくか分岐状のアルキル基またはアルコキシ基を意味し、Ｒｆおよびｎは前記と同じ意味を有する）
で表される多環化合物である蛍光誘導体化試薬を提供する。

(Wherein R ₁ represents a linear or branched alkyl group or alkoxy group having 1 to 10 carbon atoms, and Rf and n have the same meaning as described above)
A fluorescent derivatization reagent which is a polycyclic compound represented by the formula:

  As a further preferred embodiment of the present invention, the chemical structural formula [Ia]:

で表されるF-trap ピレンまたは化学構造式［IIa］：

F-trap pyrene represented by the chemical structure [IIa]:

で表されるF-trap クマリンであることからなる蛍光誘導体化試薬を提供する。

A fluorescent derivatization reagent comprising the F-trap coumarin represented by:

  Another object of the present invention is to provide a method for measuring amines or amino acid-related substances, which comprises separating and measuring amines or amino acid-related substances using the fluorescent derivatization reagent as another embodiment. Yes.

  Furthermore, as another form of the present invention, when an amine or an amino acid-related substance in a sample is derivatized by using a fluorescent derivatization reagent or a derivatization method, excess remaining unreacted reagent is adsorbed by fluorinated adsorption. A method for selective removal by an agent is provided.

  The fluorescent derivatization reagent and the fluorescent derivatization method according to the present invention include a fluorescent derivatization reagent in which amines and amino acid-related substances contained in a sample such as bioactive amines, amino acids, and peptides remain in the sample. It has a great effect that it can be separated and detected with high sensitivity and comprehensiveness without receiving unreacted interference.

  The fluorescent derivatization reagent according to the present invention comprises a fluorescent site, an amine reaction site, and a fluoro site that is a fluorous tag.

Specifically, the fluorescent derivatization reagent of the present invention is:
General formula [I]:

（式中、Ｒｆは炭素原子数４ないし１０個の直鎖状もしくか分岐状のフッ素原子で完全にもしくは部分的に置換されたフルオラスアルキル基を意味し、ｎは０または１ないし６の整数を意味する）
または一般式［II］：

Wherein Rf represents a fluoroalkyl group completely or partially substituted with a linear or branched fluorine atom having 4 to 10 carbon atoms, and n is 0 or 1 to 6 Means an integer)
Or general formula [II]:

（式中、Ｒ_１は炭素原子数１ないし１０個の直鎖状もしくか分岐状のアルキル基またはアルコキシ基を意味し、Ｒｆおよびｎは前記と同じ意味を有する）
で表される多環化合物である。

(Wherein R ₁ represents a linear or branched alkyl group or alkoxy group having 1 to 10 carbon atoms, and Rf and n have the same meaning as described above)
It is a polycyclic compound represented by these.

  More specifically, the fluorescent derivatization reagent of the present invention has the chemical structural formula [Ia]:

で表されるF-trap ピレンまたは化学構造式［IIa］：

F-trap pyrene represented by the chemical structure [IIa]:

で表されるF-trap クマリンである。

It is F-trap coumarin represented by.

  The derivatization reagents [Ia] and [IIa] according to the present invention are, for example, 1-pyrenebutyric acid or 7-methoxycoumarin-4-acetic acid, 4- (3,3,4,4,5,5,6, Using 6,7,7,8,8,9,9,10,10,10-heptadecafluorodecylthio) phenol, N, N'-diisopropylcarbodiimide and N, N-dimethylaminopyridine (DMAP) In the presence of a solvent such as tetrahydrofuran, it can be synthesized under conditions such as room temperature.

  Further, in the fluorescent derivatization method according to the present invention, the fluorescent derivatization reagent of the present invention is, for example, the following chemical reaction formula [Ib]:

（式中、Ｒ_２は炭素原子数１ないし２０個の直鎖状もしくは分岐状アルキル基を意味し、Ｒ_１、Ｒｆならびにｎは前記と同じ意味を有する）
または下記化学反応式 [IIb]：

(Wherein R ₂ represents a linear or branched alkyl group having 1 to 20 carbon atoms, and R ₁ , Rf and n have the same meaning as described above)
Or the following chemical reaction formula [IIb]:

（式中、Ｒ_１、Ｒ_２、Ｒｆならびにｎは前記と同じ意味を有する）
に従って、試料中のアミン類またはアミノ酸関連物質のアミノ基と反応して、試料中のアミン類またはアミノ酸関連物質が誘導体化され疎フッ素性の蛍光性アミン誘導体を生成すると同時に、フルオラスタグであるフッ素性部位が脱離して親フッ素性のフルオラスタグ含有反応副生成物が生成する。

(Wherein R ₁ , R ₂ , Rf and n have the same meaning as described above)
The amines or amino acid-related substances in the sample react with the amino group in the sample to produce a fluorophobic fluorescent amine derivative at the same time as the fluorinated fluorescent amine derivative. The chemical site is eliminated and a fluorous tag-containing reaction by-product is produced.

  The fluorescent derivatization reagent thus obtained and the derivatization method using the same can derivatize amines and amino acid-related substances in a sample under mild conditions, and simultaneously with the derivatization, fluorous. Remove the tag. Since the fluorous tag is thus detached, only the unreacted reagent has the fluorous tag. Therefore, the compound having a fluorous tag can be easily and selectively separated by adsorption with an adsorbent such as a commercially available fluorinated silica gel. As a result, since the fluorescent derivatization reagent and derivatization method of the present invention can selectively derivatize only amines and amino acid-related substances, the amines and amino acid-related substances can be selectively separated with high sensitivity.・ Can be analyzed.

Examples of amines and amino acid-related substances that can be derivatized with high sensitivity and high selectivity by the fluorescent derivatization reagent and the derivatization method according to the present invention include, for example, ethylamine (C ₂ ), propyl Amine (C ₃ ), n-butylamine (C ₄ ), n-pentylamine (C ₅ ), n-hexylamine (C ₆ ), n-heptylamine (C ₇ ), n-octylamine (C ₈ ), n-nonylamine (C ₉ ), n-decylamine (C ₁₀ ), n-aminoundecane (C ₁₁ ), n-dodecylamine (C ₁₂ ), n-tetradecylamine (C ₁₄ ), n-hexadecylamine ( C ₁₆ ) and the like, and examples of amino acid-related substances include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, and histidine. , Isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, valine and the like.

(Synthesis of F-trap pyrene)
The synthesis scheme of F-trap pyrene is shown in the following chemical reaction formula [Ic]. 1-Pyrenebutyric acid (288
mg, 1 mmol) and 4- (3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecylthio) phenol (286 mg, 0.5 mmol) in 7 mL dehydrated tetrahydrofuran (THF), N, N'-diisopropylcarbodiimide (155 μL, 1 mmol), and N, N-dimethylaminopyridine (DMAP) (61 mg, 0.5 mmol) was added and stirred at room temperature for 20 hours. After adding 100 mL of ethyl acetate to the reaction solution, it was washed with 30 mL of 0.1 M hydrochloric acid and water three times each. The ethyl acetate layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography (developing solvent; hexane: ethyl acetate = 4: 1), and white powdered F-trap pyrene [4- (3,3,4,4,5,5,6,6,7 , 7,8,8,9,9,10,10,10-heptadecafluorodecylthio) phenyl 4- (pyren-1-yl) butanoate] (236 mg, 56.1% yield).

The analysis results of the nuclear magnetic resonance spectrum of the obtained F-trap pyrene were as follows.
¹ H-NMR (600
MHz, CDCl ₃ ) δ 2.32
(2H, m), 2.69 (2H, t, J = 7.0 Hz), 3.08 (2H, t, J = 8.2 Hz), 3.49 (2H, t, J = 7.6
Hz), 7.03 (2H, d, J = 4.6 Hz), 7.36 (2H, d, J = 4.6 Hz), 7.88 (1H, d, J = 7.6 Hz),
7.99 (1H, t, J = 7.6 Hz), 8.03 (4H, s), 8.12 (1H, d, J = 4.6 Hz), 8.16 (1H, d, J = 6.7
Hz), 8.31 (1H, d, J = 9.2 Hz).

(Synthesis of F-trap coumarin)
The synthesis scheme of F-trap coumarin is shown in the following chemical reaction formula [IIc]. 7-methoxycoumarin-4-acetic acid (117 mg, 0.5 mmol) and 4- (3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10 , 10-Heptadecafluorodecylthio) phenol (286 mg, 0.5 mmol) dissolved in 7 mL of dehydrated tetrahydrofuran (THF), N, N'-diisopropylcarbodiimide (93 μL, 0.6 mmol), and DMAP (61 mg 0.5 mmol), and stirred at room temperature for 3 hours. After adding 100 mL of ethyl acetate to the reaction solution, it was washed with 30 mL of 0.1 M hydrochloric acid and water three times each. The ethyl acetate layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography (developing solvent; hexane: ethyl acetate = 2: 1) and white powdered F-trap coumarin [4- (3,3,4,4,5,5,6,6,7 , 7,8,8,9,9,10,10,10-heptadecafluorodecylthio) phenyl 2- (7-methoxy-2-oxo-2H-chromen-4-yl) acetate] (99 mg, yield 25.1%).

The analysis results of the nuclear magnetic resonance spectrum of the obtained F-trap coumarin were as follows.
¹ H-NMR (600
MHz, CDCl ₃ ) δ 2.39
(2H, m), 3.08 (2H, t, J = 7.9 Hz), 3.10 (2H, s), 3.95 (3H, s), 6.33 (1H, s), 6.87
(1H, s), 6.88 (1H, d, J = 8.9 Hz), 7.02 (2H, d, J = 8.9 Hz), 7.35 (2H, d, J = 8.9 Hz),
7.56 (1H, d, J = 8.9 Hz).

(Analysis of amine derivatization with F-trap pyrene)
Add 1.4 mL of pyridine and 400 μL of a 5 mM F-trap pyrene THF solution to 200 μL of the amine solution as the test sample, and heat at 90 ° C. for 1 hour. Add 100 μL of the reaction solution to a fluorous solid-phase extraction cartridge (F-SPE) (2 g, manufactured by Fluorous Technologies). The cartridge was washed with 4 mL of water, eluted with 4 mL of 80% (v / v) methanol, and 20 μL thereof was injected into the HPLC. The HPLC analysis conditions are as follows. Column: COSMOSIL 5C18-AR-II (inner diameter 4.6
mm x length 15 cm, manufactured by Nacalai Tesque), mobile phase: 80% (v / v) acetonitrile (0-10 min), acetonitrile-water-THF (8: 1: 1, v / v) (10- Gradient elution at 40 min), flow rate 1.0 mL / min, fluorescence detection: excitation wavelength 345 nm, fluorescence wavelength 375 nm.

13 types of aliphatic amines (ethylamine (C ₂ ), propylamine (C ₃ ), n-butylamine (C ₄ ), n-pentylamine (C ₅ ), n-hexylamine (C ₆ ), n-heptylamine (C ₇ ), n-octylamine (C ₈ ), n-nonylamine (C ₉ ), n-decylamine (C ₁₀ ), n-aminoundecane (C ₁₁ ), n-dodecylamine (C ₁₂ ), n- FIG. 4 shows a chromatogram when tetradecylamine (C ₁₄ ) and n-hexadecylamine (C ₁₆ )) are derivatized and analyzed according to the above-described procedure.

Within 40 minutes, fluorescence peaks of all 13 amine derivatives were detected. At this time, a peak of the unreacted reagent was slightly observed around 26 minutes, but it did not interfere with separation and quantification of the amine derivative. On the other hand, FIG. 4 shows a chromatogram when the same operation was performed except that the fluorous solid phase extraction operation was not performed. Unlike FIG. 3, a large unreacted reagent peak appeared around 26 minutes, greatly hindering the separation and quantification of n-tetradecylamine (C ₁₄ ). From the above results, it was shown that only the unreacted reagent can be selectively removed by the fluorous solid phase extraction operation. The removal rate of the unreacted reagent was calculated by the fluorous solid phase extraction operation from the ratio of the peak areas in the chromatograms of FIGS. At this time, at least 99.95% or more of the unreacted reagent was removed.

  Table 1 shows the removal rate of F-trap pyrene when the composition of the eluate used for the fluorous solid-phase extraction was changed. By combining a fluorinated solvent (methanol, acetonitrile, dimethylformamide, etc.) and a phophophobic solvent (water, dimethylsulfoxide), only the unreacted reagent is adsorbed in the solid-phase extraction cartridge with a maximum removal rate of 99.97% or more. It was possible.

(Analysis of amine derivatization with F-trap coumarin)
Add 200 mL of amine solution (200 μL) as a test sample to 20% pyridine in acetonitrile (1.4 mL) and 5 mM F-trap coumarin in THF (400 μL) and heat at 90 ° C. for 10 minutes. Add 100 μL of the reaction solution to a fluorous solid-phase extraction cartridge (F-SPE) (2 g, manufactured by Fluorous Technologies). The cartridge was washed with 4 mL of water, eluted with 4 mL of 70% (v / v) acetonitrile, and 20 μL thereof was injected into the HPLC. The HPLC analysis conditions are as follows. Column: COSMOSIL 5C18-AR-II
(Inner diameter 4.6 mm x length 15 cm, manufactured by Nacalai Tesque), mobile phase: gradient elution of acetonitrile aqueous solution (0-30 min) (40-90% (v / v)), flow rate 1.0 mL / min, fluorescence detection : Excitation wavelength 345 nm, fluorescence wavelength 375 nm.

13 types of aliphatic amines (ethylamine (C ₂ ), propylamine (C ₃ ), n-butylamine (C ₄ ), n-pentylamine (C ₅ ), n-hexylamine (C ₆ ), n-heptylamine (C ₇ ), n-octylamine (C ₈ ), n-nonylamine (C ₉ ), n-decylamine (C ₁₀ ), n-aminoundecane (C ₁₁ ), n-dodecylamine (C ₁₂ ), n- FIG. 5 shows a chromatogram when tetradecylamine (C ₁₄ ) and n-hexadecylamine (C ₁₆ )) are derivatized and analyzed according to the above operation.

Within 40 minutes, fluorescence peaks of all 13 amine derivatives were detected. At this time, a peak of unreacted reagent was slightly observed at around 32 minutes, but it did not interfere with separation and quantification of the amine derivative. On the other hand, FIG. 6 shows a chromatogram when the same operation is performed except that the fluorous solid phase extraction operation is not performed. Unlike FIG. 5, a peak derived from the reagent at 25 minutes and a large unreacted reagent peak appeared at around 32 minutes, greatly hindering the separation and quantification of n-tetradecylamine (C ₁₂ ). From the above results, it was shown that only the unreacted reagent can be selectively removed by the fluorous solid phase extraction operation. The removal rate of the unreacted reagent was calculated by the fluorous solid phase extraction operation from the ratio of the peak areas of the chromatograms of FIGS. At this time, it was found that at least 99.99% or more of the unreacted reagent was removed.

  Table 2 shows the removal rate of F-trap coumarin when the composition of the eluate used for fluorous solid phase extraction was changed. Combining a fluorinated solvent (methanol, acetonitrile, dimethylformamide, etc.) and a fluorophobic solvent (water, dimethyl sulfoxide), adsorbs only unreacted reagents in a solid-phase extraction cartridge with a removal rate of 99.99% or more. .

  The fluorescent derivatization reagent and fluorescent derivatization method according to the present invention can separate and detect bioactive amines, amino acids, peptides and other amines and amino acid-related substances contained in a sample. It can be used in fields such as food and environment.

The figure which shows the chromatogram which shows the derivatization analysis result of 13 types of aliphatic amines by F-trap pyrene (Example 3). The figure which shows the chromatogram which compared the case where the fluorous solid-phase extraction operation was not performed in the derivatization analysis operation of 13 types of aliphatic amines by F-trap pyrene (Example 3). The figure which shows the chromatogram which shows the derivatization analysis result of 13 types of aliphatic amines by F-trap coumarin (Example 4). The figure which shows the chromatogram which compared the case where a fluorous solid-phase extraction operation was not performed in the derivatization analysis operation of 13 types of aliphatic amines by F-trap coumarin (Example 4).

Claims (7)

  A fluorescent derivatization reagent comprising a fluorescent site, an amine reaction site, and a fluoro moiety. The fluorescent derivatization reagent according to claim 1, wherein the fluorescent derivatization reagent has the general formula [I]:

Wherein Rf represents a fluoroalkyl group completely or partially substituted with a linear or branched fluorine atom having 4 to 10 carbon atoms, and n is 0 or 1 to 6 Means an integer)
Or general formula [II]:

(Wherein R ₁ represents a linear or branched alkyl group or alkoxy group having 1 to 10 carbon atoms, and Rf and n have the same meaning as described above)
A fluorescent derivatization reagent, which is a polycyclic compound represented by the formula: The fluorescent derivatization reagent according to claim 1 or 2, wherein the fluorescent derivatization reagent is:
Chemical structural formula [Ia]:

F-trap pyrene represented by the chemical structural formula [IIa]:

A fluorescent derivatization reagent characterized by being an F-trap coumarin represented by: General formula [I]:

Wherein Rf represents a fluoroalkyl group completely or partially substituted with a linear or branched fluorine atom having 4 to 10 carbon atoms, and n is 0 or 1 to 6 Means an integer)
Or general formula [II]:

(Wherein R ₁ represents a linear or branched alkyl group or alkoxy group having 1 to 10 carbon atoms, and Rf and n have the same meaning as described above)
Is reacted with amines or amino acid-related substances,
Reaction formula [Ib]:

(Wherein R ₂ represents a linear or branched alkyl group having 1 to 20 carbon atoms, and R ₁ , Rf and n have the same meaning as described above)
Or chemical formula [IIb]:

(Wherein R ₁ , R ₂ , Rf and n have the same meaning as described above)
A fluorescent derivatization method, characterized by derivatizing according to The fluorescent derivatization method according to claim 4, wherein the fluorescent derivatization reagent comprises:
General formula [IIa]:

F-trap pyrene represented by the general formula [IIa]:

A fluorescent derivatization method characterized by being an F-trap coumarin represented by:   Using the fluorescent derivatization reagent according to any one of claims 1 to 3 or the derivatization method according to claim 4 or 5 to derivatize and separate and measure amines and amino acid-related substances in a sample. A method for measuring amines and amino acid-related substances in a sample characterized by   Using the fluorescent derivatization reagent according to any one of claims 1 to 3 or the derivatization method according to claim 4 or 5, when an amine or an amino acid-related substance in a sample is derivatized, an excess amount is obtained. A removal method, wherein the remaining unreacted reagent is selectively removed with a fluorinated adsorbent.

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