JPH06179653A - Naphthalimide compound and labeling agent using the same - Google Patents

Abstract

PURPOSE:To obtain a new naphthalimide compound, capable of separating and determining an optically active amine agent, having a strong fluorescent intensity and useful as a labeling agent for an amino acid or amines. CONSTITUTION:This naphthalimide derivative of formula I (R is H or lower alkyl; X is halogen), e.g. a compound of formula II. The compound of formula II is obtained by reacting 2,3-naphthalenedicarboxylic anhydride with glycine and then reacting the resultant intermediate compound with cyanuric fluoride. This compound is useful as a labeling agent and capable of deriving a fluorescent derivative from an amino acid or amines, fractionating and separating the resultant derivative with a high sensitivity by the fluorometry. When R is the alkyl group; the compound has optical activity and can be used as a reagent for converting optically active amines into a diastereomeric derivative to rapidly separate and determine the optically active amines by the high- performance liquid chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a naphthalimide derivative used for fluorescence detection of an organic compound having an amino group (hereinafter referred to as “the compound of the present invention”).
And a use of the compound of the present invention as a labeling agent in high performance liquid chromatography. Labeling here means fluorescence labeling and diastereomeric derivatization. The above-mentioned compound of the present invention is a novel substance which has not been published in the literature and is useful as a fluorescent labeling agent for amino acids and amines. When R is an alkyl group, it has optical activity and can be used as a diastereomer derivatization reagent for optically active amines, and the optically active amines can be rapidly separated and quantified by high performance liquid chromatography. As a fluorescent labeling agent for amines, 7-methoxycoumarin-3-carbonyl fluoride in which fluorinated coumarin is bound with an acid fluoride as a functional group has been known. (For example, Fujino et al., Analytical Science Vol. 6 465-466 1990, Analyt.
ical Sciences 6 , 465-466 1
990. The detection limit of amine of this known compound is 100.
Although reported as femtomoles, optically active amines cannot be separated and quantified. The present inventors have conducted extensive studies to develop a labeling agent capable of separating and quantifying optically active amines with higher fluorescence intensity, and as a result, a chiral acid halide having a naphthalimide group has the object. The present invention has been completed based on this finding. That is, the present invention adds the reagent of the present invention to a sample to be tested containing an amino acid or an amine, introduces the amino acid or the amine into a fluorescent derivative, and conducts a fluorescence measurement to quantify with high sensitivity and its application. Regarding The structural formulas of the compounds obtained by this study are exemplified below. Next, the present invention will be described in more detail by way of examples. Example 1, Synthesis of compound (II). The synthetic scheme is shown in FIG. 5 g of 2,3-naphthalenedicarboxylic acid anhydride, 1.9 g of glycine, 0.4 ml of triethylamine and 200 ml of dehydrated xylene are placed in a 500 ml eggplant-shaped flask equipped with a Dean-Stark apparatus and heated in an oil bath for 2 hours. After cooling, the precipitate is filtered by a 3G glass filter and recrystallized from an ethanol-water mixed solvent to obtain an intermediate (I) with a yield of 83%. 300m
0.127 g of the intermediate (I), 30 μl of cyanuric fluoride, 45 μl of dehydrated pyridine and 250 ml of dehydrated acetonitrile are placed in a l-shaped eggplant-shaped flask, and reacted for 2 hours while stirring with a magnetic stirrer while flowing argon at room temperature. After distilling off acetonitrile under reduced pressure, the obtained white solid is recrystallized from n-hexane. Melting point 186-
The compound (II) of 189 degreeC was obtained. Yield 10% Example 2 Synthesis of compound (III). The synthetic scheme is shown in FIG. In a three-necked flask equipped with a condenser, 11.9 g of thionyl chloride and compound (I)
(2.55 g) and dehydrated pyridine (50 μl) are taken, reacted for 4 hours in an argon stream, and thionyl chloride is distilled off under reduced pressure. The obtained solid matter is heated and dissolved in 50 ml of dehydrated benzene and recrystallized. White crystals with a melting point of 211-214 ° C. were obtained. Yield 34% Example 3 Synthesis of compound (V) The synthetic scheme is shown in FIG. In a 300 ml eggplant flask equipped with a Dean-Stark apparatus, 5 g of 2,3-naphthalenedicarboxylic acid anhydride and 2.25 of L-alanine were added.
g, triethylamine 0.35 ml, dehydrated toluene 20
Take 0 ml and heat in an oil bath for 1 hour. After cooling, the precipitate is filtered off with a glass filter and recrystallized from an ethanol-water mixed solvent. Intermediate (IV) is obtained with a yield of 56%. Intermediate (IV) in a 200 ml eggplant-shaped flask
0.592 g, 120 μl of cyanuric fluoride, 180 μl of dehydrated pyridine, and 80 ml of dehydrated acetonitrile are taken, and reacted for 2 hours in an argon stream at room temperature while stirring with a magnetic stirrer. Acetonitrile is distilled off under reduced pressure, and the obtained white solid is recrystallized from n-hexane. Melting point 195-197 ° C, yield 15% Example 4 100 μm of aliphatic amines (C ₁₀ -C ₁₆ ) dissolved in acetonitrile in a 2 ml plastic tube
l (10 ⁻⁴ M), derivatization reagent (II) 200 μl
(10 ⁻⁴ M), triethylamine 250 μl (10
^-3 M) are taken in this order, reacted at room temperature for a certain period of time, and then 10 μl is injected into HPLC. Using a mixed solvent of acetonitrile / water (9: 1) as the eluent,
Fluorescence detection is performed at ex = 259 nm and λem = 384 nm. The obtained chromatogram is shown in FIG. As shown here, 2 long-chain alkylamines from decylamine (C ₁₀ ) to dodecylamine (C ₁₂ ), tetradecylamine (C ₁₄ ), hexadecylamine (C ₁₆ ) are used.
Good separation was possible within 0 minutes. Detection limit is 4
High sensitivity with femtomole. Example 5 50 μl of DL-phenylethylamine (10 ⁻⁵⁾ dissolved in acetonitrile in a 2 ml plastic tube.
M), 50 μl of triethylamine (2.5 × 10
^-4 M), 50 µl of the reagent (V) of the present invention (2 x 10
^-4 M) respectively, and after reacting for 5 minutes, 50 μl (5 × 10 ⁻³ M) of ethylamine is added and reacted for 5 minutes at room temperature. Inject 10 μl of the reaction solution into the HPLC. The obtained chromatogram is shown in FIG. The two optical isomers could be well baseline separated.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a synthetic scheme of compounds (II) and (III). FIG. 2 is a synthetic scheme of compound (V). FIG. 3 is an HPLC chromatogram of aliphatic amines. FIG. 4 is a chromatogram showing that DL-phenylethylamine can be separated and quantified.

Claims (1)

[Claims] 1) General formula A naphthalimide derivative represented by the formula (wherein R is hydrogen or a lower alkyl group, and X is a halogen group). 2) The naphthalimide derivative according to claim 1, wherein R is hydrogen and X is chlorine. 3) R is hydrogen and X is fluorine.
The naphthalimide derivative according to the item. 4) The naphthalimide derivative according to claim 1, wherein R is a methyl group and X is fluorine. 5) General formula A labeling agent comprising a naphthalimide derivative represented by the formula (wherein R is hydrogen or a lower alkyl group, and X is a halogen group).