JPH112606A - Chirality analysis method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve the detecting sensitivity in chirality analysis by introducing a fluorescent functional group into the material having chirality, exciting the material with right and left circularly polarized lights respectively and measuring the intensity of the respective fluorescence. SOLUTION: A flurescent functional group is introduced into the material having chirality. It is excited with right and left circularly polarized lights. The intensity of the respective fluorescence is measured. The chirality of this material is analyzed by the fluorescence intensity difference information of the left circular polarized-light excitation and the right circular polarized-light excitation. That is to say, the fluorescent functional group is introduced in the material having the chirality such as physiological active material by using a chemical means, and the fluorescent material is obtained. Then, circular bicolor fluorescent spectrum or fluorescence detecting circular bicolor spectrum is measured, and the analysis of the chirality of the sample material as the object is performed. In this case, the fluorescent functional group is not specially limited, but it is desieble that the groups contain a naphthalene ring, an anthrascene ring, a pyrene ring, a perylene ring, a coronene ring and a porphyrin ring.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chirality analysis method, and more particularly to an improvement in measurement sensitivity.

[0002]

2. Description of the Related Art Many physiologically active substances such as drugs, toxic substances, and functional biological components existing in a living body have so-called chirality.
It is widely known that a substance strongly depends on an absolute structure, a three-dimensional structure, or a three-dimensional configuration of a substance. For example, the R form of asparagine, one of the amino acids, is sweet, while the S form is bitter. The (+) form of estrone functions as a female hormone, but the (-) form does not. Thalidomide R
It is well known that the body has a hypnotic effect but the S body has a teratogenic effect.

[0003] Therefore, search for these physiologically active substances,
In studies of synthesis, metabolism, action, and the like, it has been considered extremely important to understand the chirality of the substance. However, optical isomers that differ only in this chirality
In general, the chemical properties are the same or very similar. Therefore, the means for grasping and analyzing the chirality is a single crystal X
It is limited to line structure analysis or chirooptical spectroscopy (general term such as circular dichroism spectrum (CD) and optical rotatory dispersion spectrum (ORD)). Among them, CD
Has been widely used for this kind of research because the sample can be measured in a solution state and the measurement operation is relatively easy.

[0004]

However, the CD, which is the difference between the left-handed circularly polarized light absorption and the right-handed circularly polarized light absorption exhibited by the sample material, has the following two limitations in application. In order for an appropriate CD spectrum to be observed, the target sample molecule needs to contain a chromophore that is a source of appropriate light absorption. In the case of a substance containing no chromophore, no appropriate CD spectrum is observed, so that it cannot be applied.

[0005] On the other hand, although ORD is observed even in a sample without CD, the ORD spectrum in such a case is generally monotonous and is not very useful for structural analysis. Conversely, if there is a CD, the ORD spectrum is more complex in shape than the CD, although the information is only equivalent to the CD.
Moreover, the sensitivity is low. Therefore, ORD does not compensate for the limitations of CD. The sensitivity of the CD is 1/100 even in the case of the optimum sample, usually 1/1000 to 1/1000 compared to the light absorption itself.
It is as low as 0, and a relatively high concentration of the measurement sample is required to make up for it.

However, it is generally difficult to collect a large amount of a target physiologically active substance, and a great deal of effort has been made to secure a large amount of sample in measurement and analysis. In order to reduce such efforts and to open up research fields that could not be undertaken because it is difficult to collect a large amount of samples, it is desired to improve the sensitivity of measurement means in chirality analysis. . The present invention has been made in view of the above prior art, and has as its object to greatly improve the detection sensitivity in chirality analysis.

[0007]

In order to achieve the above object, a chirality analysis method according to the present invention comprises introducing a fluorescent functional group into a substance having chirality, exciting it with left and right circularly polarized light, It is characterized in that the fluorescence intensity is measured, and the chirality of the substance is analyzed from the difference information of the fluorescence intensity between the left circularly polarized light excitation and the right circularly polarized light excitation. Further, in the present invention, the left and right circularly polarized light is irradiated by switching at substantially the same wavelength, and the irradiation wavelength is sequentially changed, and the fluorescence intensity difference between left circularly polarized light excitation and right circularly polarized light excitation is correlated with the wavelength. It is preferable to obtain a spectrum and analyze the chirality of the substance from the spectrum.

[0008]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will be described below with reference to the drawings. In the present invention, as described above, a fluorescent functional group is introduced into a substance having chirality, such as a physiologically active substance, using a chemical means to form a fluorescent substance. The color spectrum is measured and used for chirality analysis of the target sample substance. Here, the fluorescent functional group is not particularly limited, but a naphthalene ring,
Anthracene ring, pyrene ring, perylene ring, coronene ring,
A group containing a porphyrin ring or the like has high fluorescence intensity and high symmetry of itself, and the electronic state and the direction of the transition moment of absorption / fluorescence have been sufficiently studied, which is convenient for later analysis.

Circular dichroic fluorescence excitation spectrum refers to the measurement of the fluorescence intensity when the sample is excited by alternately irradiating the sample with monochromatic left and right circularly polarized light, and when the sample is excited with the left circularly polarized light,
The difference in fluorescence intensity when excited by the right circularly polarized light is recorded with respect to the wavelength. The fluorescence detection circular dichroism spectrum is obtained by dividing the difference between the fluorescence intensities by the average fluorescence intensity with respect to the wavelength.

When these spectra are collected, monochromatic light sequentially wavelength-scanned by a spectroscope is linearly polarized, and then introduced into a photoelastic modulator, and left and right circularly polarized light is modulated according to the modulation frequency. Preferably, it is generated and irradiated on the sample. The sensitivity of these two spectra is the conventional C
Compared with D and ORD, the sensitivity is one to three orders of magnitude higher, and the desired sensitivity improvement is realized. In addition, by these methods, a spectrum reflecting the chirality of a substance originally having no chromophore can be obtained, and measurement and analysis can be performed.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a more specific embodiment of the present invention will be described with reference to the drawings. 2R, 3R-butanediol Although this 2R, 3R-butanediol is not a physiologically active substance, it is a model substance selected in order to confirm the effects of the present invention, and has a structure as shown in FIG.

The CD spectrum and the ORD spectrum of this substance are shown in FIGS. CD spectrum measurement conditions Cell length: 10 mm Concentration: 0.1% Solvent: H ₂ O Measurement temperature: room temperature Range: 600-200 nm Band width: 1.0 nm Integration: twice Response: 2 seconds Scanning speed: 50 nm / min

ORD spectrum measurement conditions Cell length: 10 mm Concentration: 0.8% Solvent: H ₂ O Measurement temperature: room temperature Range: 600-200 nm Band width: 1.0 nm Integration: twice Response: 2 seconds Scanning speed: 50 nm / min No CD spectrum is observed since this molecule does not contain a chromophore. The ORD spectrum is also monotonous and is not useful for structural analysis.

Therefore, the O contained in this butanediol is
Using H group, react with β-naphthoyl chloride,
A naphthyl group, which is a fluorescent functional group, is introduced. Then, this reagent undergoes a condensation reaction with 2R, 3R-butanediol to give 2R, 3R-bisnaphthoylbutanediol (FIG. 4).

FIG. 5 shows the CD spectrum of a 1.78 ppm acetonitrile solution of this sample. The introduced chromophore causes the chromophore to have a CD, obtains an exciton splitting type spectral structure, and enables analysis of the chirality of the molecule. CD spectrum measurement conditions Cell length: 10 mm Concentration: 0.000178% Solvent: CH ₃ CN Measurement temperature: room temperature Range: 350-190 nm Band width: 1.0 nm Integration: twice Response: 2 seconds Scanning speed: 50 nm / min

Further, this sample was diluted to 1/100 to 17.8 ppb, and its CD spectrum, fluorescence detection CD
The measured spectrum and circular dichroic fluorescence excitation spectrum are shown in FIGS. Fluorescence detection CD spectrum measurement conditions Concentration: 1.78 × 10 ^-6 % Solvent: CH ₃ CN Measurement temperature: room temperature Range: 310-190 nm Band width: 5.0 nm Integration: 6 times Response: 2 seconds Scanning speed: 50 nm / min

Fluorescence excitation CD spectrum measurement conditions Concentration: 1.78 × 10 ⁻⁶ % Solvent: CH ₃ CN Measurement temperature: room temperature Range: 310-190 nm Band width: 5.0 nm Integration: 6 times Response: 2 seconds Scanning speed: 50nm / min

As is clear from FIG. 6, the CD spectrum has the same size as the noise and is difficult to discriminate, but the fluorescence detection CD spectrum (FIG. 7) and the circular dichroic fluorescence excitation spectrum (FIG. 7). According to FIG. 8), sufficient S /
Observed with N ratio. The sensitivity ratio of the circular dichroism fluorescence excitation spectrum to CD calculated in this example is about 20 times. As described above, when the present invention is applied, spectral information that reflects chirality can be obtained with a sensitivity that could not be expected in the past with respect to a substance that did not originally have a chromophore and could not measure a CD spectrum. If the sensitivity is not sufficient even if a CD can be observed due to the presence of a chromophore, application of the present invention can improve the sensitivity by an order of magnitude.

Further, in the present invention, the chirality of the sample substance is observed with fluorescence having wavelength selectivity higher than that of light absorption. For example, if there are multiple asymmetric centers in a molecule and CD measurement is performed as it is, it is difficult to determine which asymmetric center is reflected in the obtained CD band, and analysis Is often difficult. Even in such a case, when a fluorescent functional group is introduced at a position that reflects the structure of the chiral center whose chirality is to be examined, and the spectrum of the present invention is measured, the spectrum is determined by the high selectivity of fluorescence detection. It is simple, and it is possible to obtain local structural information, and it is possible to analyze the structure of a substance molecule with positional selectivity.

Example of Introduction of Fluorescent Functional Group In order to chemically introduce a fluorescent functional group, a relatively chemically active functional group of a compound to be introduced is used. For example, a hydroxyl group (-OH), a carboxyl group (-CO
OH) can be introduced by utilizing an esterification reaction. Amino group is (-NH _2), OPA and fluorosilicone fluorescamine, 5-dimethylamino-1-naphthalenesulfonyl chloride (aka dansyl chloride)
Reactions such as can be used.

As one example of the present invention, a procedure for introducing a naphthoyl group into two 1,2-OH groups of a sample 1,2-dihydroxycyclohexane by esterification will be described. 1. A glass reaction vessel (5 ml eggplant type flask) dried with warm air was prepared, and 5 mg of the dried sample was placed therein.
The air inside is replaced with an inert gas (argon gas). 2.5 day dry catalyst in a desiccator over phosphorus oxide (0.1 equivalent of 4-dimethylaminopyridine; 0.53 mg) and activator (4 equivalent of silver trifluoromethanesulfonate; 44 mg) in the glass container.

3. 0.5 ml of refluxed pyridine is added using a glass syringe, and the mixture is stirred and dissolved with a magnetic stirrer. 4. 66 mg of 2-naphthoyl chloride as a derivatization reagent
(4 equivalents), wrap the whole container with aluminum foil, shield from light, and react at room temperature with stirring for 10 hours. 5. After completion of the reaction, add one drop of distilled water, stir for 30 minutes,
Decompose excess derivatizing reagent. 6. Remove the aluminum foil, add 2 ml of benzene and stir vigorously.

7. Add 1 tablespoon of anhydrous sodium sulfate. This is passed through a column of anhydrous sodium sulfate and eluted with benzene to remove water and solids. 8. The benzene is removed by evaporation using a rotary evaporator,
Further, the pressure is reduced by a vacuum pump to remove pyridine. 9. 1 ml of hexane / ethyl acetate (2: 1) is added to the solid to form a suspension, which is applied to an alumina column and eluted with 2 ml of ethyl acetate to remove acidic components. Ten. The eluate is evaporated to dryness, dissolved in 0.5 ml of chloroform, and purified by thin-layer chromatography on silica gel using benzene / ethyl acetate (9: 1) as a developing solution.

Next, examples of introducing fluorescent functional groups into various substances will be described. Introduction Example 1, 1,2-trans-cyclohexane-dio
As shown in FIG. 9, the 1,2-trans-cyclohexane-diol (a) was reacted with naphthoyl chloride (Naph-Cl), and the 1,2-trans-cyclohexane-diol bisnaphthoate (a- 1)
Anthroyl chloride (Anth-C
l) and reacting with 1,2-trans-cyclohexane-
A method for preparing diol bisanthroate (a-2),
6-methoxynaphthoyl chloride (MeONaph-
Cl) to give 1,2-trans-cyclohexane-diol bis-6-methoxy-2-naphthoate (a-3).

Introduction Example 2 5-Cholestane As shown in FIG. 10, 5-cholestane (b) is reacted with anthroyl chloride (Anth-Cl) to give 5-cholestane bisnaphthoate (b-2). Method, 1-pyrene carbonyl chloride (Py-C
1) to give 5-cholestanebispyrenecarboxylate (b-4). Introduction Example 3 Ouagegenin As shown in FIG. 11, for ouagegenin (c),
Reacting with naphthoyl chloride (Naph-Cl),
Oubergenin 1,3,19-trinaphthoate (c-
1) and the like.

[0026]

As described above, according to the chirality analysis method of the present invention, a fluorescent functional group is introduced into a substance having chirality, and information on the difference in fluorescence intensity between left circularly polarized light excitation and right circularly polarized light excitation is obtained. Since the chirality is analyzed, it is possible to significantly improve the sensitivity.

[Brief description of the drawings]

FIG. 1 shows 2R, 3 used as a sample in one embodiment of the present invention.
It is explanatory drawing of the three-dimensional structure of R-butanediol.

FIG. 2 is a CD spectrum diagram of the 2R, 3R-butanediol.

FIG. 3 is an ORD spectrum diagram of the 2R, 3R-butanediol.

FIG. 4 shows 2R, 3 used as a sample in one embodiment of the present invention.
2R, 3 which is a modified fluorescent functional group of R-butanediol
It is explanatory drawing of the three-dimensional structure of R-bis-naphthoylbutanediol.

FIG. 5 is a CD spectrum diagram of 2R, 3R-bis-naphthoylbutanediol (1.78 ppm).

FIG. 6 is a CD spectrum diagram of 2R, 3R-bis-naphthoylbutanediol (17.8 ppb).

FIG. 7 is a fluorescence detection CD spectrum of 2R, 3R-bis-naphthoylbutanediol (17.8 ppb).

FIG. 8 is a diagram showing a circular dichroic fluorescence excitation spectrum of 2R, 3R-bis-naphthoylbutanediol (17.8 ppb).

FIG. 9 is an explanatory diagram of an example of introducing a fluorescent functional group into 1,2-trans-cyclohexane-diol.

FIG. 10 is an explanatory diagram of an example of introduction of a fluorescent functional group into 5-cholestane.

FIG. 11 is an explanatory diagram of an example of introduction of a fluorescent functional group into ouabagenin.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Koji Nakanishi United States 10027 New York, New York Riverside Drive 560 (72) Inventor Nina Verova United States 10027 New York, New York 119 Street West 400 (72) Inventor Tong Founding United States 11737 New York Elmhurst Province 42 Avenue 94-16

Claims (2)

[Claims] 1. A fluorescent functional group is introduced into a substance having chirality, and the fluorescent functional group is excited with left and right circularly polarized light to measure the respective fluorescence intensities. A chirality analysis method characterized by further analyzing the chirality of the substance. 2. The method according to claim 1, wherein the left and right circularly polarized lights are illuminated while being switched at substantially the same wavelength, and the irradiation wavelengths are sequentially changed, so that the fluorescence intensity of the left circularly polarized light excitation and the right circularly polarized light excitation is changed. A chirality analysis method characterized by obtaining a spectrum by correlating the difference with a wavelength and analyzing the chirality of the substance from the spectrum.