JPS63295548A - Novel pyrene derivative and high-performance liquid chromatography using said derivative as labeling agent - Google Patents

Abstract

NEW MATERIAL:Pyrene-1-carbonylnitrile of formula I. USE:A fluorescent labeling agent for high-performance liquid chromatography. For example, it is extremely useful for the high-sensitivity analysis of a minor component of living body such as bile acid or corticoid. PREPARATION:Pyrene-1-carboxylic acid is converted into an acid halide according to conventional method, e.g. by treating with a halogenation agent such as oxalyl chloride. The objective compound of formula I can be produced by reacting the acid halide with trialkylsilylcyanide in the presence of a catalyst such as zinc iodide or aluminum chloride. Both reaction processes are carried out in a solvent (e.g. benzene) at room temperature or under slight heating for several hr-10hr. It is essential to use an organic solvent absolutely free from alcohols because the presence of even the slightest amount of alcohols in the system inhibits the reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel pyrene derivative useful as a labeling agent (derivatizing agent) for high performance liquid chromatography (HPLC).

[Background of the invention]

High performance liquid chromatography (HPLC) with fluorescence detection
) It is extremely useful in the analysis of trace components in living organisms such as bile acids, steroid hormones, and prostaglandins, and various fluorescent labeling agents have been proposed and put into practical use. However, when it comes to fluorescent labeling agents for alcoholic hydroxyl groups, all of the ones known so far have advantages and disadvantages, and they are excellent enough to satisfy all four reactivity, sensitivity, and stability characteristics. So far, not many agents have been discovered.

Among the fluorescent labeling agents with alcoholic hydroxyl groups known so far, the one with excellent reactivity, sensitivity, and stability is 1 or 9-
Anthroylnitrile is a typical example. However, even with these compounds, it cannot be said that the sensitivity is still sufficiently high, and the emergence of a fluorescent labeling agent for alcoholic hydroxyl groups with even higher sensitivity is awaited.

[Purpose of the invention]

The present invention was made in view of the above-mentioned situation, and is extremely useful in, for example, highly sensitive analysis of trace biological components such as bile acids and corticoids by HPLC.

The object of the present invention is to provide a novel fluorescent labeling agent for alcoholic hydroxyl groups with high detection sensitivity.

[Structure of the invention]

The pyrene-1-carbonylnitrile represented by the formula of the present invention and the pyrene-1-carbonylnitrile of the present invention can be easily synthesized, for example, according to the following synthetic route.

That is, first, pyrene-1-carboxylic acid is treated according to a conventional method with an oxalyl chloride or the like to give an acid 9-dendecide, which is then treated with zinc iodide, aluminum chloride, or the like. The desired pyrene-1-carbonylnitrile can be easily obtained by reacting with trialkylsilylcyani r (eg, trimethylsilyl cyanide, triethylsilyl cyanide IF kJ ) in the presence of a catalyst. The reaction in both steps is usually carried out in a solvent commonly used in this type of reaction, such as benzene, dichloromethane, dichloroethane, or chloroform, but the presence of even a trace amount of alcohol in the system can interfere with the reaction. So,
An essential requirement is that the solvent be an organic solvent that does not contain any alcohols. Further, as is common practice, these organic solvents must be used in an anhydrous state. The reaction temperature for both steps is usually room temperature or slightly warmed, and the reaction time varies slightly depending on the reaction temperature and the reaction reagent used, but usually several hours to 10 hours is sufficient for both steps. It is. The amounts of reaction reagents, catalysts, solvents, etc. to be used may be appropriately determined according to common knowledge regarding chemical reactions. After the reaction, post-treatment may be performed according to a conventional method, and if necessary, purification may be carried out by recrystallization with an appropriate recrystallization solvent.

Pyrene-1-carboxylic acid is used as a raw material.

For example, J, Am, Chem, Sac, 63.249
4 (1941), pyrene and acid anhydride are subjected to a Friedel-Crafts reaction to obtain 1-acylpyrene.
em, Soe, 78, 17] 6 (1956)
Since it can be easily obtained by oxidizing with hypochlorous acid according to the method described in , it is sufficient to use the product obtained in this way.

The thus obtained pyrene derivative of the present invention is extremely useful as a fluorescent labeling agent for alcoholic hydroxyl groups in HPLC analysis, and is useful for labeling bile acids, which are trace components in living organisms, for example.
It is used for various purposes such as Colchik 41 and other hypothermic steroids (P) (as a highly sensitive labeling agent in PLO analysis).

Regarding the method of labeling a test sample such as steroid r using the pyrene derivative of the present invention, examples of the solvent include acetonitrile and chloroform.

Using an aprotic organic solvent such as benzene or ethyl acetate, an organic base such as triethylamine, trimethylamine, or tertiary alkylamine such as quinuclidine is added to the solvent in an amount of about 0.05 to 2%. A test sample is dissolved, the pyrene derivative of the present invention is added thereto, and the mixture is allowed to react for several tens of minutes at room temperature or, if necessary, with heating (50 to 70°C).

During the labeling reaction, it is desirable to react the pyrene derivative of the present invention, which is a labeling agent, in an excess amount to the test sample in order to cause the test sample to react sufficiently. That is, even if the pyrene derivative of the present invention is not particularly removed after the reaction, the fluorescence of the reagent itself is extremely weak and the fluorescence properties are different, so it will not affect the fluorescence analysis of the test sample.

The pyrene derivative of the present invention has a 7th alcoholic hydroxyl group,
It reacts specifically with secondary alcoholic hydroxyl groups and phenolic hydroxyl groups. However, secondary alcoholic hydroxyl groups with steric hindrance (for example, the 11α position of steroid r, 1
It hardly reacts with the 7β position, axial hydroxyl group) or tertiary alcoholic hydroxyl group.

In this way, the test sample labeled with the pyrene derivative of the present invention remains in the reaction solution during the labeling reaction.

or other solvents (e.g. ethyl acetate, chloroform).

After being dissolved in benzene, etc.), and further subjected to treatments such as washing with water and drying if necessary, it is subjected to IPLC. The HPLC operating method, measurement conditions, etc. can be carried out according to conventional methods.
Detection is of course carried out using a fluorometer.

When a hydroxyl group is labeled with the pyrene-1-carbonylnitrile of the present invention, the fluorescence wavelength is 420 nm and the maximum excitation wavelength is 370 nm.

When HPLC analysis was performed using the pyrene derivative of the present invention as a labeling agent and steroid P as a test sample, the detection sensitivity was 8 pII (isoane P rotosterone) in a normal phase system.
In addition, it is 500 fg (S/N = 10 X3β-hydroxy-5-cholenoincidence ratio) in the interpreter system, and both are far superior to conventional ones.

The present invention will be explained in more detail with reference to Examples below.
The present invention is not limited in any way by these Examples.

〔Example〕

Example 1. Synthesis of pyrene-1-carbonylnitrile Pyrene-1-carboxylic acid 200 myt Hensen anhydride 2
0WLl! oxideryl chloride r11
1 Lt was added thereto, and the mixture was stirred and reacted at room temperature for 4 hours. After the reaction, the reaction mixture was concentrated under reduced pressure to remove benzene and excess oxychloride P to obtain a yellow residue containing acid chloride L and 0. Dissolve this in 20 WLl of anhydrous benzene and
．． 5 m/ml and 1 inch of zinc iodide were added, and the mixture was stirred and reacted at room temperature for 6 hours. The reaction wave solvent was distilled off, and the residue was recrystallized from hexane-acetone to obtain 136 d of orange needle-like crystals of pyrene-1-carbonylnitrile. Yield 65.6%, mp189
~191°C Elemental analysis value: Cl8H7N.

Calculated value (@c, 84.69; H, 3,55; N, 5.4
9 actual measurements (C, 84, 47; H, 3, 28; N, 5.
40°IR CHC an-': 2240 (C=N
), 1675 (C=O).

ix MS m/z: 255 (M”), 229 (
IBM-CN:) 勺, 201 ([M-C0CN″l+)
.

Example 2. Labeling of hydroxysteroid type 1 with pyrene-1-carbonylnitrile Add 1 μl of hydroxysteroid to 1% triethylamine/
It was dissolved in 100 μl of acetonitrile solution, 100 μyt of pyrene-1-carbonylnitrile was added thereto, and the mixture was reacted at 25° C. for 30 minutes. After the reaction, the mixture was dissolved in ethyl acetate, washed with water, and then subjected to HPLC to determine the production rate (esterification rate) of the derivative.

(HPLC device and measurement conditions) Device: Quarters 6000A (manufactured by Waters Association) Detector: 650-10LC fluorescence detector (manufactured by Hitachi, Ltd.) (Ex: 350 nm, E!m: 42
Set to 0nm. ) Column: Coamosil 5SL
5μm (4+mφxt5crn) Mobile phase: Hexane/ethyl acetate (100/~lO0/75) Mobile phase flow rate:
2 mfmin.

Results> Table 1 shows the results of investigating the relative reactivity of 10 types of hydroxysteroids with pyrene-1-carbonylnitrile.
Shown below.

In the table, a stands for axial and e stands for equatorial.

qe is quashie cattorial, prim is first class, p
As is clear from Table 1, hen is pyrene-1 of the present invention.
-Carbonyl nitrile reacts quantitatively with primary hydroxyl groups, and also reacts well with equatorial secondary alcoholic hydroxyl groups and phenolic hydroxyl groups, but sterically hindered secondary alcoholic hydroxyl groups (11α-position , 17β-position and axial hydroxyl group).

Actual example 3. Labeling of hydroxysteroids with -1-carbonylnitrile (using quinuclidine as the organic base) (1) 3β-hydroxy-5-cholenoinacid P
(3β-0H-Δ5)10n, 9 is quinuclidine 160
Acetonitrile 200μ containing μ#! and add 200μ9 of pyrene-1-carbonylnitrile to it.
The mixture was heated to 60° C., and the derivative production rate at 5, 10, 15, 20, and 30 minutes was measured by fluorescence detection HPLC.

(HPLC conditions) (The device and detector are the same as in Example 2.) Column: Co
smogfl 5C18 Mobile phase: 0.1% potassium phosphate buffer (pH 7,0)/methanol (1/15) Mobile phase flow rate: 1.5 mVmin Detection: Maximum excitation wavelength 350 nm Fluorescence wavelength 420 nm Note that after derivatization IJ) Pyrenoyl derivative of cholic acid 5
3β-0H was added to serve as an internal standard, and the peak height ratio was determined.
- The derivative production rate of Δ5 was calculated. FIG. 1 shows a time course showing the relationship between reaction time and reaction rate.

As is clear from Figure 1, the reaction was completed within 10 minutes,
It was found that the fluorescent ester was produced quantitatively.

(2) Dissolve 1 μl of methyl lithochlate in 200 μl of 0.08% quinuclidine/acetonitrile solution, add 100 μl of pyrene-1-carbonylnitrile to
The reaction was carried out at 0°C, and the production rate of the derivative was measured by fluorescence detection HPLC in the same manner as above.

Results> The reaction was completed in 5 minutes, and the secondary hydroxyl group of methyl cholate was quantitatively converted into a fluorescent ester.

From the above results, it can be seen that when quinuclidine is used as an organic base in the labeling reaction of hydroxyl groups with male non-1-carbonyl nitrile, the reaction rate is significantly increased.

Example 4. Detection limit 3β-0H-Δ5 according to method (1) of Example 3
-(1-birenoyl) derivatives and 3-(1-anthroyl) derivatives (1-anthroylnitrile was used instead of pyrene-1-carbonylnitrile. - Comparative example) were prepared, and each was detected by fluorescence detection HPLC. I asked for limits.

<HPLC conditions> (The device and detector are the same as in Example 2.) i) 3- (1
-birenoyl) derivative column: Cosmosil 5C18 mobile phase: 0.1
% potassium phosphate buffer (PH7,0)/methanol (
1/15) Mobile phase flow rate: 1.5 mVmin Detection: Maximum excitation wavelength 350 nm Fluorescence wavelength 420 nm Sample: 4 p# 1i) 3-(1-Anthroyl) derivative column: 1)
Same mobile phase as: 0.1 tyric acid weak solution (pH 7,0)/methanol (1/9) Mobile phase flow rate: 1.5*vmin Detection: Maximum excitation wavelength 370 nm Fluorescence wavelength 470 nm Sample: 50 p&Results> The detection limit of the 3-(1-pyrenoyl) derivative is S/N
= 5 and the detection limit for the 3-(1-anthroyl) derivative was the same (4βg at S/N = 5).

That is, the measurement sensitivity of 3β-0H-Δ5 by HPLC when the pyrene-1-carbonylnitrile of the present invention is used as a labeling agent is higher than that when using the existing labeling agent 1-anthroylnitrile. It turns out that it is more than 10 times higher.

Example 5. Analysis of bile acids by derivatized HPLC Using the pyrene-1-carbonylnitrile of the present invention as a labeling agent, the hydroxyl groups of various bile acids were labeled, and then HPLC
Separation measurements were carried out using C. The obtained liquid chromatogram L is shown in FIG. 2. In the figure, Aid cholic acid, B the peaks of ursodeoxycholic acid, C the peaks of chenodeoxycholic acid, and D the peaks of deoxycholic acid, respectively.

However, the I(PLO conditions are as follows.

(The device and detector are the same as in Example 2.) Column: Co
smosil 5C18 mobile phase: 0.3% potassium phosphate buffer (p) 16.0)/methanol ('/6) Mobile phase flow rate: 1.8 mVmin As is clear from Figure 2, the pyrene-1 of the present invention - Derivatization using carbonyl nitrile as a labeling agent) I
Various bile acids can be separated and measured extremely well using PLC.

Example 6. Analysis of bile acids by derivatized HPLC As in Example 5, three types of bile acids each having one hydroxyl group were separated and measured by derivatized HPLC using pyrene-1-carbonylnitrile as a labeling agent. The obtained liquid chromatogram is shown in FIG.

In the figure, E indicates norlithocholic acid, F indicates lithocholic acid, and G indicates the peak of 3β-0H-Δ5.

However, the HPLC conditions were the same as in Example 3.

As is clear from FIG. 3, the derivatized HPLC of the present invention using pyrene-1-carbonylnitrile as a labeling agent allows very effective separation and measurement of various bile acids having one hydroxyl group.

〔Effect of the invention〕

As described above, the present invention provides a novel pyrene derivative useful as a fluorescent labeling agent for HPLC and derivatized HPLC using the same as a labeling agent. By using it as a hydroxyl group, it can be used for HPLC analysis of various compounds with hydroxyl groups, such as bile acids, hydroxysteroids such as steroid P hormone, and prostagranone, which are trace components in the body. This has had an extremely significant effect in that it has made it possible to perform detailed analysis.

[Brief explanation of the drawing]

FIG. 1 shows the time course of the derivatization reaction of 3β-hydroxy-5-cholenoic acid (3β-0H-Δ5) with pyrene-1-carbonylnitrile obtained in Example 3 (1), The derivatization reaction rate (− value) at each reaction time (A) on the horizontal axis is connected to the points plotted along the vertical axis. The liquid chromatogram obtained in Example 6 is shown. However, A is cholic acid, B
indicates ursodeoxycholic acid, c indicates chenodeoxycholic acid, D indicates deoxycholic acid, E indicates norlithocholic acid, F indicates lithocholic acid, and G indicates the peak of 3β-0H-Δ5, respectively. Patent applicant: Wako Pure Chemical Industries, Ltd. Figure 2 (minutes)

Claims (2)

[Claims] (1) Formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ Pyrene-1-carbonylnitrile represented by (2) High-performance liquid chromatography characterized by using pyrene-1-carbonylnitrile represented by the formula ▲ Numerical formulas, chemical formulas, tables, etc. ▼ as a labeling agent for hydroxyl groups.