JPH11228550A - Isothiocyanate compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound useful as a fluorescent labeling reagent enabling the accurate judgment of the kind and configuration of an amino acid constituting a fine amount of a peptide compound. SOLUTION: A compound of formula I [R is (substituted) phenyl], for example, a compound of formula II. The compound of formula II among the compound of formula I is obtained by reacting a compound of formula III with thiophenol in the presence of triethylamine in acetone, converting the nitro group of the obtained product into an amino group, subjecting the reaction product to other reactions and finally reacting the reaction product with CSCl2 under thermal refluxing. Since the new compound can separate an optically active amino acid, the compound is useful for determining the configuration of the amino acid in a peptide compound containing the D-amino acid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel isothiocyanate compound useful as a fluorescent labeling reagent.

[0002]

2. Description of the Related Art In the field of biotechnology, it is very important to rapidly determine the amino acid sequence of peptide compounds such as polypeptides and oligopeptides. As a method for determining the amino acid sequence of a peptide compound, Edman (Edm) using phenylisothiocyanate (PITC) is used.
an) Decomposition method is known (Edman, P., Acta Chim. Sc
and., 4, 283, 1950; Edman, P., Acta Chim. Scand.,
10, 761, 1956; Edman, P. and Begg, G., Eur. J. Bioc
hem., 1, 80, 1967). The Edman degradation method involves labeling only the amino terminal residue of the peptide compound and removing it as a phenylthiohydantoin derivative, separating the derivative by high performance liquid chromatography (HPLC), and identifying the type of the amino acid. In. The Edman degradation method is widely used in the fields of biochemistry and biotechnology because the entire amino acid sequence of a peptide compound can be easily determined by sequentially removing and identifying amino acids that are constituents of the peptide compound.

In recent years, as a labeling agent used in the Edman degradation method, a fluorescent labeling reagent such as fluorescein isothiocyanate [FITC: Maeda, H. and Kawauchi, H., Bioc.
him.Biophys. Res. Commun., 31, 188, 1968] and 4-N, N-
Dimethylamino-1-naphthylisothiocyanate [DNS
-NCS: Ichikawa, H. and Tanimura, T., Chem. Pharm.
Bull., 18, 1498, 1970], N-dansylanilinophenyl isothiocyanate [DNSA-PITC: Jin, S.-W., et a
l., FEBS Lett., 198, 150, 1986; Hirano, H. and Witt
man-Liebold ,, B., Biol. Chem. Hoppe-Seyler, 367, 1
259, 1986] have been used in place of phenylisothiocyanate. Also, 7-substituted-4- (2,1,3-benzoxdiadialyl) isothiocyanate has been developed as an excellent fluorescent labeling reagent (Japanese Patent Publication No. 6-94461).

[0004] By using these fluorescent labeling reagents, the detection sensitivity of derivatized amino acids has been greatly improved, so that the amino acid sequence of a trace amount of a peptide compound can be determined. However, recently, various new peptide compounds containing D-amino acids have been discovered, and as their importance has been elucidated, a sequencing method capable of determining whether an amino acid is a D-amino acid or an L-amino acid has been developed. It has become necessary. Since the conventional fluorescent dye has a very large molecular structure itself, there is a problem that a slight structural difference between labeled optically active amino acids cannot be distinguished by the HPLC method. Therefore, development of a labeling reagent capable of discriminating the type and configuration (D-form or L-form) of amino acids constituting a trace amount of a peptide compound is desired.

[0005]

An object of the present invention is to provide a compound useful as a fluorescent labeling reagent. More specifically, it is an object of the present invention to provide an isothiocyanate compound that is useful as a labeling reagent that can accurately determine the type and configuration of amino acids constituting a trace amount of a peptide compound. Another object of the present invention is to provide a labeling reagent having the above characteristics.

[0006]

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, the following isothiocyanate compounds are extremely useful as fluorescent labeling reagents for determining the amino acid sequence of peptide compounds. In particular, they have found that they are useful as highly sensitive fluorescent labeling reagents for accurately determining the configuration of amino acids. The present invention has been completed based on these findings.

That is, the present invention provides a compound represented by the formula (I):

Embedded image (Wherein R represents a phenyl group which may have a substituent). According to a preferred embodiment of the present invention, there is provided the above compound, wherein R is an unsubstituted phenyl group.

[0008] In another aspect, the present invention provides,
A fluorescent labeling reagent comprising the compound represented by (I) is provided. This fluorescent labeling reagent can be used for the amino acid sequence analysis of a peptide compound, the fluorescence quantitative analysis of an amine compound, an amino acid compound, or a peptide compound, and can be preferably used for determining the configuration of the amino acid constituting the peptide compound. . In yet another aspect, there is provided an amino acid compound fluorescently labeled with a compound of formula (I). This compound can be used as a standard substance when performing an analysis such as high performance liquid chromatography on the amino acid sequence of the peptide compound.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In the above formula (I), R represents a phenyl group which may have a substituent. The number of substituents on the phenyl group is not particularly limited, but is, for example, about 1 to 3, preferably about 1 or 2. When a plurality of substituents are present, they may be the same or different. In addition, the position of the substituent on the phenyl group is not particularly limited, and it is possible to substitute at an arbitrary position.

For example, as a substituent on a phenyl group, C
_1-4 alkyl group (methyl group, ethyl group, etc.), C _1-4 alkoxy group (methoxy group, ethoxy group, etc.), carboxyl group, halogen atom (fluorine atom, chlorine atom, bromine atom, etc.), C _1-4 An alkylcarbonyl group (such as an acetyl group), a C _1-4 alkoxycarbonyl group (such as an ethoxycarbonyl group), an amino group, a mono- or di-substituted amino group (such as a mono- or di-C _1-4 alkyl-amino group), a nitro group, And one or more substituents selected from the group consisting of cyano groups.

The compound of the present invention may form a salt such as a hydrochloride, but it goes without saying that the compound in the form of a salt is also included in the scope of the present invention. Also, any hydrate or solvate is included in the scope of the present invention. Since the method for producing the compound of the present invention is specifically and specifically described in the Examples of the present specification, those skilled in the art can appropriately refer to the descriptions and use the starting materials, reaction conditions, reagents, and the like as appropriate. The compound of the present invention included in the above general formula can be easily produced by modification or alteration. A compound in which R is a phenyl group having a substituent can be easily produced by using a thiophenol derivative having a corresponding substituent as a starting material. In carrying out the reaction, the substituent may be protected with an appropriate protecting group, if necessary.

The compound of the present invention represented by the formula (I) can efficiently react with the terminal amino acid of the peptide compound under mild conditions, and after the reaction, the peptide compound can be reacted with boron trifluoride or trifluoroacetic acid. , A fluorescent amino acid derivative (thiocarbamyl amino acid) labeled with a 2,1,3-benzoxadiazole derivative can be cleaved from the peptide chain. This labeled amino acid is
For example, it can be easily separated by means such as high performance liquid chromatography, and its optical isomer can be identified. The specific method is, for example,
This is described in detail in the examples of 269617.

Accordingly, the compounds of the present invention are useful as fluorescent labeling reagents for analyzing amino acid sequences of peptide compounds. The operation itself of the above analysis method is widely used in the art as the Edman method using a fluorescent labeling reagent, and those skilled in the art can use the compound of the present invention as a fluorescent labeling reagent instead of a conventional fluorescent labeling reagent. Extremely sensitive analysis can be easily performed. As used herein, the term “peptide compound” refers to two or more peptide-bonded amino acid residues, including proteins (polypeptides) and oligopeptides having about 10 amino acid residues or less. It is used as a concept that encompasses compounds that include and should be interpreted in the broadest sense.

The fluorescent labeling reagent containing the compound of the present invention represented by the formula (I) can also be used for the quantitative analysis of amine, amino acid and peptide compounds at the nanomolar or picomolar level. In addition, detection of amine compounds, amino acid compounds, or peptide compounds under a microscope in vitro on biological tissue samples such as tissue sections or body fluids; or production of amine compounds, amino acid compounds, or peptide compounds for the purpose of detection. It can also be used for staining living tissue performed in vivo using an endoscope or the like.

Since the fluorescent labeling reagent of the present invention has high reactivity with isothiocyanate, it can react with the amino acid at the terminal of the peptide very easily. In addition, since the fluorescent group is compact, the D / L optical isomer The feature is that the body can be separated by high performance liquid chromatography. In addition, the excitation and emission wavelength of the fluorescent group is a long wavelength range that is hardly affected by noise, and a fluorescent derivative having excellent fluorescence intensity and high stability is provided.

[0016]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to the following Examples. The compound numbers in the examples correspond to the compound numbers in the following scheme.

Embedded image

Example 1 Synthesis of (3) 1.0 g of (2) (NBD-Cl) was dissolved in 10 ml of acetonitrile, 1.0 ml of thiophenol was added, and 0.8 ml of triethylamine was added dropwise with stirring, followed by 2 hours at room temperature. Stirred.
The reaction solution was concentrated under reduced pressure, and the resulting precipitate was collected by filtration and yellow crystals
1.2 g were obtained. ^{APCl-MS; M - = 273} (4) Synthesis 500 mg of (3) 20 ml of methanol and methylene chloride 1
The solution was dissolved in 0 ml, and 2.5 ml of concentrated hydrochloric acid was added. To this solution was added 350 ml of iron powder, and the mixture was vigorously stirred at room temperature. Further, a small amount of iron powder was added, and the mixture was stirred for 30 minutes. Then, the reaction solution was filtered to remove iron powder. Silica gel column (methylene chloride: hexane = 2:
And eluted with 1) to give 170 mg of yellow crystals.

Synthesis of (5) 300 mg of (4) is dissolved in methylene chloride and 3 equivalents of m-CPB
A was added and stirred at room temperature. Until the ingredients disappear
Add m-CPBA, stir at room temperature, and wash the reaction with saturated NaHCO
_After washing with _three solutions, drying with Na ₂ SO ₄ and distilling off. Purify on silica gel column (eluted with methylene chloride) to obtain yellow crystals.
mg was obtained. APCl-MS; Synthesis of (M + H) ⁺ = 276, (MH) ^- = 274 (1) Dissolve 150 mg of (5) in 15 ml of acetonitrile and add CSCl ₂
A solution in which 0.3 ml of benzene was dissolved in 1.0 ml of benzene was added, and the mixture was refluxed for 5 hours. After completion of the reaction, the reaction solution was distilled off under reduced pressure.
Purification by a silica gel column (hexane: ethyl acetate = 3: 1) gave 60 mg of yellow crystals. APCl-MS; (M + H) ⁺ = 31
8, M ^- = 317

[0019]

The compound of the present invention does not exhibit fluorescence by itself, and has the property of emitting remarkable fluorescence after selectively reacting and bonding with amino groups of amine compounds, amino acid compounds, peptide compounds and the like. . Therefore, the compounds of the present invention can be used as fluorescent labeling reagents for the fluorescence analysis of these compounds and the amino acid sequencing of peptide compounds. In particular, since the compound of the present invention can separate optically active amino acids, it is useful for determining the amino acid sequence of peptide compounds containing D-amino acids.

Claims (7)

[Claims] (1) Formula (I): (Wherein, R represents a phenyl group which may have a substituent). 2. The compound according to claim 1, wherein R is an unsubstituted phenyl group. 3. A fluorescent labeling reagent comprising the compound according to claim 1 or 2. 4. The fluorescent labeling reagent according to claim 3, which is used for amino acid sequence analysis of a peptide compound. 5. The method according to claim 3, which is used for determining the configuration of amino acid residues constituting the peptide compound.
The reagent according to 1. 6. The reagent according to claim 3, which is used for a fluorimetric analysis of an amine compound, an amino acid compound, or a peptide compound. 7. An amino acid compound fluorescently labeled with the compound of the formula (I) according to claim 1.