JPH08198873A - Compound-selective fluorescent labeling reagent with sulfhydryl group - Google Patents

Abstract

(57) [Abstract] [Objective] A compound-selective fluorescent labeling reagent having a sulfhydryl group, which has a high fluorescence quantum effect, is position-selective, does not change the properties of the compound to be labeled, and can be easily labeled. [Structure] The following general formula: (In the formula, R ¹ and R ² are each independently a hydrogen atom, an alkyl group, an alkoxyl group or a sulfonate group, and R ³
Is an alkyl group, an alkyl sulfonate group or an ammonioalkyl group, n is an integer of 1 to 3, X is an anion species, and Y is an alkyl chain having 1 to 10 carbon atoms or an oxygen atom, a nitrogen atom and An alkyl chain containing 1 to 10 carbon atoms and having 1 to 10 carbon atoms. ) A compound-selective fluorescent labeling reagent having a sulfhydryl group consisting of a water-soluble indocyanine compound represented by the formula (1). [Effect] Having one maleimide group in the molecule, it reacts with a sulfhydryl group of a protein or the like to obtain a highly sensitive fluorescent label. It does not crosslink the labeled substance and does not impair the properties of the protein.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a compound-selective fluorescent labeling reagent having a sulfhydryl group capable of highly sensitively detecting a fluorescently labeled compound by reacting with a sulfhydryl group.

The fluorescent labeling reagent of the present invention is used for fluorescence detection of polymers such as proteins and nucleic acids and detection of low molecular weight compounds by high performance liquid chromatography (HPLC).

[0003]

2. Description of the Related Art Many fluorescent labeling reagents capable of reacting with sulfhydryl groups are known, and maleimide group, iodoacetamide or bromoacetamide are typical reactive groups.

Fluorescein isocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC) and the like are well known as fluorescent labeling compounds and are widely used in the fields of protein analysis and nucleic acid analysis. In the nucleic acid analysis, a DNA oligomer labeled with FITC or TRITC is used when the base sequence is analyzed by an automatic DNA sequencer, or it is used when a dynamic analysis of DNA is performed. In the case of base sequence analysis, generally, the number of molecules of the DNA fragment contained in each band after electrophoresis is about 1
It is in the range of × 10 ^-16 to 1 × 10 ^-18, which is the limit that can be detected by combining an optical system using a laser as a light source.

Japanese Unexamined Patent Publication (Kokai) No. 2-191674 describes an indocyanine type highly fluorescent compound, which is water-soluble by directly adding a sulfonic acid group to an aromatic ring. Since the compound has a plurality of reactive groups, the compound to be labeled is likely to be modified by crosslinking during labeling.

Further, the compound described as a fluorescent labeling dye in JP-A-5-40097 also has a problem of cross-linking, and since it cannot react with various functional groups by itself, it is previously activated. It requires an additional step to obtain the desired labeled substance.

In recent years, attempts have been made to analyze the behavior of minute components such as proteins and nucleic acids in a system using a fluorescence microscope. To do so, it is necessary to detect one molecule, which is a level that cannot be achieved by sensitivity with conventional fluorescent labeling reagents. Also,
In the case of identifying the type of virus or bacterial cell in serum or sample extract, it is now possible to amplify only a specific gene several thousand to several tens of thousands times using gene amplification technology (PCR). Since noise may be included in the process, it became necessary to analyze without using PCR.

[0008]

[Problems to be Solved by the Invention] Highly sensitive analytical methods such as fluorescence and chemiluminescence have been applied to the detection of minute amounts of biological components such as proteins and nucleic acids. The current fluorescent labeling reagent is insufficient in sensitivity to measure the above, and a fluorescent agent having high molar extinction coefficient and fluorescence quantum yield is required. Further, since it is generally used in an aqueous system, high water solubility is required. In addition, a regioselective label is required so that the properties of the labeled body do not change before and after the cover, and it is a labeling agent that can modify sulfhydryl groups rather than amino groups, and that it does not crosslink the labeled bodies. It is required to have one maleimide group in the molecule.

[0009]

The compound of the present invention for solving the above-mentioned problems is a compound-selective fluorescent labeling reagent having a sulfhydryl group consisting of a water-soluble indocyanine compound represented by the following general formula.

Embedded image (In the formula, R ¹ and R ² are each independently a hydrogen atom, an alkyl group, an alkoxyl group or a sulfonate group, and R ³
Is an alkyl group, an alkyl sulfonate group or an ammonioalkyl group, n is an integer of 1 to 3, X is an anion species, and Y is an alkyl chain having 1 to 10 carbon atoms or an oxygen atom, a nitrogen atom and An alkyl chain containing 1 to 10 carbon atoms and having 1 to 10 carbon atoms. )

In the compounds of the present invention, X in the above general formula is an anionic species such as halogen, acetic acid, perchloric acid, carbonic acid and the like.

The compound of the present invention can react with a sulfhydryl group of a substance to be labeled to add spectroscopic characteristics of indocyanine having a high molar extinction coefficient and a high fluorescence quantum yield to the substance to be labeled. 780nm, fluorescence wavelength 560 ~ 800n
The compound can have a fluorescence property of m 2.

The spectral characteristics of the compounds of the present invention are R
^{When 1} , R ² is a hydrogen atom, R ³ is an ethyl group, Y is an alkyl chain having 5 carbon atoms, and X is a chlorine ion, the spectral characteristics of n of 1 to 3 are shown in Table 1.

[0013]

[Table 1]

All of the compounds of the present invention are novel compounds which have not been published in the literature and have a water-soluble group in the side chain. When labeling a protein or the like, the compound can be labeled without impairing the water solubility of the protein. . Further, by having a piperazino ring in the side chain, the side chain becomes rigid, the protein and the labeled dye moiety do not interact hydrophobically, and the property of the protein is maintained as it is before and after labeling. Furthermore, since the maleimide group is one asymmetric compound, it does not crosslink the proteins.

The compound of the present invention is synthesized by using 2,3,3-trimethylindolenine or a derivative thereof as a raw material.
These raw materials were produced by the Fischer synthesis method (E. Fischer and
O. Hess: Berichte, 17 , 559 (1883), etc.) or commercially available. After introducing an alkyl group into the raw material nitrogen, it is reacted with triethyl orthoformate to give a cyanine dye, and then with piperazino maleimide to give a sulfhydryl group-selective fluorescent labeling reagent.

Compounds (A) to (H) are shown below as typical examples of the compounds thus obtained.

[0017]

[Chemical 4]

[0018]

Embedded image

[0019]

[Chemical 6]

[0020]

[Chemical 7]

[0021]

Embedded image

[0022]

[Chemical 9]

[0023]

[Chemical 10]

[0024]

[Chemical 11]

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to the following Examples as long as the gist thereof is satisfied.

Example 1 Synthesis of Compound (A) 5.0 g (31.4 mmol) of 2,3,3-trimethylindolenine was dissolved in 100 ml of acetonitrile, and 5.9 g of iodoethane (37.
(8 mmol) was added and the mixture was heated under reflux overnight. The reaction solution was concentrated under reduced pressure, ether was added to the residue, and crystals of N-ethyl-2,3,3-trimethylindolenium iodide salt were collected by filtration. The crystals were gray-red and the yield was 9.1 g, 92%.

Separately, 5.0 g of 2,3,3-trimethylindolenine
(31.4 mmol) was dissolved in 100 ml of dimethylformamide, 8.4 g (37.6 mmol) of ethyl 6-bromohexanoate was added, and the mixture was heated at 100 ° C overnight. The reaction mixture was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (solvent: methanol / chloroform) to give N- (5-ethyloxycarbonylpentyl) -2,3,3-trimethylindolenium bromine salt. It was Reddish brown oil, yield 3.4g, yield
It was 40%.

2.0 g (6.3 mmol) of N-ethyl-2,3,3-trimethylindolenin iodine salt and 1.8 g of N- (5-ethyloxypentyl) -2,3,3-trimethylindolenium bromine salt
(6.3 mmol) was dissolved in 10 ml of pyridine, heated to 110 ° C., 1.6 ml (10.0 mmol) of triethyl orthoformate was added, and the mixture was heated for 30 minutes. The reaction solution was concentrated under reduced pressure, dissolved in chloroform, and washed with water three times. The chloroform solution was concentrated, separated and purified by silica gel column chromatography (solvent: methanol / chloroform), and N-ethyl-N '-(5-carboxypentyl) -3,3,3', 3'-tetramethyl-trimethyl. Got chin indocyanine. It was a green metallic luster crystal, and the yield was 860 mg and the yield was 29%.

160 mg (0.34 mmol) of this compound was dissolved in 2 ml of dimethylformamide, and 100 mg (0.35 mmol) of piperazinoethylmaleimide and 49 of triethylamine.
μl (0.36 (mmol) was added, and dicyclohexylcarbodiimide (75 mg, 0.36 mmol) was added while cooling to 0 ° C), and the mixture was reacted overnight at room temperature. The reaction solution was filtered,
The filtrate was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (solvent: methanol / chloroform) to obtain compound (A). Black-red powder with a yield of 120 mg
The yield was 51%.

The obtained compound (A) was added to a PBS buffer of pH 7.4 (NaCl: 8.0g, KCl: 0.2g, Na ₂ HPO ₄ : 11.5g, KH ₂ PO _4).
Fig. 1 shows the fluorescence spectrum of the solution dissolved in / LL of water).

Example 2 Synthesis of compound (B) N-ethyl-N '-(5-carboxypentyl) -3,3,3', 3'-tetramethyl-trimethine indocyanine 320 mg (0.68 mmol) 5 ml Of 30% fuming sulfuric acid was added little by little. After stirring for 1 hour as it is, it is poured into water containing sodium hydrogen carbonate, and the precipitated sodium sulfate is removed by filtration.
The filtrate was concentrated under reduced pressure. Remaining sodium sulfate is suspended in methanol, filtered off, and separated by Sephadex LH.
Separation and purification using 20 / water to give N-ethyl-N '-(5-carboxypentyl) -3,3,3', 3'-tetramethyl-trimethine indocyanine-5,5'-disulfonic acid Obtained. A black-red solid,
The yield was 310 mg, and the yield was 70%.

N-ethyl-N '-(5-carboxypentyl) -3,
3,3 ', 3'-Tetramethyl-trimethine indocyanine-5,
Dissolve 120 mg (0.19 mmol) of 5'-disulfonic acid in a water / tetrahydrofuran (THF) mixed solvent, add 65 mg (0.22 mmol) of piperazinoethylmaleimide, adjust the pH of the solution to 6.5 with sodium hydrogen carbonate, and then dicyclohexyl. Carbodiimide 48 mg (0.23 mmol) in THF
Was added to the solution, and the mixture was stirred overnight at room temperature. The precipitated dicyclohexylurea was removed by filtration, the filtrate was concentrated under reduced pressure, and separated and purified with Sephadex LH20 / water to obtain compound (B). It was a black-red solid and the yield was 145 mg and the yield was 92%.

Example 3 Synthesis of compound (C) 2.0 g of N-ethyl-2,3,3-trimethylindolenin iodine salt
(6.3 mmol) and N- (5-ethyloxycarbonylpentyl) -2,3,3-trimethylindolenium bromine salt 1.8 g
(6.3 mmol) was dissolved in 10 ml of pyridine, heated to 110 ° C., 1.3 g (9.8 mmol) of 1,3,3-trimethoxypropene was added, and the mixture was heated for 1 hour. The reaction solution is concentrated under reduced pressure,
It was dissolved in chloroform and washed with water three times. The chloroform solution was concentrated, separated and purified by silica gel column chromatography (solvent: methanol / chloroform), and N-ethyl-N '-(5-carboxypentyl) -3,3,3', 3'-tetramethyl-penta. I got methine indocyanine. It was a green metallic luster crystal, and the yield was 710 mg and the yield was 23%.

As in the synthesis of compound (A), N-ethyl
-N '-(5-carboxypentyl) -3,3,3', 3'-tetramethyl
-Pentamethine indocyanine 200 mg (0.43 mmol)
And piperazinoethylmaleimide were reacted and isolated and produced by silica gel column chromatography to obtain compound (C). It was a black-blue solid and the yield was 180 mg and the yield was 58%.

Example 4 Labeling of protein with compound (A) 12.5 mg of bovine serum albumin (BSA) was dissolved in 2.5 ml of 0.1 M phosphate buffer of pH 8 and 10 ml of 0.3 M dithiothreitol aqueous solution was added, It was left at ℃ for 5 hours. Using a Pharmacia packed column PD-10, PBS buffer at pH 7.4 (NaCl: 8.0g, KCl: 0.2g, Na ₂ HPO ₄ : 11.5g, KH ₂ PO ₄ / water 1L)
L) and separated and purified. 50 ml of a 100 mM aqueous solution of the compound (A) was added to 3.0 ml of the obtained reduced BSA solution, and the mixture was left at 25 ° C. overnight. The labeled protein fraction was separated with a PBS buffer using a PD-10 column. The obtained 3.0 ml fraction was freeze-dried.

BSA labeled with compound (A) was adjusted to pH 7.4.
Fig. 2 shows the fluorescence spectrum of the solution dissolved in PBS buffer.
Shown in

Intracellular introduction of labeled protein and fluorescent observation NIH3T3 cells cultured in Dulbecco's modified MEM medium (hereinafter abbreviated as DMEM) were used to attach the cells to the glass of a Meridian glass bottom dish. 1 mg of BSA labeled with the compound (A) was dissolved in 10 ml of PBS buffer, passed through a 0.2 μm filter and diluted 1/10 with sterile water.

Compound (A) was labeled with BSA and adjusted to pH 7.4.
Figure 2 shows the absorption spectrum of the solution dissolved in PBS buffer.
Shown in

This solution was passed through a 0.2 μm filter again, put into a silica capillary for microinjection, and the dye-labeled BSA was introduced into the cells while applying a pressure of about 2 kg / cm ² . By this operation, 0 per cell.
That is, 1 pl, that is, 15 zeptomoles of the dye BSA was introduced.

After discarding DMEM and repeating the linear pattern four times with PBS, an epifluorescence microscope (Olympus OM was used in PBS buffer).
T-2) was used for fluorescence observation. Fluorescence was detected using a high sensitivity CCD camera after passing through a G excitation filter. As a result, only cells microinjected with BSA could be detected.

[0035]

EFFECTS OF THE INVENTION The fluorescent labeling reagent of the present invention has indocyanine in the mother nucleus and can detect zeptomole level, and since it has one maleimide group in the molecule, it reacts selectively with the sulfhydryl group of the substance to be labeled. However, the labeled bodies do not cross-link. Also, the piperazino group in the side chain is acidic to the compound.
It imparts water-solubility in the neutral range, and the side chain is made rigid so that the interaction between the dye and the protein is suppressed and the protein properties are not impaired.

[Brief description of drawings]

FIG. 1 shows an absorption spectrum of a solution obtained by dissolving the compound (A) of the present invention in PBS buffer having a pH of 7.4.

FIG. 2: BSA labeled with the compound (A) of the present invention, pH 7.
The absorption spectrum of the solution of 4 in PBS buffer is shown.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C07D 207: 448 209: 10)

Claims (2)

[Claims] 1. A compound-selective fluorescent labeling reagent having a sulfhydryl group, which comprises a water-soluble indocyanine compound represented by the following general formula. Embedded image (In the formula, R ¹ and R ² are each independently a hydrogen atom, an alkyl group, an alkoxyl group or a sulfonate group, and R ³
Is an alkyl group, an alkyl sulfonate group or an ammonioalkyl group, n is an integer of 1 to 3, X is an anion species, and Y is an alkyl chain having 1 to 10 carbon atoms or an oxygen atom, a nitrogen atom and An alkyl chain containing 1 to 10 carbon atoms and having 1 to 10 carbon atoms. ) 2. The following general formula: (In the formula, R ¹ and R ² are each independently a hydrogen atom, an alkyl group, an alkoxyl group or a sulfonate group, and R ³
Is an alkyl group, an alkyl sulfonate group or an ammonioalkyl group, n is an integer of 1 to 3, X is an anion species, and Y is an alkyl chain having 1 to 10 carbon atoms or an oxygen atom, a nitrogen atom and An alkyl chain containing 1 to 10 carbon atoms and having 1 to 10 carbon atoms. ) The method of reacting a water-soluble indocyanine compound represented by the formula (4) with a sulfhydryl group of an object and measuring with high sensitivity by fluorescence detection.