JPH09124599A - Indocyanine compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject compound represented by a specific formula, capable of emitting fluorescence by exciting light of near-infrared rays or far- infrared rays, having high water-soluble property, capable of labeling a compound to be labeled selectively to functional group and useful as a fluorescent labeled reagent. SOLUTION: This compound is represented by formula I [A<1> and A<2> are each benzene ring or naphthalene ring; R<1> and R<2> are each H, an alkyl, an aryl, etc.; R<3> is an alkyl, an isolated or dissociated alkyl sulfonate, an aminoalkyl, etc.; (n) is 1-3; X<-> is an anion; Z is formula II, etc.; M<+> is an alkali metal ion; Y is a 1-10C alkylene, etc.], e.g. a compound of formula III. The compound is preferably obtained by introducing an alkyl group onto a nitrogen atom of 2, 3, 3-trimethylindonylene and reacting the resultant compound with trimethyl orthoformate, etc., to form a cyanine dye skeleton and then introducing a labeling group represented by Z thereto.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an indocyanine derivative. The compound of the present invention is useful as a fluorescent labeling reagent for highly sensitively detecting a compound having an amino group, a sulfhydryl group, and / or a carbonyl group.

[0002]

2. Description of the Related Art A labeling reagent is a reagent that imparts a specific property to a target compound so that it can be detected. Representative labeling methods using a labeling reagent include, for example, a fluorescent labeling method, a method of introducing a radioisotope, and a method of using an enzyme. Although the method using a radioisotope enables a highly sensitive measurement, there is a risk of exposure to an operator due to the use of an unsealed radioisotope.
In addition, facilities that use radioisotopes are limited,
There are problems in the treatment of radioactive waste and the management of radioactive materials, and enormous investment is required for measuring equipment and facility management.
The ELISA method is known as a method using an enzyme, but a sufficient sensitivity cannot be obtained by the conventional colorimetric method, and therefore, higher sensitivity measurement is attempted using a fluorescent labeling reagent together.

For the above reasons, labeling methods using fluorescent labeling reagents have become the mainstream of labeling methods for microanalysis. The fluorescent labeling reagent is a reagent for binding a fluorophore to a specific functional group in the labeled compound and detecting the labeled compound by utilizing the added fluorescent property. Conventionally, various fluorescent labeling reagents have been proposed, and in detection using high performance liquid chromatography (HPLC), detection sensitivity has been increased to about several femtomoles (injection amount of sample). However, it cannot be said that the sensitivity is still sufficient for the detection of minute amounts of biopolymers such as proteins and nucleic acids, and that single molecule measurement is possible by image processing using a fluorescence microscope or the like. Therefore, there is a demand for a highly sensitive fluorescent labeling reagent.

Fluorescent labeling reagents used for labeling biopolymers such as proteins have not only high fluorescence intensity but also selective binding to specific functional groups, and properties of the labeled biopolymer after binding. It is required to have various properties such as not changing. Further, a labeling reagent having a plurality of binding sites in the molecule may change the structure or property of the compound to be labeled by cross-linking formation, so the labeling reagent should have only one labeling group in the molecule. There must be. For example, JP-A-2-91674 discloses an indocyanine-type highly fluorescent compound, but the compound has a plurality of reactive groups, and the crosslinking reaction may proceed during labeling. is there. The fluorescent labeling dye disclosed in JP-A-5-40097 may also cause a crosslinking reaction. Further, since the compound itself cannot react with various functional groups, it is necessary to activate the reagent in advance, which causes a problem that the operation is complicated.

In addition to the above requirements, the labeling reagent is also required to have high water solubility. Indocyanine green, which is a type of indocyanine compound, is a substance that exhibits specific absorption by infrared irradiation and emits fluorescence, and is used for liver function tests (indocyanine green load test) and the like.
Conventionally, attempts have been made to fluorescently label proteins using the fluorescence of indocyanine green, and various derivatives in which the mother nucleus of indocyanine green is modified or a reactive group is introduced have been proposed. However, the reactive indocyanine green derivative having a reactive group introduced therein is extremely insoluble in water, and when used as a labeling reagent, the compound needs to be dissolved in an organic solvent such as dimethyl sulfoxide. Therefore, when an aqueous protein solution is added to the reaction system, there arises a problem that the reagent is precipitated and the labeled protein is precipitated.

For example, the compound disclosed in Japanese Patent Application No. 6-222059 has poor water solubility, and it is necessary to dissolve the compound in an organic solvent before use. The compound disclosed in JP-A-2-91674 is a compound having improved water solubility by directly introducing a sulfonic acid group into an aromatic ring, but even with this compound, protein labeling can be performed in a pure aqueous system. Does not have sufficient water solubility.

On the other hand, when a protein is labeled with an indocyanine green derivative, it is necessary to label it so as not to inhibit the physiological activity of the protein itself. Previously reported indocyanine green derivatives have a short distance between the labeled protein and the indocyanine green label, which may impair some physiological activity of the labeled protein. is there. For example, Patney
The indocyanine green derivative developed by (Patoney) et al. Has an isothiocyano group directly introduced into the benzene ring, and the distance between the label and the protein is extremely short.

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to provide a fluorescent labeling reagent that alleviates or eliminates the above problems. More specifically, a fluorescent label having a high fluorescence quantum effect can be simply and functionally introduced into a labeled compound in a functional group-specific manner, and a fluorescent labeling reagent that does not change the properties of the labeled compound can be used. It is an object of the present invention to provide. Another object of the present invention is to provide a fluorescent labeling reagent having the above characteristics and excellent in water solubility.

[0009]

Means for Solving the Problems The inventors of the present invention have made diligent efforts to solve the above problems, and by synthesizing various derivatives of indocyanine green, fluorescence is generated by excitation light of near infrared rays or far infrared rays, We succeeded in producing an indocyanine green derivative with high water solubility.
The present inventors have also proved that the above indocyanine green derivative is useful for labeling biopolymers such as antibodies. Furthermore, when sodium iodide or the like is allowed to act on the above compound capable of forming an intramolecular counter ion (Zwitter ion), the anion of the salt is added to the cation part of the molecule and the cation of the salt is added to the anion part. It was found that as a result of the inhibition of the formation of the intramolecular counterion, the ionicity of the whole molecule is maintained and the water solubility is remarkably increased. The present invention has been completed based on these findings.

That is, the present invention provides the following formula:

Embedded image (In the formula, A ¹ and A ² each independently represent a benzene ring or a naphthalene ring, R ¹ and R ² are each independently a hydrogen atom,
An alkyl group, an aryl group, a free or dissociated sulfonic acid group, or an alkoxy group, R ³ represents an alkyl group, a free or dissociated sulfonic acid alkyl group, or an aminoalkyl group or an ammonioalkyl group, and n is
Represents an integer of 1 to 3, X ⁻ represents an anion species as necessary, and Z represents the following formula:

Embedded image Is a group selected from the group consisting of, M ⁺ is an alkali metal ion, W and Y each independently have 1 to 1 carbon atoms.
Represents an alkylene group of 0, or W and Y are 1 selected from the group consisting of oxygen atom, nitrogen atom, and sulfur atom.
And an alkylene group having 1 to 10 carbon atoms containing one or more atoms).

According to a preferred embodiment of the present invention, A ¹ and A ²
Are both naphthalene rings, R ¹ and R ² are both hydrogen atoms, R ³ is a dissociated linear alkyl sulfonate group having 2 to 5 carbon atoms, n is 3 and Y is 2 carbon atoms. A linear alkylene group of -5, Z is a succinimidooxy group or an alkali metal sulfosuccinimidooxy group; and X ^- is an iodine ion, R ³ is a sodium ion having a salt number of 2 There is provided the above compound, which is ˜5 linear alkyl sulfonate groups.

According to another aspect of the present invention, a fluorescent labeling reagent comprising the above compound; a compound comprising at least one functional group selected from the group consisting of an amino group, a hydroxyl group, a carbonyl group and a sulfhydryl group. The above-mentioned reagent used for fluorescent labeling of the above; the above-mentioned reagent in which the compound having a functional group is a biopolymer; and the biopolymer is selected from the group consisting of proteins, glycoproteins, lipids, phospholipids, polysaccharides, and nucleic acids. The above reagents are provided.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION In the above general formula, A ¹ and A ² each independently represent a benzene ring or a naphthalene ring, and preferably a naphthalene ring can be used. A ¹ and / or A ²
Is a naphthalene ring, the condensation position of the naphthalene ring with respect to the pyrrole ring to which A ¹ and / or A ² is condensed is not particularly limited. When A ¹ and / or A ² represents a naphthalene ring, R ¹ and R ² form a naphthalene ring 2
It can be substituted at any position of any of the phenyl rings. When A ¹ and / or A ² represents a phenyl ring, R ¹ and R ² may be substituted at any position on the phenyl ring.

R ¹ and R ² each independently represent a hydrogen atom, an alkyl group, an aryl group, a free or dissociated sulfonic acid group, or an alkoxy group. As the alkyl group,
A linear or branched lower alkyl group having about 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, can be used. For example, methyl group, ethyl group, isopropyl group, n-butyl group, sec-butyl group, tert-butyl group and the like can be preferably used. Examples of the aryl group include a phenyl group, a naphthyl group, a pyridyl group, an imidazolyl group, a pyrrolyl group and the like. In the dissociated sulfonic acid group, an alkali metal ion or the like such as sodium ion or potassium ion may be present as a counter ion. As the alkoxy group, it is possible to use a linear or branched lower alkoxy group having about 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, and more specifically, a methoxy group,
It is possible to use an ethoxy group, a propoxy group, an n-butoxy group, a sec-butoxy group, a tert-butoxy group and the like.

R ³ represents an alkyl group, a free or dissociated sulfonate alkyl group, or an amino or ammonioalkyl group. As the alkyl group, a linear or branched lower alkyl group having about 1 to 6 carbon atoms, preferably having 1 to 4 carbon atoms can be used. The sulfonic acid of the alkyl sulfonate group, or the amino group or the ammonio group of the amino or ammonioalkyl group can be substituted at any position of the alkyl group, but it is preferable to use one substituted at the terminal of the alkyl group. is there. The amino group or ammonio group may have one or two (1 to 3 in the case of ammonio group) the same or different lower alkyl groups (preferably alkyl groups having about 1 to 6 carbon atoms). Good.

For example, as R ³ ,-(CH ₂ ) _k -SO _3- (k is 3
Groups having an integer of from 5 to 5) are suitable. R ³
When is a dissociated alkyl sulfonate group, an alkali metal ion such as sodium ion or potassium ion may be present as a counter ion of the sulfonate ion. When R ³ is an ammonioalkyl group, an anion such as a halogen ion or a perchlorate ion, preferably a halogen ion may be present as a counter ion.

X ⁻ represents an anion species which may be optionally present, for example, halogen ion, acetate ion, perchlorate ion, carbonate ion, or thiocyanate ion. The anionic species represented by X ⁻ acts to cancel the positive charge on the nitrogen atom in the mother nucleus and keep the compound as a whole neutral. For example, when a dissociated sulfonic acid moiety is present in any one of R ¹ , R ² , and R ³ , the negative charge of the dissociated sulfonic acid and the positive charge on the nitrogen atom in the nucleus cancel each other out. because, X ^- may not be present. On the other hand, for example, when ^one of R ¹ and R ² is a dissociated sulfonic acid group and R ³ is an ammonioalkyl group, the charges of these groups are balanced, and thus X ^- may be needed to counteract the positive charge on the nitrogen atom. As X ⁻ , chlorine ion, bromine ion, iodine ion and the like can be preferably used. Of these, iodine ion is particularly desirable. n
Represents an integer of 1 to 3, but n is preferably 3.

As Y, a linear or branched alkylene group having 1 to 10 carbon atoms, preferably a linear or branched alkylene group having 2 to 5 carbon atoms can be suitably used. Particularly preferably, a trimethylene group, a tetramethylene group or a pentamethylene group can be used. Y represents a linear or branched alkylene group having 1 to 10 carbon atoms and containing at least one atom selected from the group consisting of oxygen atom, nitrogen atom, and sulfur atom.

Specifically, as a group represented by -Y-CO-,
For _{example, -CH 2 -CO-; - (CH} 2) 2 -CO-; - (CH 2) 3 -CO-; - (CH 2)
₄ -CO-;-(CH ₂ ) ₅ -CO-; -CH ₂ -CO-NH- (CH ₂ ) ₅ -CO-;-(CH ₂ ) ₂
-CO-NH- (CH ₂ ) ₅ -CO-;-(CH ₂ ) ₃ -CO-NH- (CH ₂ ) ₅ -CO-;-(C
H ₂ ) ₄ -CO-NH- (CH ₂ ) ₅ -CO-; -CH ₂ -CO-NH- (CH ₂ ) ₅ -CO-NH- (CH
_{2) 2 -CO-; - (CH} 2) 4 -CO- (N, N'-piperadinyl) - (CH 2) 2 -CO
(N, N'-piperadinyl is-(CH ₂ ) ₄ -CO- in the 1-position of piperazine.
The group is substituted, and the 4- (CH ₂ ) _2- Z group is substituted at the 4-position. Hereinafter, the same applies. ); Or-CH _{2
-CO-NH- (CH 2) 5} -CO- (N, N'-piperadinyl) - (CH 2) 2 -CO- , etc. can be utilized.

In the group represented by Z, W is as described above.
A group corresponding to Y in the example of Y-CO 2 can be preferably used. Further, the counter ion of M ⁺ and X sulfonic acid groups of the N- sulfosuccinimidyl group ^- the M ⁺ of the counterion, may be the same or different, that both a sodium ion preferable. Furthermore, R ¹ and / or R ²
Represents a dissociated sulfonic acid group, and / or R ³ represents a dissociated alkyl sulfonate group, the counter ion of the sulfonic acid is the M ⁺ and / or N-sulfosuccinimine of the X ⁻ counter ion. M of the counter ion of the sulfonic acid group of the diloxy group
^It may be the same as or different from ⁺ , but both are preferably sodium ions.

Without wishing to be bound by any particular theory, for example, when R ³ is a dissociated alkyl sulfonate group, the negative charge of the sulfonate group and the positive charge on the nitrogen atom (wherein N ⁺ is Represents each other, and a so-called inner salt (Zwitter ionic compound) is formed. In such an intramolecular salt type compound, the ionicity of the entire molecule is lowered, and the water solubility of the compound may be impaired. On the other hand, in such a compound, an alkali metal ion exists as a counter ion against the negative charge of the sulfonic acid group,
And, X as counterions with respect to the positive charge on the nitrogen atom ^-
When the anionic species represented by the formula (3) is present, the production of an intramolecular salt due to the negative charge of the sulfonic acid group and the positive charge on the nitrogen atom is hindered, so that the water solubility may be significantly increased. Therefore, X ⁻ is an anion such as iodine ion, and
A compound in which R ³ is an alkyl sulfonate group forming a salt with sodium ion is a preferred embodiment of the present invention.

In the above general formula, the compounds of the following preferred embodiments and the compounds of the schemes in Examples,
For convenience, the positive charge is described as being fixed on one nitrogen atom in the nucleus, but it is understood by those skilled in the art that the positive charge can be transferred to another nitrogen atom in the nucleus through a conjugated double bond. Easy to understand. Therefore, it goes without saying that all such isomers based on conjugation are included in the scope of the present invention.

The compound of the present invention can be synthesized using 2,3,3-trimethylindolenine, 2,3,3-trimethylbenzoindolenine or their derivatives as raw materials. These raw materials are used in the Fischer synthesis method (E. Fischer and O.
Hess: Berichte, 17, 559, 1883, etc.) or commercially available. For example, it can be produced by introducing an alkyl group onto the nitrogen atom of the starting compound, reacting triethyl orthoformate or the like to form a cyanine dye skeleton, and then introducing a labeling group represented by Z 1.

In order to produce a compound which does not form an intramolecular salt, such as a compound in which X ^- is an anion such as iodide ion and R ³ is an alkyl sulfonate group which forms a salt with sodium ion, , compounds and positive charges formed a each other by intramolecular salt cancellation on the nitrogen atom of the dissociated anionic charge and the mother nucleus of the alkyl sulfonate group ^- after producing the (X nonexistent) of compound, the compound e.g. It may be dissolved at a high concentration in an organic solvent such as dimethylformamide or dimethylsulfoxide, and a salt such as sodium iodide may be added to the solution. However, the method for producing the compound of the present invention is not limited to these methods.

Compound (A) is a typical example of the compound of the present invention.
~ (P) is shown below.

Embedded image

[0026]

[Chemical 6]

[0027]

Embedded image

[0028]

Embedded image

[0029]

Embedded image

The compound of the present invention can be used, for example, for labeling biopolymers derived from animals and plants having a reactive amino group, hydroxyl group, thiol group, carbonyl group, sulfhydryl group and the like. Examples of biopolymers include complex proteins such as proteins and glycoproteins, complex lipids such as lipids and phospholipids, polysaccharides, and nucleic acids. For example, antibodies and enzymes having high specificity for cancer and the like are suitable targets for labeling the compound of the present invention. Further, the compound of the present invention is a reactive amino group, a hydroxyl group, a thiol group,
Since it easily reacts with a low molecular weight compound having a carbonyl group, a sulfhydryl group and the like, it is possible to label an amplification system substance such as biotin-avidin used in the amplification system.

For example, when an N-succinimidyloxy group and an N-sulfosuccinimidyloxy group are used as Z, these groups, together with the carbonyl group to which Z is bonded, form a reactive active ester. Reacts with an amino group contained in the compound to be labeled to form an amide bond. When an N-maleimidoalkyl group is used as Z, for example, a thiol group (H
SR) reacts to form a thioether. Furthermore, Z
Is -NH-NH ₂ , the aldehyde group (OHC-R) at the terminal of the reducing sugar contained in the compound to be reacted reacts with -Y-CO-NH-N.
H-CO-R is formed. Therefore, by appropriately selecting and using the compound of the present invention, it is possible to label the compound to be labeled with a functional group specific. However, the use of the compound of the present invention is not limited to those exemplified above.

The compound of the present invention is a compound in which an active site is introduced into an indocyanine compound having a high molar extinction coefficient and fluorescence quantum yield, and can add the spectroscopic properties of indocyanine to the labeled compound. The labeled compound labeled with the compound of the present invention has an excitation wavelength of 500 to 800 n.
Since it has fluorescence characteristics of m 2 and a fluorescence wavelength of 550 to 850 nm, it is possible to detect with extremely high sensitivity by using a high-power semiconductor laser or He / Ne laser as an excitation light source.

[0033]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples. In addition, those skilled in the art who referred to this embodiment,
It will be easily understood that the desired compound within the scope of the present invention can be similarly easily produced by appropriately modifying or modifying the starting materials, reagents, reaction conditions and the like.
The compound numbers in the examples correspond to the compound numbers in the scheme, and the compound (A) and the like correspond to the numbers of the compounds shown as the above preferable compounds.

Example 1: Preparation of compounds (A) and (B)

Embedded image

5.0 g (31.4 mmol) of compound 1 (2,3,3-trimethylindolenine) was dissolved in 100 ml of acetonitrile, 5.9 g (37.8 mmol) of iodoethane 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 gray red crystals of compound 2 were collected by filtration (9.1 g,
92%). Separately, Compound 1 (5.0 g, 31.4 mmol) was dissolved in 100 ml of dimethylformamide (DMF), 20.0 g (89.6 mmol) of ethyl 6-bromohexanoate was added, and the mixture was heated at 80 ° C. under a nitrogen atmosphere overnight. The reaction mixture was concentrated under reduced pressure, and the obtained oil was washed with 50 ml of ether. Next, the obtained oil was dissolved in 100 ml of methanol, and 11.5 g (287.5 mmol) of sodium hydroxide was dissolved in water and added,
It hydrolyzed by stirring at room temperature for 5 hours. After distilling off methanol under reduced pressure, the mixture was neutralized with 1N hydrochloric acid. After washing with chloroform to remove the water-insoluble oil, the mixture was concentrated to dryness under reduced pressure. The inorganic salt was removed by dissolving in ethanol and filtering, and the obtained filtrate was concentrated to dryness under reduced pressure and further dried under reduced pressure to obtain a pale red powder of Compound 3 (3.2 g,
37.3%).

Compound 2 (3.2 g, 10.2 mmol) and compound 3 (2.8 g, 10.2 mmol) were dissolved in 60 ml of pyridine and heated to 110 ° C., then 2.5 ml of triethyl orthoformate was added.
(15.0 mmol) was slowly added dropwise and the mixture was heated for 1 hour. The reaction solution was concentrated under reduced pressure, dissolved in chloroform and washed with water. The chloroform solution was concentrated under reduced pressure, and the obtained residue was subjected to silica gel column chromatography (solvent: methanol / chloroform) for separation and purification. The eluent was concentrated to dryness under reduced pressure, and the obtained solid was dried under reduced pressure to obtain a green-red metallic luster crystal of compound 4 (1.6 g, 33%).

Compound 4 (20 mg, 0.042 mmol) and N-hydroxysuccinimide (NHS) 6.4 mg (0.056 mmol)
Was dissolved in 100 μl of anhydrous DMF and kept at 0 ° C., 180 mg (0.874 mmol) of dicyclohexylcarbodiimide (DCC) was dissolved in 50 μl of anhydrous DMF, and the mixture was reacted at 0 ° C. overnight. Hydrogen chloride gas was blown into the reaction solution, and ether was added to obtain the target compound (A) (18.0 mg, 70.1
%). Compound (A) was added to PBS (pH 7.4) (NaCl: 8.0 g, KCl: 0.2
g, Na ₂ HPO ₄ : 11.5 g, KH ₂ PO ₄ : 0.2 g / water 1000 ml) shows the absorption spectrum of the solution.

Compound 4 (300 mg, 0.64 mmol) was added to DMF.
It was dissolved in 20 ml, 242 mg (0.81 mmol) of piperazinoethylmaleimide dihydrochloride monohydrate (PEM) and 120 μl (0.88 mmol) of triethylamine were added, and the mixture was cooled to 0 ° C. To this solution was added 680 mg (3.30 mmol) DCC and
The reaction was carried out at 0 ° C overnight. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, and the obtained residue was subjected to silica gel column chromatography (solvent: methanol / chloroform) for separation and purification to obtain a green-red metallic luster crystal of compound (B). Was
(350 mg, 78.6%).

Example 2: Preparation of compound (C)

Embedded image

Compound 1 (2.0 g, 6.3 mmol) was added to DMF 10
Dissolve in 0 ml of ethyl 6-bromohexanoate 8.4 g (37.6
Was added and the mixture was heated at 80 ° C. under a nitrogen atmosphere overnight. The reaction solution was concentrated under reduced pressure, and the residue was subjected to silica gel chromatography (solvent: methanol / chloroform) for separation and purification to obtain a reddish brown oil of compound 5 (3.4g, 4
0%). Compound 2 (2.0 g, 6.3 mmol) and Compound 5 (2.4
g, 6.3 mmol) in 10 ml of pyridine and 110 ℃
After heating, the mixture was slowly added dropwise with 1.6 ml (10.0 mmol) of triethyl orthoformate and heated for 1 hour. The reaction solution was concentrated under reduced pressure, the residue was dissolved in chloroform and washed with water.
The chloroform solution was concentrated under reduced pressure, and the obtained residue was subjected to silica gel column chromatography (solvent: methanol / chloroform) for separation and purification. The eluent was concentrated to dryness under reduced pressure, and the obtained solid was dried under reduced pressure to give compound 6
To give a green-red crystal (960 mg, 26.1%).

Compound 6 (200 mg, 0.35 mmol) was dissolved in 10 ml of acetonitrile to give 10 ml of hydrazine monohydrate.
(205.7 mmol) was added and the mixture was stirred at room temperature overnight. The reaction mixture was made weakly acidic with 1N hydrochloric acid, concentrated under reduced pressure, and the residue was purified with Sephadex LH20 / methanol to give compound (C).
Was obtained (130 mg, 72.3%).

Example 3: Preparation of compound (H)

Embedded image

Compound 2 (3.2 g, 10.2 mmol) and compound 3 (2.8 g, 10.2 mmol) were dissolved in 60 ml of pyridine, heated to 110 ° C., and then 1,3,3-trimethoxypropene.
2.0 g (15.1 mmol) was added and heated for 1 hour. The reaction solution was concentrated under reduced pressure, and the obtained residue was dissolved in chloroform and washed with water. The chloroform solution was concentrated under reduced pressure,
The obtained residue was subjected to silica gel column chromatography (solvent: methanol / chloroform) for separation and purification. The eluent was concentrated to dryness under reduced pressure, and the obtained residue was dried under reduced pressure to obtain a green metallic luster crystal of compound 7 (1.2 g,
23.8%). Compound 7 (200 mg, 0.40 mmol) was reacted with NHS 60 mg (0.52 mmol) in the same manner as in Example 1, and the reaction product was isolated and purified to obtain compound (H) (1.2 g, 23.8%). .

Example 4: Preparation of compound (J)

Embedded image

Compound 8 (4.0 g, 19.1 mmol) was added to DMF.
Dissolve in 60 ml, and add 13.0 g of ethyl 6-bromohexanoate (5
8.3 mmol) was added and the mixture was heated at 80 ° C. under a nitrogen atmosphere overnight. The reaction mixture was concentrated under reduced pressure, and the residual oil was washed with 50 ml of ether. Then, the obtained oil was dissolved in 100 ml of methanol and 7.0 g (175 g of sodium hydroxide) was added.
(Mmol) was dissolved in water and added, and the mixture was stirred at room temperature for 5 hours for hydrolysis. Methanol was distilled off under reduced pressure and then neutralized with 1N hydrochloric acid. After washing with chloroform to remove the water-insoluble oil, the mixture was concentrated to dryness under reduced pressure. The residue was dissolved in ethanol and filtered to remove inorganic salts, and the filtrate was concentrated to dryness under reduced pressure and dried under reduced pressure to obtain a pale blue powder of compound 9 (4.2 g, 67.9%).

Compound 8 (7.0 g, 19.1 mmol) was dissolved in 100 ml of acetonitrile to give 4.5 g (2 g of iodoethane).
8.9 mmol) was added and the mixture was heated under reflux overnight. The reaction solution was concentrated under reduced pressure, and ether was added to the residue to obtain gray-blue crystals of compound 10 (7.4 g, 84.8%). Glutaconaldehyde dianyl hydrochloride 6.0 g (24.2 mmol) was added to acetic anhydride 100 m.
Compound 10 (7.4 g, 20.3 mmol) was added under stirring. The mixture was heated to 100 ° C. and reacted for 1 hour, the reaction mixture was concentrated under reduced pressure, and ether was added to the residue to give green crystals.

The above green crystals were dissolved in 60 ml of pyridine, compound 9 (5.6 g, 17.3 mmol) was added, and the mixture was mixed at 60 ° C.
Reacted for 5 hours. The reaction solution was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography (solvent: methanol / chloroform) for separation and purification. The eluent was concentrated to dryness under reduced pressure, and the obtained solid was dried under reduced pressure to give a compound.
12 green crystals were obtained (3.8 g, 35.2%). Compound 12 (20 mg,
0.032 mmol) and NHS 4.9 mg (0.043 mmol) were dissolved in 100 ml of anhydrous DMF and kept at 0 ° C.
(Mmol) was dissolved in 50 ml of anhydrous DMF and added, and the mixture was reacted overnight. Hydrogen chloride gas was blown into the reaction solution, and ether was added to obtain the target compound (J) (10.5 mg, 43.2%).

Example 5: Preparation of compounds (K) and (L)

Embedded image

Compound 8 (5 g, 23.9 mmol) / DMF 100 ml
6-Bromohexanoic acid ethyl ester (6.32 ml, 35.
5 mmol) was added and the mixture was heated at 80 ° C. for 16 hours. The reaction mixture was concentrated under reduced pressure, 200 ml of ether was added to the residue for crystallization, and the obtained crystals were filtered and washed with ether,
Drying under reduced pressure gave compound 8 '. 1M for compound 8 '
Aqueous sodium hydroxide solution / methanol = 1/1 mixed solution
30 ml was added and the mixture was stirred at room temperature for 2 hours. Methanol was distilled off under reduced pressure, the aqueous solution was neutralized with a 4 M aqueous hydrochloric acid solution, and then washed three times with chloroform. The aqueous solution was concentrated under reduced pressure,
A red solid of compound 9 was obtained. Yield 5.75 g (Yield 74.4
%).

Compound 13 (5.0 g, 14.5 mmol) and glutaconaldehyde dianyl hydrochloride (4.23 g, 14.5 mmol) were suspended in a mixed solution of 20 ml of acetic anhydride and 300 ml of acetic acid and heated at reflux temperature for 5 hours. . The red solution was concentrated under reduced pressure, 200 ml of a mixed solution of ethyl acetate and water was added to the obtained residue to suspend it, and a black red solid was collected by filtration. After washing with water, it was dried under reduced pressure to obtain a black-red powder of compound 14. Yield 5.20
g (yield 66.1%).

Compound 14 (300 mg, 0.553 mmol) and compound 9 (179 mg, 0.553 mmol) were dissolved in 5 ml of pyridine,
The mixture was stirred at 50 ° C for 1 hour. The reaction mixture is concentrated under reduced pressure,
The obtained residue was dissolved in 10 ml of water to adjust the pH of the solution to 3, and then purified by a Sephadex LH20 column (solvent: methanol) to obtain Compound 15 as a black-green solid. yield
105 mg (yield 25.9%).

Compound 15 (106 mg, 0.145 mmol) / 50 v / v%
Sodium N-hydroxysuccinate sulfonate (65.2 mg, 0.287 mmol) and N, N'-dicyclohexylcarbodiimide (129 mg, 0.596 mmol) were added to 3 ml of an aqueous THF solution, and the mixture was reacted overnight at 4 ° C and then filtered. The filtrate was concentrated under reduced pressure at 20 ° C or lower. Ethyl acetate (10 ml) was added to the residue, and the generated crystals were washed with ether three times to give a black green solid of compound (K) (110 mg, 80.7%). MS (FAB) m / e = 906
(M ^-); fluorescence _{spectra: λ ex = 768 nm, λ} em = 807 nm (H
₂ O)

100 mg (0.11 mmol) of the above compound (K) was dissolved in 2 ml of methanol, and 1 g of sodium iodide (6.7
(5 mmol) methanol solution in 5 ml was added. Methanol
After concentrating to 1/2 under reduced pressure and cooling at 4 ° C. overnight, the precipitated crystals were collected by filtration. The obtained green crystals were dried with a vacuum desiccator to obtain 85 mg of compound (L) (yield 72%). The water solubility and infrared absorption spectrum data (IR) of the compound (K) before the salt formation of sodium iodide (intramolecular counter ion type) and the compound (L) after the salt formation were measured. Further, the iodine content was determined by the Schöniger method.

Infrared absorption spectra of the compound (L) incorporating sodium iodide as a salt and the counterion type compound (K) used as a raw material changed, which was observed in the intramolecular counterion type compound. Absorption at 2360 and 1740 cm ^{-1 was} abolished in compound (L) (see Fig. 5: a) compound (L): after salt formation, b) is compound (K): intramolecular counterion type, ie salt (Before formation). From these results, it is clear that the crystal structure is changed between the case where the intramolecular counterion is formed and the case where the salt of sodium iodide is formed.

Example 6: Synthesis of compounds (Q) and (R) According to the method of Example 5, indocyanine green-N-butanoic acid sulfosuccinimide ester was produced. 150 mg (0.17 mmol) of this compound was dissolved in 3 ml of methanol, and a solution of 1.5 g (10 mmol) of sodium iodide in 8 ml of methanol was added. Methanol was concentrated to 1/2 under reduced pressure, cooled at 4 ° C. overnight, and the precipitated crystals were collected by filtration. The obtained green crystals were dried with a vacuum desiccator to obtain 117 mg of compound (Q).
(66%). Water solubility data and infrared absorption spectrum data (IR) before and after sodium iodide salt formation were measured.
Further, the iodine content was determined by the Schöniger method.

Similarly, indocyanine green-N-hexanoic acid sulfosuccinimide ester 100 mg (0.11 mmo
l) in 2 ml of methanol and add sodium perchlorate.
A solution of 0.3 g (2.5 mmol) in 20 ml of methanol was added.
Methanol was concentrated to 1/4 under reduced pressure, cooled at 4 ° C. overnight, and the precipitated crystals were collected by filtration. The obtained green crystals were dried with a vacuum desiccator to obtain 23 mg of compound (R) (yield 21
%). The water solubility data and IR before and after salt formation of sodium perchlorate were measured.

[0057]

[Table 1] ─────────────────────── Example No. Intramolecular counterion type After double salt formation ─────────── ───────────── Example 5 Compound (K) Compound (L) Water solubility: 1 mg / 10 ml or more 1 mg / 0.8 ml IR (cm ^-1 ): 2360, 1740 Disappearing I content: -12.3% Example 6 Raw material Compound (Q) Water solubility: 1 mg / 10 ml or more 1 mg / 0.9 ml IR (cm ^-1 ): 2370, 1790 Absorption disappeared I content: -13.5% Example 6 Raw material Compound (R) Water solubility: 1 mg / 10 ml or more 1 mg / 5 ml IR (cm ^-1 ): 2520 Absorption disappeared ────────────────── ──────

Example 7: Synthesis of compound (I)

Embedded image

Compound 16 (5.0 g, 15.1 mmol) and glutaconaldehyde dianyl hydrochloride (4.40 g, 15.1 mmol) were suspended in a mixed solution of 20 ml of acetic anhydride and 300 ml of acetic acid and heated at reflux temperature for 5 hours. . The red solution was concentrated under reduced pressure, 200 ml of a mixed solution of ethyl acetate and water was added to the obtained residue to suspend it, and a black red solid was collected by filtration. After washing with water, it was dried under reduced pressure to obtain a black-red powder of compound 17. Yield 5.33
g (yield 65.2%).

Compound 17 (167 mg, 0.308 mmol) and compound 9 (100 mg, 0.309 mmol) obtained in Example 6 above were treated with pyridine 3
It was dissolved in ml and stirred at 50 ° C. for 1 hour. The reaction mixture was concentrated under reduced pressure, the resulting residue was dissolved in 10 ml of water to adjust the pH of the solution to 3, and then the Sephadex LH20 column was used.
Purification with (solvent: methanol) gave a black-green solid of compound 18. Yield 51 mg (yield 23.1%).

Compound 18 (30 mg, 41.8 μmol) / 50 v / v%
Sodium N-hydroxysuccinate sulfonate (16.8 mg, 77.4 μmol) and N, N'-dicyclohexylcarbodiimide (25.8 mg, 0.125 mmol) were added to 1 ml of an aqueous THF solution, and the mixture was reacted overnight at 4 ° C and then filtered. The filtrate was concentrated under reduced pressure at 20 ° C or lower. Add 10 ml of ethyl acetate to the residue,
The generated crystals were further washed with ether 3 times to obtain a black green solid of compound 19. Yield 34 mg (yield 88.8%). MS (FAB)
^{m / e = 892 (M -} ); fluorescence _{spectra: λ ex = 771nm, λ em} = 8
22 nm (water)

Compound 19 above was treated with a solution of sodium iodide in methanol according to the method of Example 6 to give compound (I)
I got Infrared absorption spectrum of compound 19 and compound (I)
Comparing the infrared absorption spectra of the compounds, the absorptions at 2360 and 1740 cm ^-1 observed in compound 19 were
It was not observed in (I).

Example 8: Labeling of protein with compound (A) and fluorescence detection 3.0 mg of bovine serum albumin (BSA) was dissolved in 5.0 ml of 0.1 M carbonate buffer of pH 8.5 to obtain 50 mM DMS of compound (A).
5 ml of O 2 solution was added and left at 37 ° C. for 1 hour. HPLC (gel column, fluorescence detection: excitation wavelength 550 nm, detection wavelength 569n
When this solution was measured using m), a peak of labeled BSA was observed in addition to the peak of compound (A). Fig. 2 shows the fluorescence spectrum of a solution prepared by dissolving the compound (A) in PBS at pH 7.4, and Fig. 3 shows the BSA labeled with the compound (A).
The fluorescence spectrum of the solution melt | dissolved in PBS of pH 7.4 is shown.
Furthermore, when the same measurement was carried out by labeling with different amounts of BSA, a good proportional relationship was observed between the fluorescence intensity and the amount of BSA as shown in Fig. 4, and detection of about 0.1 femtomolar was detected for BSA. Turned out to be the limit. The signal / noise ratio at this time was 3.

Example 9: Labeling of antibody with the compound of the present invention 1 mg of the compound (L) was dissolved in 0.94 ml of water to give a 1 mM solution. 1 mg of human IgG was dissolved in 1 ml of 50 mM sodium carbonate buffer having a pH of 8.5, 0.2 ml of the above solution containing the compound (L) was added to this solution, and the mixture was reacted at 30 ° C. for 1 hour. The reaction solution was applied to a Sephadex G-25 column to separate labeled human IgG. As a separation buffer, 50 mM phosphate buffer (pH 7.4) was used. The unreacted compound (L) remained on the upper end of the Sephadex gel, and the separation from the labeled IgG was good. Obtained labeled IgG
Was freeze-dried and stored frozen at -20 ° C. This labeled IgG
Fig. 6 shows the result of measuring 1 mg of the fluorescent substance by dissolving it in 100 ml of 50 mM phosphate buffer (pH 7.4).

[0065]

Industrial Applicability The compound of the present invention has a high fluorescence quantum yield, and thus is highly fluorescent and highly water-soluble. Further, the compound of the present invention is characterized in that the compound to be labeled can be labeled with a functional group selectively. Furthermore, since it has only one labeling group in the molecule, it does not crosslink the compound to be labeled, and the characteristic of the compound to be labeled can be maintained as it is even after labeling. Therefore, when the compound of the present invention is used, fluorescent labeling can be carried out while retaining the three-dimensional structure and physiological activity of biopolymer such as protein, and the spectroscopic property of indocyanine can be added to the target molecule.

[Brief description of the drawings]

FIG. 1 was prepared by adding the compound (A) of the present invention to PBS (NaCl:
8.0g, KCl: 0.2g, Na ₂ HPO ₄ : 11.5g, KH ₂ PO ₄ : 0.2g / water
It is a figure which shows the absorption spectrum of the solution melt | dissolved in 1000 ml.

FIG. 2 is a diagram showing a fluorescence spectrum of a solution prepared by dissolving the compound (A) of the present invention in PBS having a pH of 7.4.

FIG. 3 BSA labeled with the compound (A) of the present invention
FIG. 3 is a diagram showing a fluorescence spectrum of a solution in which is dissolved in PBS of pH 7.4.

FIG. 4 Labeling using the compound (A) of the present invention
It is a figure which shows the correlation between the fluorescence intensity of BSA, and the amount of BSA used for reaction.

FIG. 5 is a diagram showing infrared absorption spectra of a compound (L) incorporating sodium iodide as a salt and an intramolecular counterion type compound (K). In the figure, a) shows the infrared absorption spectrum of the compound (L), and b) shows the infrared absorption spectrum of the compound (K).

FIG. 6 Human labeled with the compound (L) of the present invention
It is a figure which shows the fluorescence spectrum of IgG.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location G01N 21/78 G01N 21/78 C // (C07D 403/14 207: 404 209: 60) (C07D 403/14 207: 444 209: 60)

Claims (7)

[Claims] 1. The following formula: (In the formula, A ¹ and A ² each independently represent a benzene ring or a naphthalene ring, R ¹ and R ² are each independently a hydrogen atom,
An alkyl group, an aryl group, a free or dissociated sulfonic acid group, or an alkoxy group, R ³ represents an alkyl group, a free or dissociated sulfonic acid alkyl group, or an aminoalkyl group or an ammonioalkyl group, and n is
Represents an integer of 1 to 3, X ⁻ represents an anion species as necessary, and Z represents the following formula: Is a group selected from the group consisting of, M ⁺ is an alkali metal ion, W and Y each independently have 1 to 1 carbon atoms.
Represents an alkylene group of 0, or W and Y are 1 selected from the group consisting of oxygen atom, nitrogen atom, and sulfur atom.
A compound having 1 or more atoms and having 1 to 10 carbon atoms). 2. A ¹ and A ² are both naphthalene rings,
R ¹ and R ² are both hydrogen atoms, and R ³ is the number of carbon atoms dissociated
A linear alkyl sulfonate group of 2 to 5, n is 3, Y is a linear alkylene group of 2 to 5 carbon atoms, and Z is a succinimidooxy group or an alkali metal sulfosuccinimidooxy group. The compound according to 1. 3. The compound according to claim 1, wherein X ⁻ is an iodine ion, and R ³ is a linear alkyl sulfonate group having 2 to 5 carbon atoms which forms a salt with a sodium ion. 4. A fluorescent labeling reagent comprising the compound according to any one of claims 1 to 3. 5. The reagent according to claim 4, which is used for fluorescent labeling of a compound having one or more functional groups selected from the group consisting of an amino group, a hydroxyl group, a carbonyl group, and a sulfhydryl group. 6. The reagent according to claim 5, wherein the compound having a functional group is a biopolymer. 7. The reagent according to claim 6, wherein the biopolymer is selected from the group consisting of proteins, glycoproteins, lipids, phospholipids, polysaccharides, and nucleic acids.