JP5319130B2 - Keiko solvatochromic dye and method of use thereof - Google Patents

Description

  The present invention relates to a fluorescent solvatochromic dye whose emission wavelength (that is, emission color) changes depending on the polarity of a solvent, and a method for using the same.

Fluorescent solvatochromic dye is a dye whose color changes depending on the solvent polarity around the molecule, and is currently used as a probe for observing the dynamics of biomolecules (especially lipid molecules) in real time with a fluorescent microscope. ing. Since these discoloration mechanisms do not require contact with a specific chemical species, antigen-antibody reactions and single nucleotide polymorphisms, which are difficult with other fluorescent dyes, can be highly sensitive.
Examples of this fluorescent solvatochromic dye include NBD, dansyl, prodan, and Dapoxyl. Among them, Dapoxyl has the best performance. It is a rare solvatochromic dye, and has an absorption band that can be photoexcited in the long wavelength region (near 370 nm), a large Stokes shift, and a quantum yield of fluorescence. High (Non-Patent Document 1).
On the other hand, a thiophene derivative approximate to the fluorescent solvatochromic dye of the present invention is known as a nonlinear optical material (Non-patent Document 2).

Photochemistry and Photobiology, 1997, 66 (4): 424-431 J. Heterocyclic Chem., 31, 1005-1009 (1994)

However, commercially available fluorescent solvatochromic dyes (Non-patent Document 1) have a shorter excitation wavelength than other fluorescent dyes, so that they are photodamaged when used with cells and biomolecules. In addition, there is a possibility that strong charge transfer interaction between molecules tends to work, so that the solubility is poor and synthesis and purification are difficult.
Accordingly, the present invention provides (1) a long excitation wavelength, (2) a large molar extinction coefficient, (3) a high fluorescence quantum yield, and (4) a new fluorescence solver that greatly shifts the emission wavelength even with a slight change in polarity. The object was to provide a tochromic dye.

The fluorescent solvatochromic dye can be synthesized mainly by condensing two kinds of raw materials, but its synthesis is difficult, and even if synthesized, it does not necessarily show fluorescent solvatochromism. Therefore, the present inventors separately synthesized a chromophore skeleton (thiophene skeleton or furan skeleton), an electron donating site, and an electron withdrawing site to produce a Suzuki-Miyaura cross-coupling method (Chemical Reviews, Vol. 95, p2457-2483). , 1995), and various thiophene derivatives or furan derivatives having sulfonic acid as an electron withdrawing site were synthesized by directly connecting them.
In particular, by protecting the sulfonic acid group as a bulky alkyl ester, the solubility in organic solvents was greatly improved, facilitating mass synthesis and purification. This protecting group could be quantitatively deprotected by reacting with a quaternary ammonium salt in DMF, and it was possible to synthesize various derivatives that are water-soluble and exhibit fluorescent solvatochromism. Furthermore, from this derivative, it is possible to synthesize a sulfonic acid derivative that can be labeled, and various derivatives that have increased electron withdrawing property to increase the emission wavelength shift width.

（式中、Ｘは酸素原子（−Ｏ−）又は硫黄原子（−Ｓ−）を表し、
ｍは１〜４の整数を表し、
Ｒ^１は、下記（ａ）〜（ｅ）のいずれかを表し、
（ａ）−Ｏ−Ｒ^８（式中、Ｒ^８は、アルキル基の側鎖を有するアルキル基を表す。）
（ｂ）−Ｙ（式中、Ｙはハロゲン原子又はハロゲン化アルキル基を表す。）
（ｃ）−ＮＨ（ＣＨ_２）_ｎＮＨ_２（式中、ｎは１〜１４の整数を表す。）
（ｄ）−ＮＨ（ＣＨ_２）_ｎＮＨＣＯ（ＣＨ_２）_ｏＹ（式中、ｎ及びｏはそれぞれ１〜１４の整数を表し、Ｙはハロゲン原子を表す。）
（ｅ）−ＮＨ（ＣＨ_２）_ｎＣＨ_３（式中、ｎは１〜２０の整数を表す。）
Ｒ^２及びＲ^３は、それぞれ同じであっても異なってもよく、水素原子、アルコキシ基、アシルアミノ基、アルキル基、ハロゲン置換アルキル基、アミノ基、ヒドロキシ基又はハロゲン原子を表し、但し、Ｒ^２及びＲ^３は共同して芳香族若しくはエーテル結合を含んでもよい脂肪族の５、６又は８員環を形成してもよく、Ｒ^４及びＲ^５は、それぞれ同じであっても異なってもよく、水素原子又はアルキル基を表し、Ｒ^６及びＲ^７は、それぞれ同じであっても異なってもよく、水素原子、又は置換基を有していてもよいアルキル基を表し、但し、Ｒ^４及びＲ^６は共同して芳香族若しくは脂肪族の５員環又は６員環を形成してもよく、Ｒ^５及びＲ^７は共同して芳香族若しくは脂肪族の５員環又は６員環を形成してもよい。）で表されるケイ光ソルバトクロミック色素、又はこのケイ光ソルバトクロミック色素から成る極性検査薬である。 That is, the present invention has the following formula:

(In the formula, X represents an oxygen atom (—O—) or a sulfur atom (—S—),
m represents an integer of 1 to 4,
R ¹ represents any of the following (a) to (e):
(A) ^{-O-R 8} (wherein, ^{R 8} is. Represents an alkyl group having a side chain alkyl group)
(B) -Y (wherein Y represents a halogen atom or a halogenated alkyl group)
(C) —NH (CH ₂ ) _n NH ₂ (wherein n represents an integer of 1 to 14)
_{(D) -NH (CH 2)} n NHCO (CH 2) o Y ( wherein, n and o each represents an integer of 1 to 14, Y represents a halogen atom.)
(E) -NH (CH ₂ ) _n CH ₃ (wherein n represents an integer of 1 to 20)
R ² and R ³ may be the same or different and each represents a hydrogen atom, an alkoxy group, an acylamino group, an alkyl group, a halogen-substituted alkyl group, an amino group, a hydroxy group, or a halogen atom, provided that R ² And R ³ together may form an aliphatic 5-, 6- or 8-membered ring which may contain an aromatic or ether bond, and R ⁴ and R ⁵ may be the same or different, respectively. Represents a hydrogen atom or an alkyl group, and R ⁶ and R ⁷ may be the same or different and each represents a hydrogen atom or an alkyl group which may have a substituent, provided that R ⁴ and R ⁶ may jointly form an aromatic or aliphatic 5-membered ring or 6-membered ring, and R ⁵ and R ⁷ together form an aromatic or aliphatic 5-membered ring or 6-membered ring May be. ) Or a polar test agent comprising this fluorescent solvatochromic dye.

The present invention also provides a fluorescent solvatochromic dye formed by linking this fluorescent solvatochromic dye to a protein or peptide containing amino acids containing amino groups, hydroxyl groups, thiol groups or carboxyl groups, or poly or oligonucleotides. It is a dye complex.
Furthermore, the present invention provides a step of irradiating ultraviolet light to the fluorescent solvatochromic dye, the fluorescent solvatochromic dye complex or a solution containing them, and the emission wavelength or emission color from the fluorescent solvatochromic dye. It is a method for inspecting the polarity around the fluorescent solvatochromic dye comprising measuring.

The fluorescent solvatochromic dye of the present invention has improved solubility in various solvents. As a result, since purification becomes easy, it is possible to easily obtain a large amount of a fluorescent solvatochromic dye having high purity.
In addition, since the fluorescent solvatochromic dye of the present invention has a longer excitation wavelength than conventional dyes, it can prevent photodamage of cells and biomolecules as much as possible. Can be observed.
Since this fluorescent solvatochromic dye can be linked to lipids, proteins, nucleic acids, etc., it is used as a fluorescent probe that detects changes in microscopic environments such as biomolecules with fluorescent color change with high sensitivity. be able to.

Ｘは酸素原子（−Ｏ−）又は硫黄原子（−Ｓ−）を表す。
色素母骨格として、チオフェン骨格又はフラン骨格を用意して、鈴木−宮浦クロスカップリング法で電子供与部位及び電子吸引部位を連結することにより、電子吸引部位としてスルホン酸を持つチオフェン誘導体やフラン誘導体を容易に合成することができる。
また、色素母骨格として、複数のチオフェン骨格又はフラン骨格を含んでもよく、ｍは１〜４、好ましくは１又は２の整数を表す。 The fluorescent solvatochromic dye used in the present invention is represented by the following formula.

X represents an oxygen atom (—O—) or a sulfur atom (—S—).
Prepare a thiophene skeleton or furan skeleton as the chromophoric skeleton, and connect the electron donating site and the electron withdrawing site by the Suzuki-Miyaura cross-coupling method to obtain a thiophene derivative or furan derivative having sulfonic acid as the electron withdrawing site. It can be easily synthesized.
In addition, the chromophore skeleton may include a plurality of thiophene skeletons or furan skeletons, and m represents an integer of 1 to 4, preferably 1 or 2.

R ¹ is first represented by (a) below.
(A) -O-R ⁸
In the formula, R ⁸ represents an alkyl group having an alkyl group side chain. R ⁸ is preferably represented by — (CH ₂ ) _o —C (R ⁹ ) ₃ , and each R ⁹ is independently at least two of which are alkyl groups, preferably having 1 to 4 carbon atoms. It is a linear alkyl group, the remainder represents a hydrogen atom, and o represents an integer of 0-2.
In general, a fluorescent solvatochromic dye has high planarity, a strong electromigration transfer interaction between molecules, and low solubility in a solvent. For example, a compound (compound 25) in which R ⁸ is a methyl group described in Non-Patent Document 2 does not dissolve in DMF. However, in the fluorescent solvatochromic dye of the present invention, since R ⁸ is an alkyl group that is not linear (that is, a bulky alkyl group), a steric hindrance that hinders such intermolecular interaction occurs, Increased solubility in As a result, there are advantages such as easy measurement of fluorescence in a solution and easy purification at the time of manufacture.

R ² and R ³ may be the same or different and each represents a hydrogen atom, an alkoxy group, an acylamino group, an alkyl group, a halogen-substituted alkyl group, an amino group, a hydroxy group, or a halogen atom. The alkoxy group, acylamino group and alkyl group each preferably have 1 to 20 carbon atoms, more preferably 1 to 4 carbon atoms. The halogen atom is preferably a chlorine atom or a fluorine atom.
R ² and R ³ are preferably electron donating groups. Examples of such an electron donating group include an alkoxy group, an acylamino group, an alkyl group, an amino group, and a hydroxy group, preferably an alkoxy group, an alkyl group, and a hydroxy group.
R ² and R ³ may jointly form an aliphatic 5-, 6- or 8-membered ring which may contain an aromatic or ether bond. For example, R ² and R ³ on the same thiophene ring or furan ring may jointly form an aliphatic 5- or 6-membered ring that may contain an aromatic or ether bond (—O—). it may, further, when m is 2 or more, R ² or R ³ on one thiophene ring or a furan ring cooperates with R ² or R ³ on adjacent thiophene ring or a furan ring, aromatic or ether An aliphatic 5-, 6- or 8-membered ring which may contain a bond (—O—) may be formed.

R ⁴ and R ⁵ may be the same or different and each represents a hydrogen atom or an alkyl group. This alkyl group is preferably an alkyl group having 1 to 4 carbon atoms.
R ⁶ and R ⁷ may be the same or different and each represents a hydrogen atom or an alkyl group that may have a substituent. This alkyl group is preferably an alkyl group having 1 to 4 carbon atoms, and examples of the substituent include a hydroxyl group, an amino group, a thiol group, and a sulfonic acid group.
However, R ⁴ and R ⁶ may jointly form an aromatic or aliphatic 5-membered ring or 6-membered ring containing a nitrogen atom, and R ⁵ and R ⁷ together form an aromatic group containing a nitrogen atom. Alternatively, an aliphatic 5-membered ring or 6-membered ring may be formed.

Various derivatives can be easily obtained by a known method from the dye in which R ¹ is (a) —O—R ⁸ . These derivatives can be used for various polarity tests as they are or by binding them to proteins or nucleotides. Here are some examples.

A dye wherein R ¹ is (a) —O—R ⁸ (R ⁸ is a neopentyl group, R ^{2 to} R ⁵ are hydrogen atoms, and R ⁶ and R ⁷ are methyl groups is referred to as “compound 1”. An example of modification will be described below. The reaction is shown in FIG. However, in the following, Compound 1 was used for ease of explanation, but the same reaction is possible for other compounds of the present invention.
Compound 1 (neopentyl ester) is protected with sulfonic acid, but can be deprotected by stirring in DMF at 160 ° C. in the presence of tetramethylammonium chloride to give sulfonic acid (Compound 5) (Tetrahedron). Letters, vol. 38 (3), 355-358 (1997)). Sulfonic acid (Compound 5) can be derivatized to acid chloride (Compound 6) by reaction with phosphorus pentachloride. Acid chloride (compound 6) is easily condensed with an amino group.

Various functional groups can also be introduced into this acid chloride (compound 6) via a sulfonic acid amide bond. For example, a compound having an amino group at the terminal can be obtained by linking with diamine (for example, ethylenediamine) (compound 7).
Moreover, this compound 7 is made to react with bromoacetyl bromide etc., and the haloacetylamide body (compound 8) which has a halogen atom at the terminal can be synthesize | combined.
Furthermore, an acid chloride (compound 6) can be reacted with an alkylamine to link alkyl chains (compound 9).
In general, acid chloride and acid amide have stronger electron withdrawing properties than esters. In general, when the electron withdrawing property and electron donating property of the dye are strong, both the absorption maximum wavelength and the fluorescence maximum wavelength are greatly shifted by a long wavelength, and the Stokes shift is increased (Non-patent Document 1). Therefore, the fluorescent dyes of compounds 6 to 9 have longer absorption and fluorescence wavelengths than compound 1, and a large Stokes shift.
As described above, the functional substituent can be condensed by the above-described method to obtain a derivative capable of further improving optical properties and providing a labeled site.

That is, the dye compound in which R ¹ is the following (b) to (e) can be derived from the dye in which R ¹ is (a) —O—R ⁸ .
(B) -Y (wherein Y represents a halogen atom. The halogen atom is preferably a fluorine atom or a chlorine atom).
(C) —NH (CH ₂ ) _n NH ₂ (wherein n represents an integer of 1 to 14, preferably 1 to 4)
(D) —NH (CH ₂ ) _n NHCO (CH ₂ ) _o Y (wherein n and o independently represent an integer of 1 to 14, preferably 1 to 4, Y represents a halogen atom or an alkyl halide) The halogen atom is preferably a fluorine atom or a chlorine atom, and the halogenated alkyl group is a perfluoroalkyl chain having 1 to 34 carbon atoms, preferably 1 to 2 carbon atoms, such as perfluoromethane. .)
(E) -NH (CH ₂ ) _n CH ₃ (wherein n represents an integer of 1 to 20, preferably 1 to 4)

Furthermore, by binding such a dye compound to various amino acids and nucleotides constituting a protein, a complex can be formed and labeled. Examples thereof are shown in FIGS. 2 (1) to (4).
(1) A diamine compound can be linked to the compound 6 (acid chloride) and linked to an amino group-containing amino acid (asparagine, glutamine, lysine, arginine, etc.) of the protein by an enzyme (transglutaminase) reaction. As a result, the protein can be a labeled protein in which R ¹ is the following (f).
(F) —NH—R ¹⁰ (wherein R ¹⁰ is a protein residue obtained by removing the amino group from a protein containing an amino group-containing amino acid, or poly or oligo from which the amino group of the base of poly or oligonucleotide is removed. Represents a nucleotide residue.)
(2) A compound (compound 7) obtained by linking this diamine compound can be linked to an amino group-containing amino acid (asparagine, glutamine, lysine, arginine, etc.) of a protein by an enzyme (transglutaminase) reaction. As a result, the protein can be a labeled protein in which R ¹ is the following (g).
_{(G) -NH (CH 2)} n NH-R 10 ( ^{wherein, R 10} is a protein comprising an amino group-containing amino acids, .n representing the protein residue obtained by removing the amino group is, for example, 1 14 and preferably represents an integer of 1 to 4.)

(3) The dye compound can be linked to a thiol group-containing amino acid (cysteine or the like) by reacting with bromoacetyl bromide. As a result, the protein can be a labeled protein in which R ¹ is the following (h).
_{(H) -NH (CH 2)} n NHCO (CH 2) o -S-R 11 ( ^{wherein, R 11} is a protein containing a thiol group-containing acids, represents a protein residue obtained by removing the thiol group. n and o each represent, for example, an integer of 1 to 14, preferably 1 to 4.)
(4) When the compound 7 (dye having an amino terminus) is reacted with a protein having a carboxyl group activated with carbodiimide such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, a carboxyl group-containing amino acid (asparagine) Acid) and the like. As a result, the protein can be a labeled protein in which R ¹ is the following (i).
(I) —NH (CH ₂ ) _n NHSO ₂ —R ¹² (wherein R ¹² represents a protein residue obtained by removing the carboxyl group from a protein containing a carboxyl group-containing amino acid. -14, preferably an integer of 1-4.
In addition, amino acids having a hydroxyl group (serine, threonine, tyrosine, etc.) can be labeled by binding to these compounds by inducing to an aldehyde group.

In such a dye complex, the change in polarity around the dye can be known, so that it can be used for the following applications, for example.
(1) It is useful as a biological membrane probe in the field of fluorescent dyes for bioimaging. That is, it can be used for a molecular membrane probe that is linked to a lipid molecule such as an alkyl chain and whose fluorescence color changes in response to a local environment. As a synthesis method, a sulfonic acid chloride compound (for example, compound 6 in FIG. 1) is reacted with a lipid molecule such as alkyl having an amino group at the terminal to react with a target probe (for example, compound 9 in FIG. See)). This molecule emits long-wavelength fluorescence in hydrophilic environments and short-wavelength fluorescence in hydrophobic environments, so it can be used for highly sensitive real-time measurement of biological membrane dynamics, such as detection of bacterial cell membranes, evaluation of antibacterial activity, and evaluation of drug delivery systems. it can.

(2) It is useful for highly sensitive detection of antigen / antibody reaction in the field of immunoassay. That is, a probe capable of labeling a specific amino acid residue of an antigen or antibody and measuring the antigen-antibody reaction with high sensitivity and quantitative (for example, FIG. 2 (1) to (4), the above (f) to (i ) Reference.) Can be used. If a specific amino acid site in the vicinity of the antigen-antibody recognition site can be labeled, it is exposed on the protein surface before the reaction, so that it has a long wavelength, and after the reaction, it is incorporated into a hydrophobic field, so it emits a short wavelength fluorescence. I think that. Since fluorescent solvatochromic dyes do not require recognition of specific substituents, they can be used universally for various antigen-antibody reactions. In addition, since the fluorescent solvatochromic dye emits multiple wavelengths of fluorescent light, the antigen-antibody reaction can be detected quantitatively by calculating the intensity ratio of the wavelengths.

(3) In the detection of single nucleotide polymorphism, it can be used as a probe for identifying the base sequence of a specific site with a fluorescent color with high sensitivity. For example, as shown in FIG. 3, a derivative having an amino group at the terminal is reacted with a sulfonic acid chloride compound (for example, compound 6 in FIG. 1) from a base in a nucleic acid via an appropriate spacer (for example, a methylene chain). Then, the vicinity of the base can be fluorescently labeled. Nucleic acid amidites are synthesized by a conventional method, and a nucleic acid polymer incorporated into an arbitrary base sequence is synthesized by solid phase synthesis. Hybridization of a completely complementary nucleic acid polymer to this arbitrary sequence will form a hydrophobic field, so the fluorescence will be short wavelength, but hydrophilic if base pair mismatches are present in the vicinity of the dye. Therefore, the fluorescent light has a long wavelength, and the base sequence can be identified with high sensitivity by the fluorescent color.
In these applications, since the dye of the present invention has a long absorption wavelength, photodamage of protein and DNA due to excitation light can be avoided, which is advantageous over conventional dyes.

The following examples illustrate the invention but are not intended to limit the invention.
Synthesis example 1
In this synthesis example, 4- (5- (4- (dimethylamino) phenyl) -thiophen-2-yl) -benzenesulfonic acid 2,2-dimethylpropyl ester (Compound 1) was synthesized. The synthesis route is shown in FIG.
Dissolve neopentyl alcohol (4.53 g, 51.4 mmol) in 50 mL of chloroform, add pyridine (8.33 mL, 103 mmol), and add 4-bromo-benzenesulfonic acid chloride (manufactured by Tokyo Chemical Industry Co., Ltd.) at 0 ° C. under a nitrogen stream ( A 12.0 g, 47.0 mmol) chloroform solution (20 mL) was added dropwise over 45 minutes. Thereafter, the mixture was stirred at room temperature for 23 hours, and 0.1% hydrochloric acid was added. Diethyl ether was added to extract the organic layer, which was then washed once with 0.1% hydrochloric acid, three times with deionized water and once with saturated brine, and the organic layer was dried over sodium sulfate. After distilling off the solvent, the remaining solid was recrystallized from hexane to obtain white scaly compound 2 (4.17 g, 13.6 mmol, 29%). The analysis result of the produced compound 2 (4-Bromo-benzenesulfonic acid 2,2-dimethylpropyl ester) is shown below.
¹ H-NMR (400MHz, CDCl ₃ , TMS, rt) δ 7.76 (d, J = 9 Hz, 2H), 7.69 (d, J = 9 Hz, 2H), 3.69 (s, 2H), 0.91 (s, 9H).

Compound 2 (0.80 g, 2.6 mmol) obtained above, bis (pinacolato) diboron (manufactured by Wako Pure Chemical Industries, Ltd.) (0.86 g, 3.4 mmol), [1,1′-bis (diphenylphosphino) ferrocene] -Dichloropalladium (II) dichloromethane complex (manufactured by Sigma Aldrich Japan Co., Ltd.) (0.07g, 0.09mmol) and potassium acetate (0.83g, 8.5mmol) are dissolved in 9.6mL of DMSO and stirred at 90 ° C for 1 hour and a half in a nitrogen stream did. After allowing to cool, chloroform was added and the organic layer was washed three times. After salting out, the organic layer was dried over sodium sulfate, filtered and evaporated. Purification by silica gel column chromatography (hexane: methylene chloride = 9: 1 → methylene chloride → ethyl acetate: methylene chloride = 1: 19) and evaporation of the solvent gave a white solid (compound 3) (0.75 g, 2.1 mmol, 82%) Got. The analysis results of Compound 3 (4- (4,4,5,5-tetramethyl- [1,3,2] dioxaborolan-2-yl) benzenesulfonic acid 2,2-dimethylpropyl ester) produced below are shown. .
¹ H-NMR (400MHz, CDCl ₃ , TMS, rt) δ 7.96 (d, J = 9 Hz, 2H), 7.87 (d, J = 9 Hz, 2H), 3.69 (s, 2H), 1.35 (s, 12H), 0.91 (s, 9H).

2,5-dibromothiophene (manufactured by Wako Pure Chemical Industries, Ltd.) (40.0 mg, 16.5 mmol) and compound 3 obtained above (41.0 mg, 11.6 mmol) were dissolved in a mixed solvent of 5 mL of toluene and 3 mL of methanol, 1.3 mL of 1M sodium carbonate aqueous solution and tetrakis (triphenylphosphine) palladium (0) (manufactured by Wako Pure Chemical Industries, Ltd.) (23 mg, 20 μmol) were added. Vacuum degassing and nitrogen substitution were repeated three times, and the mixture was stirred at 80 ° C. for 3 hours. After allowing to cool, it was dissolved in chloroform, washed twice with deionized water, and the organic layer was dried over sodium sulfate. After filtration to remove sodium sulfate, the filtrate was evaporated, and then purified by silica gel column chromatography (chloroform) and evaporated to give a white solid (compound 4) (32 mg, 8.2 mmol, 71%). . The analysis result of the produced compound 4 (4- (5-bromo-thiophen-2-yl) benzenesulfonic acid 2,2-dimethylpropyl ester) is shown below.
TLC; R _f = 0.09 (eluent: chloroform)
¹ H-NMR (400 MHz, CDCl ₃ , TMS, rt); δ 7.89 (d, J = 9 Hz, 2H), 7.66 (d, J = 9 Hz, 2H), 7.20 (d, J = 4 Hz, 1H ), 7.09 (d, J = 4 Hz, 1H), 3.70 (s, 2H), 0.91 (s, 9H).

4- (Dimethylamino) phenylboronic acid (Sigma Aldrich Japan Co., Ltd.) (13.6 mg, 82 μmol) and compound 4 obtained above (32 mg, 82 μmol) are dissolved in a mixed solvent of 10 mL of toluene and 5 mL of methanol. 0.66 M sodium carbonate aqueous solution 1.0 mL and tetrakis (triphenylphosphine) palladium (0) (manufactured by Wako Pure Chemical Industries, Ltd.) (12 mg, 10 μmol) were added. Vacuum degassing and nitrogen substitution were repeated three times, and the mixture was stirred at 80 ° C. for 3 hours. After allowing to cool, it was dissolved in chloroform, washed twice with deionized water, and the organic layer was dried over sodium sulfate. Sodium sulfate was removed by filtration, and the filtrate was evaporated to obtain a yellow powder (Compound 1) by silica gel column chromatography purification (chloroform) and solvent distillation. The analysis result of the produced compound 1 (4- (5- (4- (dimethylamino) phenyl) -thiophen-2-yl) benzenesulfonic acid 2,2-dimethylpropyl ester) is shown below.
TLC; R _f = 0.07 (eluent: chloroform)
¹ H-NMR (400 MHz, CDCl ₃ , TMS, rt); δ 7.86 (d, J = 9 Hz, 2H), 7.73 (d, J = 9 Hz, 2H), 7.52 (d, J = 9 Hz, 2H ), 7.39 (d, J = 4 Hz, 1H), 7.16 (d, J = 4 Hz, 1H), 3.70 (s, 2H), 3.01 (s, 6H), 0.91 (s, 9H).

Synthesis example 2
In this synthesis example, 4- (5- (4- (dimethylamino) phenyl) -furan-2-yl) -benzenesulfonic acid 2,2-dimethylpropyl ester (Compound 13) was synthesized. The synthesis route is shown in FIG.
2,5-Dibromofuran (Tokyo Chemical Industry Co., Ltd.) (0.405 g, 1.77 mmol), Compound 3 (0.6 g, 1.69 mmol), 4- (dimethylamino) phenylboronic acid (0.292 g, 1.77 mmol) in 25 ml of toluene Dissolve in 10 ml of methanol, add Na ₂ CO ₃ (0.56 g, 5.28 mmol) and Pd (PPh ₃ ) ₄ (0.097 g, 84 μmol) dissolved in 10 ml of water, and perform degassing and N ₂ substitution three times. Stir at 80 ° C. for 5 hours. After allowing to cool, the mixture was extracted 3 times with chloroform and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure, and the residue was separated and purified by silica gel chromatography (eluent; chloroform: hexane = 4: 1) to obtain a yellow solid (compound 13) (0.137 g, 20%). The analysis result of the produced compound 13 (4- (5- (4- (Dimethylamino) phenyl) -furan-2-yl) -benzenesulfonic acid 2,2-dimethylpropyl ester) is shown below.
TLC; R _f = 0.23 (chloroform: hexane = 4: 1)
¹ H-NMR (400 MHz, CDCl ₃ , TMS, rt); δ 7.88 (d, J = 9 Hz, 2H), 7.82 (d, J = 9 Hz, 2H), 7.63 (d, J = 9 Hz, 2H), 6.90 (d, J = 4Hz, 1H), 6.75 (d, J = 9Hz, 2H), 6.56 (d, J = 4Hz, 1H), 3.69 (s, 2H), 3.00 (s, 6H), 0.91 (s, 9H).

Synthesis example 3
In this synthesis example, 4- (5- (4- (dimethylamino) phenyl) -3,4-ethylenedioxythiophen-2-yl) benzenesulfonic acid 2,2-dimethylpropyl ester (Compound 16) was synthesized. . The synthesis route is shown in FIG.
3,4-Ethylenedioxythiophene (Sigma Aldrich Japan Co., Ltd.) (1.42 g, 10 mmol) dissolved in 20 ml of 50% acetic acid in THF and N-bromo dissolved in 20 ml of 50% acetic acid in THF under ice cooling Succinimide (manufactured by Wako Pure Chemical Industries, Ltd.) (3.74 g, 21 mmol) was added dropwise for 1 hour, followed by stirring at room temperature for 2 hours. After completion of the reaction, 100 ml of water was added, and the precipitated solid was separated by suction filtration. Further, 100 ml of water was added to the filtrate, and the precipitated solid was separated by suction filtration. These solids were further washed with water and dried under reduced pressure to obtain white crystals (compound 14) (2.67 g, 89%). The analysis result of the produced compound 14 (2,5-Dibromo-3,4-ethylenedioxythiophene) is shown below.
TLC; R _f = 0.49 (hexane: ethyl acetate = 3: 1)
¹ H-NMR (400 MHz, CDCl ₃ , TMS, rt); δ 4.27 (s, 4H).

Compound 14 (0.2 g, 0.66 mmol) and compound 3 (0.187 g, 0.528 mmol) obtained above were dissolved in 20 ml of toluene and 10 ml of methanol, and Na ₂ CO ₃ (0.106 g, 1.0 mmol) and Pd dissolved in 5 ml of water. (PPh ₃ ) ₄ (0.038 g, 33 μmol) was added, degassing and N ₂ substitution were performed 3 times, and the mixture was stirred at 80 ° C. for 4 hours.
After allowing to cool, the mixture was extracted 3 times with chloroform and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure, and the residue was separated and purified by silica gel chromatography (eluent: chloroform) to obtain a yellow solid (Compound 15) (0.148 g, 63%). The analysis result of the compound 15 (4- (5-Bromo-3,4-ethylenedioxythiophen-2-yl) benzenesulfonic acid 2,2-dimethylpropyl ester) produced below is shown.
TLC; R _f = 0.18 (hexane: chloroform = 1: 1)
¹ H-NMR (400 MHz, CDCl ₃ , TMS, rt); δ 7.92 (d, J = 9 Hz, 2H), 7.88 (d, J = 9 Hz, 2H), 4.45 (s, 4H), 3.67 (s, 2H ), 0.92 (s, 9H).

Compound 15 (0.148 g, 0.33 mmol), 4- (dimethylamino) phenylboronic acid (0.065 g, 0.40 mmol) obtained above was dissolved in 20 ml of toluene and 10 ml of methanol, and Na ₂ CO ₃ (0.07 dissolved in 5 ml of water). g, 0.66 mmol) and Pd (PPh ₃ ) ₄ (0.03 g, 26 μmol) were added, and degassing and N ₂ substitution were further performed three times, followed by stirring at 80 ° C. for 21 hours. After allowing to cool, the mixture was extracted 3 times with chloroform and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure, and the residue was separated and purified by silica gel chromatography (eluent; chloroform: hexane = 99: 1) to obtain a yellow solid (Compound 16) (0.0153 g, 10%). The analysis result of the produced compound 16 (4- (5- (4- (Dimethylamino) phenyl) -3,4-ethylenedioxythiophen-2-yl) benzenesulfonic acid 2,2-dimethylpropyl ester) is shown below.
TLC; R _f = 0.43 (chloroform: ethyl acetate = 99: 1)
¹ H-NMR (400 MHz, CDCl ₃ , TMS, rt); δ 7.87 (d, J = 9 Hz, 2H), 7.82 (d, J = 9 Hz, 2H), 7.63 (d, J = 9 Hz, 2H), 6.74 (d, J = 9Hz, 2H), 4.40 (d, J = 5 Hz, 2H), 4.36 (d, J = 5 Hz, 2H), 3.67 (s, 2H), 2.99 (s, 6H), 0.91 ( s, 9H).

Synthesis example 4
In this synthesis example, (2- (4- (5- (4- (dimethylamino) phenyl) thiophen-2-yl) benzenesulfonic acid amino) ethyl) carbamic acid tert-butyl ester (Compound 19) was synthesized. The synthesis route is shown in FIG.
4-Bromo-benzenesulfonic acid chloride (Tokyo Chemical Industry Co., Ltd.) (1.60 g, 6.3 mmol) dissolved in 60 ml of methylene chloride and N-Boc- dissolved in a mixed solution of 50 ml of methylene chloride and 2 ml (14 mmol) of triethylamine After adding dropwise to ethylenediamine (manufactured by Wako Pure Chemical Industries, Ltd.) (0.84 g, 5.2 mmol) for 1 hour, the mixture was stirred for 25 hours. After completion of the reaction, 50 ml of water was added for washing, followed by extraction three times with methylene chloride and drying over Na ₂ SO ₄ . The solvent was removed under reduced pressure, and the residue was separated and purified by silica gel chromatography (eluent; chloroform: ethyl acetate = 10: 1 → 6: 1) to obtain a white solid (compound 17) (1.81 g, 91%). The analysis result of the produced compound 17 ((2- (4-Bromo-benzenesulfonylamino) ethyl) carbamic acid tert-butyl ester) is shown below.
TLC; R _f = 0.38 (hexane: ethyl acetate = 3: 1)
¹ H-NMR (400 MHz, CDCl ₃ , TMS, rt); δ 7.71 (d, J = 8 Hz, 2H), 7.64 (d, J = 8 Hz, 2H), 5.30. (S, 1H), 4.78 (s, 1H), 3.22 (t, J = 5Hz, 2H), 3.07 (d, J = 5Hz, 2H), 1.43 (s, 9H).

Compound 17 (0.20 g, 0.53 mmol) obtained above and 2.5-thiophene diboronic acid (manufactured by Wako Pure Chemical Industries, Ltd.) (0.12 g, 0.7 mmol) were dissolved in a mixed solvent of 25 ml of toluene and 15 ml of methanol, and Na ₂ 5 ml of an aqueous solution of CO ₃ (0.223 g, 2.1 mmol) and Pd (PPh ₃ ) ₄ (0.04 g, 35 μmol) were added, and degassing and N ₂ substitution were performed three times, followed by stirring at 80 ° C. for 4 hours. After allowing to cool, the mixture was extracted 3 times with chloroform and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure, and the residue was separated and purified by silica gel chromatography (eluent; chloroform: ethyl acetate = 43: 7) to obtain a white powder (Compound 18) (0.267 g, 89%). The analysis result of the produced compound 18 ((2- (4- (5- (dihydroxyboryl) thiophen-2-yl) benzenesulfonylamino) ethyl) carbamic acid tert-butyl ester) is shown below.
TLC; R _f = 0.23 (chloroform: ethyl acetate = 8: 1)
¹ H-NMR (400 MHz, CDCl ₃ , TMS, rt); δ 7.71 (d, J = 9 Hz, 2H), 7.64 (d, J = 9 Hz, 2H), 7.54 (d, J = 5 Hz, 1H), 7.47 (d, J = 5Hz, 1H), 5.36. (s, 1H), 4.82. (s, 1H), 3.24 (d, J = 5Hz, 2H), 3.10 (d, J = 5Hz, 2H), 1.43 ( s, 9H).

Compound 18 (0.54 g, 1.27 mmol) obtained above and 4-bromo-N, N-dimethylaniline (manufactured by Sigma-Aldrich Japan) (0.30 g, 1.5 mmol) were dissolved in a mixed solvent of 35 ml of toluene and 25 ml of methanol. Then, 10 ml of an aqueous solution of Na ₂ CO ₃ (0.27 g, 2.54 mmol) and Pd (PPh ₃ ) ₄ (0.117 g, 101 μmol) were added, and degassing and N ₂ substitution were further performed three times, followed by stirring at 80 ° C. for 22 hours. After allowing to cool, the mixture was extracted 3 times with chloroform and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure, and the residue was separated and purified by silica gel chromatography (eluent; chloroform: ethyl acetate = 4: 1) to obtain a white powder (compound 19) (0.35 g, 56%). The analysis result of the produced compound 19 ((2- (4- (5- (4- (dimethylamino) phenyl) thiophen-2-yl) benzenesulfonylamino) ethyl) carbamic acid tert-butyl ester) is shown below.
TLC; R _f = 0.36 (chloroform: ethyl acetate = 4: 1)
¹ H-NMR (400 MHz, CDCl ₃ , TMS, rt); δ 7.84 (d, J = 8 Hz, 2H), 7.72 (d, J = 8 Hz, 2H), 7.62 (d, J = 8 Hz, 2H), 7.52 (d, J = Hz, 1H), 7.42 (d, J = 8Hz, 1H), 6.79 (d, J = 8Hz, 2H) 5.26 (s, 1H), 4.81 (s, 1H), 3.25 (d, J = 4.8Hz, 2H), 3.09 (d, J = 4.8Hz, 2H), 2.97 (s, 6H), 1.43 (s, 9H).

Synthesis example 5
In this synthesis example, 4- (5 ′-(4- (dimethylamino) phenyl) -2,2′-bithiophen-5-yl) benzenesulfonic acid 2,2-dimethylpropyl ester (Compound 21) was synthesized. The synthesis route is shown in FIG.
5,5'-Dibromo-2,2'-bithiophene (manufactured by Wako Pure Chemical Industries, Ltd.) (0.324 g, 1.00 mmol), Compound 3 (0.248 g, 0.70 mmol) in a mixed solvent of 20 ml of toluene and 12 ml of methanol Then, 1.0 M Na ₂ CO ₃ aq 5 ml and Pd (PPh ₃ ) ₄ (0.066 g, 57 μmol) were added, degassing and N ₂ substitution were performed 3 times, and the mixture was stirred at 80 ° C. for 4 hours and a half. After allowing to cool, toluene was added to extract the organic layer, washed with water and saturated brine, and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (eluent: toluene) to obtain a yellow solid (Compound 20) (0.137 g, 43%). The analysis result of the produced compound 20 (4- (5′-Bromo-2,2′-Bithiophen-5-yl) benzenesulfonic acid 2,2-dimethylpropyl ester) is shown below.
TLC; R _f = 0.07 (toluene)
¹ H-NMR (400 MHz, CDCl ₃ , TMS, rt); δ 7.89 (d, J = 8 Hz, 2H), 7.72 (d, J = 8 Hz, 2H), 7.35 (d, J = 4 Hz, 1H), 7.12 (d, J = 4 Hz, 1H), 7.00 (d, J = 4 Hz, 1H), 6.98 (d, J = 4 Hz, 1H), 3.71 (s, 2H), 0.92 (s, 9H).

Compound 20 (0.091 g, 0.20 mmol) and 4- (dimethylamino) phenylboronic acid (0.038 g, 0.23 mmol) obtained above were dissolved in a mixed solvent of 10 ml of toluene and 5 ml of methanol, and further 1.0 M Na _2. CO ₃ aq 5 ml and Pd (PPh ₃ ) ₄ (0.017 g, 15.0 μmol) were added, degassing and N ₂ substitution were performed three times, and the mixture was stirred at 80 ° C. for 19 hours. After allowing to cool, toluene was added to extract the organic layer, washed with saturated brine, and dried over Na ₂ SO ₄ . The solvent was removed under reduced pressure, and the residue was separated and purified by silica gel chromatography (eluent: chloroform) to give a yellow solid (Compound 21) (0.030 g, 29%). The analysis results of Compound 21 (4- (5 ′-(4- (Dimethylamino) phenyl) -2,2′-Bithiophen-5-yl) benzenesulfonic acid 2,2-dimethylpropyl ester) produced below are shown.
TLC; R _f = 0.36 (chloroform)
¹ H-NMR (400 MHz, CDCl ₃ , TMS, rt): δ 7.87 (d, J = 8 Hz, 2H), 7.71 (d, J = 8 Hz, 2H), 7.47 (d, J = 9 Hz, 2H ), 7.35 (d, J = 4 Hz, 1H), 7.15 (m, 2H), 7.07 (d, J = 4 Hz, 1H), 6.71 (d, J = 9 Hz, 2H), 3.69 (s, 2H ), 2.99 (s, 6H), 0.91 (s, 9H).

Example 1
In this example, fluorescence spectra of the thiophene derivatives (Compounds 1, 16, 19 and 21) and furan derivatives (Compound 13) obtained in Synthesis Examples 1 to 5 in various solvents having different polarities were measured.
These thiophene derivatives and furan derivatives are mixed with various solvents (toluene, 1,4-dioxane, tetrahydrofuran (THF), ethyl acetate, chloroform, dichloromethane, acetone, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), (Acetonitrile, ethanol, methanol). All of these dissolved well.
In addition, the solvent polarity E _T (30) (kcal / mol) of each solvent is dissolved in the solvent of the following formula betaine dye, which is an absorption type solvatochromic dye, and the absorption maximum wavelength λ (nm) is expressed by the formula E _T ( 30) = Obtained by substituting for 28591 / λ.

A solution of a standard solvent for spectrum measurement (manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 0.5 to 1.8 × 10 ⁻⁵ M was prepared from these thiophene derivatives and furan derivatives, and a visible ultraviolet spectrophotometer (V-manufactured by JASCO Corporation) 560 UV / VIS Spectrophotometer). Table 1 shows the absorption maximum wavelength (nm), and Table 2 shows the molar absorption coefficient (mol ^-1 cm ^-1 ) at the absorption maximum wavelength. In the table, the compound number is No. Represented by

Next, the fluorescence spectra of these thiophene derivatives and furan derivatives were measured. The fluorescence spectrum was measured with a fluorescence spectrophotometer (F-4500, manufactured by Hitachi, Ltd.) using the same sample as the absorption spectrum. The fluorescence quantum yield (Q) was calculated from the following equation by measuring the fluorescence emission spectrum when the sample and the reference substance were excited at 365 nm using a 0.5M sulfuric acid solution of quinine sulfate (QR = 0.546) as the reference substance. . Where I is the area of the fluorescence spectrum of the sample, IR is the area of the fluorescence spectrum of the reference substance, A is the absorbance at 365 nm of the sample, AR is the absorbance at 365 nm, n is the refractive index of the solvent in which the sample is dissolved, nR Indicates the refractive index of 0.5 M sulfuric acid.

Table 3 shows the maximum fluorescence emission wavelength (nm), and Table 4 shows the fluorescence quantum yield. Further, the fluorescence spectrum of Compound 1 is shown in FIG. In the table, the compound number is No. Represented by

  The wavelength of fluorescence emission shifted to a longer wavelength as the polarity of the solvent increased, and the fluorescence color changed so that it could be identified visually. That is, it can be seen that the compound is a fluorescent solvatochromic dye whose fluorescent wavelength changes depending on the solvent polarity around the molecule.

It is a figure which shows the manufacturing method of the thiophene derivative of this invention. The numbers in the figure indicate the compound numbers. It is a figure which shows reaction which labels protein with the pigment | dye compound of this invention. (1) and (2) are proteins containing amino group-containing amino acids, (3) is a protein containing thiol group-containing amino acids, and (4) is a method for labeling proteins containing carboxyl group-containing amino acids. It is a figure which shows reaction which labels a nucleic acid sequence with the pigment | dye compound of this invention. It is a figure which shows the manufacturing method of the thiophene derivative (compound 1) of this invention. The numbers in the figure indicate the compound numbers. It is a figure which shows the manufacturing method of the furan derivative (compound 13) of this invention. The numbers in the figure indicate the compound numbers. It is a figure which shows the manufacturing method of the thiophene derivative (compound 16) of this invention. The numbers in the figure indicate the compound numbers. It is a figure which shows the manufacturing method of the thiophene derivative (compound 19) of this invention. The numbers in the figure indicate the compound numbers. It is a figure which shows the manufacturing method of the thiophene derivative (compound 21) of this invention. The numbers in the figure indicate the compound numbers. The fluorescence spectrum of the thiophene derivative of the present invention (Compound 1) in various solvents is shown.

Claims (3)

The following formula

(In the formula, X represents an oxygen atom (—O—) or a sulfur atom (—S—),
m represents an integer of 1 to 4,
R ¹ represents any of the following (a) to (e):
(A) ^{-O-R 8} (wherein, ^{R 8} is. Represents an alkyl group having a side chain alkyl group)
(B) -Y (wherein Y represents a halogen atom or a halogenated alkyl group)
(C) —NH (CH ₂ ) _n NH ₂ (wherein n represents an integer of 1 to 14)
_{(D) -NH (CH 2)} n NHCO (CH 2) o Y ( wherein, n and o each represents an integer of 1 to 14, Y represents a halogen atom.)
(E) -NH (CH ₂ ) _n CH ₃ (wherein n represents an integer of 1 to 20)
R ² and R ³ may be the same or different and each represents a hydrogen atom, an alkoxy group, an acylamino group, an alkyl group, a halogen-substituted alkyl group, an amino group, a hydroxy group, or a halogen atom, provided that R ² And R ³ together may form an aliphatic 5-, 6- or 8-membered ring which may contain an aromatic or ether bond, and R ⁴ and R ⁵ may be the same or different, respectively. Represents a hydrogen atom or an alkyl group, and R ⁶ and R ⁷ may be the same or different and each represents a hydrogen atom or an alkyl group which may have a substituent, provided that R ⁴ and R ⁶ may jointly form an aromatic or aliphatic 5-membered ring or 6-membered ring, and R ⁵ and R ⁷ together form an aromatic or aliphatic 5-membered ring or 6-membered ring May be. ) A fluorescent solvatochromic dye represented by A fluorescent solvatochromic formed by linking the fluorescent solvatochromic dye according to claim 1 to a protein, peptide, poly or oligonucleotide containing an amino group, a hydroxyl group, a thiol group, or a carboxyl group-containing amino acid. Dye complex. The step of irradiating ultraviolet light to the fluorescent solvatochromic dye according to claim 1 or the fluorescent solvatochromic dye complex according to claim 2 or a solution containing them, and light emission from the fluorescent solvatochromic dye A method for inspecting the polarity around the fluorescent solvatochromic dye, comprising the step of measuring a wavelength or an emission color.

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