WO2013189043A1

WO2013189043A1 - Green fluorescent cyanine dyes, preparation method and uses thereof

Info

Publication number: WO2013189043A1
Application number: PCT/CN2012/077222
Authority: WO
Inventors: 彭孝军; 刘涛; 樊江莉; 王静云; 宋锋玲; 孙世国
Original assignee: 大连理工大学; 大连科荣生物技术有限公司
Priority date: 2012-06-20
Filing date: 2012-06-20
Publication date: 2013-12-27

Abstract

Disclosed is a class of green fluorescent cyanine dyes having the following structural formula (I). In the formula, X is C(CH₃)₂, O, S or Se; R₁ and R₂ are each independently selected from H, C_1-18 alkyl, OR₆, C_1-6 alkyl, OR₆ or halogen. R₃ is N(R₅)(R₇); R₄ is selected from C_1-18 alkyl, benzyl and substituted benzyl, and the substituted benzyl can be optionally substituted with the following groups: C_1-18 alkyl, CN, COOH, NH₂, NO₂, OH, SH, C_1-6 alkoxy, C_1-6 alkylamino, C_1-6 acylamino, halogen or C_1-6 haloalkyl; R₅ and R₇ are each independently selected from H or C_1-18 alkyl; R₆ is H or C_1-18 alkyl; and Y^— is an anion. The fluorescent dyes can be used in quantitative detection of DNA and biological staining, and can be applied in the fields of nucleic acid labeling, blood cell analysis, clinical diagnosis, immunoassay detection and the like.

Description

Green light fluorescent cyanine dye, preparation method and application thereof

Technical field

The invention relates to a novel fluorescent dye, a preparation method and application thereof in the field of fine chemical industry, in particular to a singly charged nitrogen-containing cyanine fluorescent dye, a preparation method thereof, and the use of the fluorescent dye, the conjugate thereof or the same The biological application of the composition.

Background technique

DNA (deoxyribonucleic acid) is a class of biological macromolecules with genetic information. The normal cells of the organism have relatively stable DNA diploid content, and only when the cancerous or malignant potential precancerous lesions occur, the abnormal changes in the DNA content of the cells are accompanied. Therefore, the specific recognition and accurate measurement of DNA, especially in living cells, is of great significance in the early diagnosis of cancer. The use of fluorescence technology for quantitative analysis of DNA has the advantages of high sensitivity, fast response, and convenient use of instruments, which have attracted the interest of researchers. At present, such dyes which are commercially available mainly include phenanthridines (EB, PI), acridines (AO), imidazoles (Hoechst, DAPI), and cyanine family (Cy, TOTO, SYTO). However, these dyes each have their own application limitations. First, a larger portion of the dye binds to DNA and exhibits fluorescence quenching, such as 10, 10'-diethyl-2, 2'-disulfo-9, 9'-biacridine, saffron T, resistant Although the detection limit of blue, methyl blue and other quenching DNA probes is as low as ng/mL, the quenched fluorescence signal is of low practical value in visualization applications such as fluorescence imaging. Second, a considerable part of the fluorescent dye excitation light is in the ultraviolet region, such as fluorescent dye DAPI, Hoechst 33258, 13⁄4^ _£ ; 1^ 34580, which can specifically identify deoxyribonucleic acid (DNA), under ultraviolet light excitation. Binding to DNA produces blue fluorescence. Since ultraviolet light can cause serious damage to components such as nucleic acids and proteins in cells, the use of such fluorescence in fluorescence microscopy is limited by the photoexcitation time [Davis SK, Bardeen C, J. Photochem Photobiol 2003; 77 :675-679] In addition, when fluorescence detection is performed in the ultraviolet region, the absorption of biological samples in this interval makes it difficult for light to enter the interior of the biological tissue, and the autofluorescence of certain components in the biological sample forms a strong background interference. The detection efficiency is greatly reduced. Third, a considerable portion of dye applications are limited to fixed cells, such as TOPRO, TOTO family dyes, ethidium bromide (EB), propidium iodide (PI), etc., which need to increase membrane permeability or similar to cause membrane collapse. The method of solution can effectively label the biological sample. However, this method of fixation often has a negative impact on the observation of the true morphology of cells and biological tissues [Kozubek S, Lukasova E, Amrichova J, Kozubek M, Liskova A, Slotova J. Anal Biochem 2000; 282: 29-38]. At the same time, acridine such as ethidium bromide, phenanthridine dyes have great toxicity and carcinogenicity. Invitrogen has developed Quant-iT PicoGreen (Ex/Em = 502/523 nm, detection range: 0.2-100 ng/mL), which can quantify ultrasensitive detection of dsDNA in solution, but the dye structure is not clear, the price It is also very expensive. Therefore, it is a challenging task to develop a fluorescent probe that satisfies both the excitation and emission wavelengths, the specificity and quantitative detection of DNA, and the permeability of living cells.

Among many kinds of fluorescent dyes, cyanine fluorescent dyes have wide wavelength range, large molar extinction coefficient and moderate fluorescence quantum yield. They are used as biomolecular fluorescent probes, CD and VCD recording materials, photosensitive materials, photosensitizers, and optoelectronics. Conversion materials and the like have been widely used. Among them, quinoline-based asymmetric cyanine fluorescent dyes have high affinity with nucleic acids, and the specificity of non-binding with other biomacromolecules makes them stand out in the fields of genomics technology, nucleic acid quantitative detection, blood cell analysis and the like. The manner in which such compounds bind to nucleic acids includes electrostatic attraction, base pair insertion, and groove bonding. The specific binding mode and binding ability depend on the structure of the marker and its ratio to the concentration of the nucleic acid. The most typical of the asymmetric cyanine compounds are TOTO and its analogs (YOYO) and derivatives (TOPRPO). TOTO (thiazole orange dimer), YOYO (oxazole yellow dimer) is a class of poly-positively charged asymmetric cyanines with high affinity for nucleic acids developed by the Glazer research group. Fluorescent dyes, different heterodimeric analogs and derivatives can be obtained by varying the length of the polymethylene chain and the structure of the aromatic nucleus (thiazole, oxazole, quinoline, pyridine and porphyrin) at both ends. These dyes have almost no fluorescence in solution, reducing the background interference of the fluorescence during detection, and the fluorescence is enhanced after binding to the nucleic acid. Jason et al. explained the double insertion of TOTO and YOYO with DNA by solution viscometry and atomic force microscopy [JA Bordelon, KJ Feierabend, SA Siddiqui, LL Wright. J. Phys. Chem. B, 2002, 106, 4838-3843]. Fiirstenberg et al. further elaborated the kinetic mechanism of fluorescence enhancement using ultrafast fluorescence conversion and time-dependent single photon counting. [A. Fiirstenberg, MD Julliard, TG Deligeorgiec, NI Gadjev. J. AM. CHEM.SOC, 2006, 128, 7661-7669] Some of these dyes have been commercialized, such as: SYTOX Blue, TOTO, ΡΟΡΟ , BOBO, YO-PRO, etc. However, most of these commercial dyes have large molecules, complex structures, and are non-permeable to living cells, and can only be used for identification and detection of nucleic acids in vitro. Summary of the invention

The present invention is directed to the deficiencies of the prior art and provides a new class of compounds which are simple in structure, high in sensitivity, suitable in excitation wavelength, and have good cell membrane permeability.

One of the objects of the present invention is to provide a class of green photochromic fluorescent dyes having the following structural formula I:

In formula I

X is C(CH ₃ ) ₂ , 0, S or Se;

Ri and R ₂ are each independently selected from gH, d- ₁₈ alkyl, 0, d- ₆ alkyl or halogen.

R ₃ is specific ₅ ) (R ₇ );

R4 is selected from _ ₁₈ alkyl, benzyl and substituted benzyl, said benzyl group optionally substituted by the following substituent groups: d_ ₁₈ _{alkyl, CN, COOH, NH 2,} N0 2, OH, SH, Ci_ 6 alkoxy An oxy group, a Cw alkylamino group, a Cw acylamino group, a halogen or a C? haloalkyl group;

R ₅ and R ₇ are each independently selected from H or d- ₁₈ alkyl;

R6 is H or _ ₁₈ alkyl;

Y" is a negative ion.

The green photochromic fluorescent dye of the present invention, wherein the sum and each are independently selected from the group consisting of H, d- ₁₈ alkyl, OR ₆ and halogen. It is preferably 11 or Cl, and R ₂ is H or a methoxy group.

The green photochromic fluorescent dye of the present invention, wherein the R4 is selected from the group consisting of d- ₁₈ alkyl, benzyl and substituted benzyl, and the substituted benzyl group is optionally substituted by the following groups: COOH, NH ₂ , OH, d_ ₆ Alkoxy or halogen. Preferably, F is selected from the group consisting of d- ₆ alkyl, benzyl and halogen substituted benzyl.

The green photochromic fluorescent dye of the present invention, wherein the sum and each are independently selected from H or d- ₆ alkyl. The green light-emitting fluorescent dye according to the present invention, wherein the Y is a halogen ion, C1 (V, PF ₆ ", BF ₄ -, CH ₃ COO - or OTs". Another aspect of the present invention provides a method for preparing a green photochromic fluorescent dye according to the present invention, which comprises the following steps: 1) reacting a compound of the formula Ila or lib with R ₄ Z to prepare a first quaternary ammonium salt intermediate Ilia, respectively. Or nib, wherein Z is halogen or OTs, Z- is a halogen anion or OTs- produced by the reaction, which is halogen:

The reaction temperature is 10-180 ° C, the reaction time is 4-48 hours, and the reaction solvent is selected from the group consisting of dichloromethane, chloroform, ethanol, acetonitrile, ethyl acetate, toluene, xylene, o-dichlorobenzene, and any two of them. Or a mixed solvent of two or more kinds in any ratio, the molar ratio of the compound of the formula Ila or lib to the FZ is 1:1 to 1:10;

2) reacting a compound of the formula V with a compound R ₃ (CH ₂ ) ₄ Z to obtain a second quaternary ammonium salt intermediate VI, wherein Z is a halogen or OTs, and Z- is a halogen anion or OTs produced by the reaction:

The reaction temperature is 10-180 ° C, the reaction time is 4-48 hours, and the reaction solvent is selected from the group consisting of dichloromethane, chloroform, ethanol, acetonitrile, ethyl acetate, toluene, xylene, o-dichlorobenzene, and any two of them. Or a mixed solvent of two or more kinds in any ratio, the molar ratio of the compound of the formula V to the compound R ₃ (CH ₂ ) ₄ Z is 1:1 to 1:10;

3) reacting the first quaternary ammonium salt intermediate Ilia or nib obtained in the step (1) with the second quaternary ammonium salt intermediate VI obtained in the step (2) to obtain a compound of the formula VII

The reaction temperature is 5-50 ° C, the reaction time is 30 minutes to 24 hours, and the reaction solvent is selected from the group consisting of dichloromethane, chloroform, methanol, ethanol, ethylene glycol monomethyl ether, and any two or more of them are optionally used. a mixed solvent composed of a ratio of a catalyst to an organic base, a compound of the formula Ilia or nib and a compound of the formula VI in a molar ratio of 1.5:1 to 1:1.5;

4) Anion exchange of the compound of the formula VII obtained in the step 3) with a sodium or potassium salt containing Y- to obtain a compound of the formula I:

The reaction temperature is 60-140 ° C, the reaction time is 10 minutes to 2 hours, the reaction solvent is DMF, DMSO or a mixed solvent thereof, and the molar ratio of the sodium salt or potassium salt containing Y- to the compound of the formula VII is 1: 1- 10: 1 ο

A further object of the present invention is to provide the use of the green photochromic fluorescent dye of the present invention in the dyeing of biological samples. Further, the present invention provides a method of dyeing a biological sample comprising the step of contacting the green light-crystal phthalocyanine dye of any of the above inventions with a biological sample.

The green photochromic fluorescent dye of the invention has a lower fluorescent background in the absence of nucleic acid, has higher fluorescence quantum yield after binding with nucleic acid, and has no affinity or affinity for biomolecules other than nucleic acid. Has a certain level of water solubility, and has good cell membrane permeability, can enter the staining of living cells; the spectrum range is appropriate, does not cause cell or tissue damage. The fluorescent dye can be used for quantitative DNA detection and biological staining, and is applied to fields such as nucleic acid labeling, blood cell analysis, clinical medical diagnosis, and immunoassay detection.

DRAWINGS

Figure 8 of the accompanying drawings, wherein:

Figure 1 is a comparison of the compound of Example 3 and the commercial dye ethidium bromide (EB) in tris(hydroxymethyl)aminomethane hydrochloride buffer at pH 7.4 in a concentration of 10 mM, before and after binding to calf thymus DNA. The fluorescence intensity is compared to the photo. The abscissa is the wavelength (nm) and the ordinate is the relative fluorescence intensity. The instrument used was an ultraviolet-visible spectrophotometer, model: Hp8453; fluorescence spectrophotometer, model: FP-6500. The concentrations of Compound A and ethidium bromide (EB) were both 0.5 μΜ and the concentration of calf thymus DNA was 50 μΜ.

Fig. 2 is a graph showing changes in fluorescence intensity of the compound of Example 4 in a buffer of ρΗ 7.4 and a concentration of 10 mM in tris(hydroxymethyl)aminomethane hydrochloride as the concentration of DNA in the calf thymus increases. The abscissa is the wavelength (nm) and the ordinate is the relative fluorescence intensity. 2B is the compound B of Example 4 in a pH 7.4, 10 mM concentration of tris(hydroxymethyl)aminomethane hydrochloride buffer, and the maximum fluorescence emission peak intensity of compound B increases with the concentration of DNA in the calf thymus. A linear plot of the concentration of bovine thymus DNA. The abscissa is the calf thymus DNA concentration (μΜ) and the ordinate is the relative fluorescence intensity. The instrument used was an ultraviolet-visible spectrophotometer, model: Ηρ8453; fluorescence spectrophotometer, model: FP-6500. The concentration of Compound B was 0.5 μΜ.

Figure 3 is a comparison of fluorescence quantum yields of compound hydrazine and commercial dye thiazole orange (TO) before and after binding to calf thymus DNA in tris(hydroxymethyl)aminomethane hydrochloride buffer at pH 7.4 and 10 mM. Figure. The instrument used is UV visible Spectrophotometer, Model: Hp8453; Fluorescence spectrophotometer, Model: FP-6500. Both Compound A and the commercial dye thiazole orange were 1.5 μΜ, and the concentration of calf thymus DNA was 150 μΜ.

Figure 4 is a comparison of the compound hydrazine and the commercial dye thiazole orange (TO) in tris(hydroxymethyl)aminomethane hydrochloride buffer at pH 7.4 and 10 mM, respectively, before and after binding to bovine serum albumin and calf thymus DNA. A comparison of the fluorescence enhancement multiples. The instrument used was a fluorescence spectrophotometer, model: FP-6500. The concentration of Compound A and the commercial dye Thiazole Orange (TO) were both 0.5 μΜ, and the concentrations of bovine serum albumin and calf thymus DNA were both 20 g/ml.

Fig. 5 is a white field micrograph of the compound of Example 3, which stains HeLa living cells (human cervical cancer cells), and Fig. 5B is a fluorescence micrograph of the staining of HeLa living cells by Compound A. The concentration of Compound A was 5 μΜ. The instrument used was a confocal laser scanning microscope, model: FV1000IX81, Japan. Excitation channel: 488 nm.

Fig. 6A is a white field micrograph of the staining of MCF-7 living cells (human breast cancer cells) by the compound B of Example 4, and Fig. 6B is a fluorescence micrograph of the staining of MCF-7 living cells by the compound B. The concentration of Compound B was 5 μΜ. The instrument used was a confocal laser scanning microscope, model: FV1000IX81, Japan. Excitation channel: 488 nm.

Fig. 7A is a white field micrograph of the staining of HeLa living cells (human cervical cancer cells) by the compound C of Example 5, and Fig. 7B is a fluorescence micrograph of the staining of HeLa living cells by the compound C. The concentration of Compound C was 5 μΜ. The instrument used was a confocal laser scanning microscope, model: FV1000IX81, Japan. Excitation channel: 488 nm.

Fig. 8A is a white field micrograph of the staining of MCF-7 living cells (human breast cancer cells) by the compound D of Example 6, and Fig. 8B is a fluorescence micrograph of the staining of MCF-7 living cells by the compound D. The concentration of Compound D was 5 μΜ. The instrument used was a confocal laser scanning microscope, model: FV1000IX81, Japan. Excitation channel: 488 nm

detailed description

Unless otherwise stated, the terms used herein have the following meanings.

The term "alkyl" as used herein includes both straight chain alkyl and branched alkyl groups. When referring to a single alkyl group such as "propyl", it is specifically referred to as a straight-chain alkyl group, and a single branched-chain alkyl group such as "isopropyl" is specifically referred to as a branched alkyl group. For example, "C ^ alkyl" includes d_ ₄ alkyl, d_ ₃ alkyl, methyl, ethyl, n-propyl, isopropyl and tert-butyl. Similar rules apply to the other groups used in this specification.

The term "halogen" as used herein includes fluoro, chloro, bromo and iodo.

The term "nodal group" as used herein refers to a -CH ₂ -Ph group. Modification of "substituted benzyl" by "optionally substituted" means that the benzyl group may be substituted at any suitable position by a suitable substituent, which may be monosubstituted or multiple substituted independently of each other. Suitable substituents include, but are not limited to H, d_ ₁₈ _{alkyl, CN, COOH, NH 2,} N0 2, OH, SH, d_ 6 alkoxy, d_ ₆ alkylamino, d_ ₆ acylamino group, halogen or d_ ₆ Haloalkyl and the like, as long as the finally formed compound has the desired properties of the present invention.

Y- denotes an anion, which may be any suitable anion, including but not limited to inorganic anions or organic anions such as halides, C10 ₄ -, PF ₆ - , BF ₄ -, CH ₃ COO - or OTs -.

The present invention provides a type of green photochromic fluorescent dye, wherein in the compound of formula I, X is preferably C(CH ₃ ) ₂ , 0 or S; more preferably X is C (CH ₃ ) ₂ or S; Preferably it is 8.

In a particular embodiment of the invention, and each independently selected from the group consisting of H, d- ₁₈ alkyl, OR ₆ and halogen; preferably, and each independently selected from the group consisting of H, d- ₁₂ alkyl, d- ₁₂ alkoxy and halogen; more preferred And independently selected from H, C^alkyl, d- ₆ alkoxy and halogen; optimally, 11 or 0, R ₂ is H or methoxy.

In one embodiment of the invention, it is _-18 alkyl; preferably d- ₁₂ alkyl; more preferably d- ₆ alkyl. In a particular embodiment of the invention, R ₄ is a substituted benzyl group; preferably a benzyl group or a benzyl group optionally substituted by C00H, NH ₂ , OH, d- ₆ alkoxy or halogen; most preferably a benzyl or halogen substituted benzyl group.

In a specific embodiment of the invention, R ₃ is N(R ₅ ) (R ₇ ), wherein R ₅ is the same as R ₇ and is selected from the group consisting of d ₁₈ alkyl, preferably C _1-12 ; most preferably C _{1- 6} yard base.

In a specific embodiment of the invention, R ₃ is N(R ₅ ) (R ₇ ), wherein, different from, one of them is H, and the other group is selected from _-18 alkyl, preferably d- ₁₂ alkyl; preferably d_ ₆ alkyl.

Y—is a halogen ion, C10 ₄ —, PF ₆ " , BF ₄ -, CH ₃ COO- or OTs-, preferably a halogen anion.

In another aspect, the present invention also provides a method for preparing the above compound, the method comprising:

Preparing a first quaternary ammonium salt intermediate and a second quaternary ammonium salt intermediate, respectively, wherein the first quaternary ammonium salt comprises preparing a quaternary ammonium salt by using a quinoline substituted compound and a 4-chloroquinoline substituent as raw materials, and then the first season The ammonium salt is reacted with a second quaternary ammonium salt intermediate in the presence of an organic base to give the final compound. The specific synthesis scheme is as follows.

The first is to prepare a first quaternary ammonium salt intermediate, that is, a compound of the formula Ila or lib, respectively, is reacted with Z to prepare a first quaternary ammonium salt intermediate Ilia or IIIb, wherein Z is a halogen or 0Ts, and Z- is a halogen formed by the reaction. Negative ions or OTs—, R ₈ is halogen:

Ilia Illb

The reaction temperature is 10-180 ° C, the reaction time is 4-48 hours, and the reaction solvent is selected from the group consisting of dichloromethane, chloroform, ethanol, acetonitrile, ethyl acetate, toluene, xylene, o-dichlorobenzene, and any two of them. Or a mixed solvent of two or more kinds in any ratio, the molar ratio of the compound of the formula Ila or lib to the FZ is 1: 1- 1: 10;

In a preferred embodiment, the reaction temperature is 40-140 ° C, the reaction time is 6-36 hours, and the reaction solvent is selected from the group consisting of: chloroform, acetonitrile, toluene, xylene, o-dichlorobenzene or, and any two of them. The molar ratio of the compound of the formula Ila or lib to R ₄ Z is 1:2 to 1:6.

In a more preferred embodiment, the reaction temperature is 60-120 ° C, the reaction time is 8-24 hours, and the reaction solvent is acetonitrile, toluene, o-dichlorobenzene or a mixture of any two or three thereof in any ratio. The solvent, the molar ratio of the compound of the formula Ila or lib to FZ is 1:2 to 1:5.

In a most preferred embodiment, the reaction temperature is 80-110 ° C, the reaction time is 8-14 hours, the reaction solvent is toluene, o-dichlorobenzene or a mixed solvent thereof, and the compound of the formula Ila or lib is mixed with the compound FUZ. The ratio is 1:3:1:5.

In a particular embodiment, it is 0 or the same halogen atom as Z.

The second quaternary ammonium salt intermediate VI, wherein Z is halogen or OTs, is obtained by reacting a compound of the formula V with a compound R ₃ (CH ₂ ) ₄ Z in a similar manner to the preparation of a compound of the formula Ilia or 111b. - a halogen anion or OT formed for the reaction: in the formula V, a 2-methylbenzothiazole having a substituent, a 2-methylbenzoxazole having a substituent, a 2-methylbenzoyl group having a substituent Selenazole, or a substituted 2,3,3-trimethyl-3H-indole

The reaction temperature is 10-180 ° C, the reaction time is 4-48 hours, and the reaction solvent is selected from the group consisting of dichloromethane, chloroform, ethanol, acetonitrile, ethyl acetate, toluene, xylene, o-dichlorobenzene, and any two of them. Or a mixed solvent of two or more kinds in any ratio, the molar ratio of the compound of the reaction material of the formula V to the compound R ₃ (CH ₂ ) ₄ Z is from 1:1 to 1:10.

In a preferred embodiment, the reaction temperature is 60-140 ° C, the reaction time is 6-36 hours, and the reaction solvent is selected from the group consisting of chloroform, acetonitrile, toluene, xylene, o-dichlorobenzene, and any two or two of them. The above is mixed according to any ratio In the solvent, the molar ratio of the compound of the formula V to the compound ^13⁄4) ₄ ∑ is 1:2 to 1:6.

In a more preferred embodiment, the reaction temperature is 80-120 ° C, the reaction time is 10-24 hours, the reaction solvent is acetonitrile, toluene, o-dichlorobenzene, and any two or three of them are formed in any ratio. The mixed solvent, the molar ratio of the compound of the formula V to the compound R ₃ (CH ₂ ) ₄ Z is 1:2 to 1:5.

In a most preferred embodiment, the reaction temperature is 90-120 ° C, the reaction time is 12-18 hours, the reaction solvent is toluene, o-dichlorobenzene or a mixed solvent thereof, and the compound of the formula V and the compound R ₃ (CH ₂ The molar ratio of ₄ Z is 1:3:1.

The prepared first quaternary ammonium salt intermediate Ilia or nib is reacted with a second quaternary ammonium salt intermediate VI under the action of an organic base to obtain a univalent nitrogen-containing cyanine compound of the formula VII:

The reaction temperature is 5-50 ° C, the reaction time is 30 minutes to 24 hours, and the reaction solvent is selected from the group consisting of dichloromethane, chloroform, methanol, ethanol, ethylene glycol monomethyl ether, and any two or more of them are optionally used. A mixed solvent of a proportional composition, and the catalyst is an organic base. The organic base is preferably diethylamine, n-propylamine, triethylamine, pyridine, piperidine or a mixture of any two or more of them in any ratio. The molar ratio of the compound of the formula Ilia or nib to the compound of the formula VI is 1.5:1 to 1:1.5; in a preferred embodiment, the reaction temperature is 15 to 50 ° C, the reaction time is 1 to 20 hours, and the reaction solvent is selected from the group 2 Methyl chloride, chloroform, methanol, ethanol, and a mixed solvent of any two or more of them in any ratio, and the catalyst is an organic base. The organic base is preferably n-propylamine, triethylamine, pyridine or a mixture of two or three of them. The molar ratio of the compound of formula Ilia or Illb to the compound of formula VI is 1.2:1 to 1:1.5;

In a more preferred embodiment, the reaction temperature is 20 to 40 ° C, the reaction time is 3 to 16 hours, the reaction solvent is dichloromethane, chloroform or a mixed solvent thereof, and the catalyst is an organic base. The organic base is preferably triethylamine, pyridine or a mixture thereof. The molar ratio of the compound of formula Ilia or Illb to the compound of formula VI is 1.2: 1- 1: 1.2;

In a most preferred embodiment, the reaction temperature is 20-30 ° C, the reaction time is 6 to 12 hours, the reaction solvent is dichloromethane, chloroform or a mixed solvent thereof, and the catalyst is an organic base. The organic base is preferably triethylamine. The molar ratio of the compound of formula Ilia or Illb to the compound of formula VI is 1: 1;

Finally, the compound VII is subjected to negative ion displacement with a sodium or potassium salt containing C1 (V, PF ₆ ", BF ₄ " or CH ₃ COO - as required to obtain a compound of the formula I according to the invention:

The reaction temperature is 60-140 ° C, the reaction time is 10 minutes to 2 hours, and the reaction solvent is selected from the group consisting of: DMF, DMSO or a mixed solvent thereof, containing C10 ₄ —, PF ₆ " , BF ₄ " or CH ₃ COO - The molar ratio of the sodium or potassium salt to the compound of formula VII is from 1 : 1 to 10:1.

In a preferred embodiment, the reaction temperature is 70-130 ° C, the reaction time is 15 minutes to 1.5 hours, and the reaction solvent is DMF, DMSO or a mixed solvent thereof. The molar ratio of the sodium or potassium salt containing C10 ₄ —, PF ₆ ", BF ₄ " or CH ₃ COO- to the compound of formula VII is from 1:1 to 7:1.

In a more preferred embodiment, the reaction temperature is 80-120 ° C, the reaction time is 20 minutes to 1 hour, and the reaction solvent is DMF, containing C10 ₄ -, PF ₆ ", BF ₄ " or CH ₃ COO - The molar ratio of the sodium or potassium salt to the compound of the formula VD is from 1:1 to 4:1.

In a most preferred embodiment, the reaction temperature is 90-110 ° C, the reaction time is 30 minutes, the reaction solvent is DMF, and the sodium or potassium salt containing C1C, PF ₆ ", BF ₄ " or CH ₃ COO- The molar ratio of the charge to the compound of formula VII is from 1:1 to 2:1.

The product of the compound of the formula I synthesized by the above method of the present invention can be confirmed by nuclear magnetic resonance spectrum or mass spectrometry.

Among the above compounds of the general formula I provided by the present invention, the most important and important structural features are: the nitrogen-containing substituent group (CH ₂ ) ₄ introduced from the preparation of the second quaternary ammonium salt intermediate VI from the starting compound of the formula V. R ₃ .

The present invention also provides a conjugate of the above compound and a composition comprising the above compound or a conjugate thereof.

The invention also provides a biological use of the above compounds, conjugates thereof or combinations thereof.

The beneficial effects of the compounds of the invention when used as fluorescent dyes are:

The introduction of a nitrogen-containing substituent into the molecule of the new compound increases the fluorescence quantum yield of the dye and the nucleic acid, thereby improving the detection sensitivity.

The nitrogen-containing substituent introduced by the new compound molecule is non-quaternized (non-positively charged), so it has good cell membrane permeability and an increased application range.

The introduction of a nitrogen-containing substituent into the molecule of the new compound appropriately increases the polarity of the molecule, reduces the binding force to the hydrophobic region of the molecule such as membrane lipids, proteins, and the like, and exhibits specific binding to the nucleic acid.

The new dye compound introduces a quinoline heterocycle at one end, and the same symmetric benzothiazole and porphyrin cyanine dye as the methine chain are equivalent to an increase in the degree of conjugation, and both the ultraviolet absorption and the fluorescence emission are red-shifted. The maximum fluorescence emission is at 530 nm.

The new compound can be applied to a common green semiconductor laser as a light source, which greatly reduces the cost of use.

The new compound product is easy to obtain and has a simple structure. Generally, the target molecule can be synthesized through two to three steps of reaction, and the yield is relatively high, and industrialization is easy.

The features and advantages of the present invention, as well as other features and advantages, will become apparent from the <RTIgt;

The compounds of the invention can be used directly in the quantitative detection of DNA as well as in biological staining applications in the form of the salts described herein. In addition, derivatives of the compounds of the invention may also be used in quantitative DNA assays as well as biological staining applications, including but not limited to conjugates.

Typically, the conjugate is used in a fluorescence activated cell sorter (FACS). As used herein, "conjugate" refers to a compound formed by the attachment of a fluorescent dye of the invention to other molecules by covalent bonds. Molecules that can be conjugated to a fluorescent dye of the invention can be molecules that specifically bind to a cell or cellular component, including but not limited to antibodies, antigens, receptors, ligands, enzymes, substrates, coenzymes, and the like. Typically, the test sample is incubated with the fluorescent conjugate for a period of time such that the fluorescent conjugate specifically binds to certain cells or cellular components in the test sample, and the binding of the fluorescent conjugate to the cell or cellular component can also be referred to. For dyeing. The staining step can be performed multiple times in sequence, or multiple dyes can be performed simultaneously with multiple conjugates. After the staining is completed, the sample is analyzed in a fluorescence activated cell sorter, wherein the excitation light source excites the fluorescent dye of the present invention in the conjugate, and the assay device determines The emitted light produced by the excited fluorescent dye.

The compositions of the present invention may comprise, in addition to the compound of formula I or a conjugate thereof, other components required for biological applications such as solvents, osmotic pressure adjusting agents, pH adjusting agents, surfactants and the like. These components are known in the industry.

The compositions of the present invention may be in the form of an aqueous solution or may be present in other suitable forms which are formulated as solutions in water prior to use.

In still another aspect, the present invention provides a method of using a compound of the above formula I, or a conjugate thereof, or a composition comprising a compound of formula I, for use in a biological application, the method comprising or a compound of the above formula I or a conjugate thereof or A step of contacting a composition comprising a compound of formula I with a biological sample. The term "contacting" as used herein may include contacting in a solution or a solid phase.

In order to demonstrate the optimized improvement of the dye properties after the introduction of the nitrogen-containing groups in the structure of the compounds of the present invention, the commercially available dyes Thiazole Orange (TO) and Bromine B were used in Examples 7, 8, 9, 10, 11 and Comparative Example 15. The ingot (EB) was used as a reference for comparison. Business

Example 1

Synthesis of the first quaternary ammonium salt intermediate 1-ethyl-4-iodoquinoline quaternary ammonium salt:

10 mmol of 4-chloroquinoline and 20 mmol of iodoethane were added to 100 ml of a round bottom flask containing 20 ml of toluene. Under nitrogen protection, the reaction was heated and refluxed for 10 h and then stopped. After the mixture was cooled to room temperature, the precipitate was filtered and washed with ethyl acetate. After drying, a yellow solid powder was obtained in a crude yield of 98%.

Example 2

Synthesis of the second quarter ammonium salt 1-diethylamine butyl-2-methylbenzothiazole quaternary ammonium salt:

10 mmol of 2-methylbenzothiazole and 20 mmol of 1-diethylamino-4-bromobutane were added to 100 ml of a round bottom flask containing 20 ml of toluene. Under nitrogen protection, the reaction was heated and refluxed for 18 h and then stopped. After the mixture was cooled to room temperature, the precipitate was filtered and washed with diethyl ether. After drying, a white solid powder was obtained in a crude yield of 75 %.

Example 3

Preparation of Compound A:

Add 1 mmol of 1-ethyl-4-iodoquinoline quaternary ammonium salt to 1 mmol of 1-diethylamine butyl-2-methylbenzothiazole quaternary ammonium salt to 100 ml of 20 ml of dichloromethane In a round bottom flask. Under a nitrogen atmosphere, 10 mmol of triethylamine was slowly added dropwise and stirred at room temperature for 12 h to stop the reaction. The resulting reaction solution was poured into diethyl ether to precipitate an orange-yellow solid dye, which was filtered and dried. The dye was separated by a neutral alumina chromatography column, and an orange component was collected using a mixed solvent of dichloromethane and methanol as an eluent, yield 67%. ¹ H-NMR (400 MHz, DMSO, TMS): δ 1.09 (t, 6H), 1.58 (t, 3H), 1.86 (m, 2H), 1.95 (m, 2H), 2.67 (m, 6H), 4.51 (t, 3H), 4.81 (tetra, 2H), 6.73 (s, 1H), 7.46-7.81 (m, 8H), 8.55 (d, 1H), 9.30 (d, 1H). MS (TOF LD+) C ₂₇ H ₃₄ IN ₃ S m/z: 432.3 [MI] +.

Example 4

Preparation of Compound B:

Add 1 mmol of 1-benzyl-4-chloroquinoline quaternary ammonium salt to 1 mmol of 1-propylamine butyl-2-methyl-5-chlorobenzothiazole quaternary ammonium salt to 100 ml containing 20 ml of dichloromethane In a round bottom flask. Under nitrogen protection, 10 mmol of triethylamine was slowly added dropwise and stirred at room temperature for 10 h to stop the reaction. The resulting reaction solution was poured into diethyl ether to precipitate an orange-yellow solid dye, which was filtered and dried. The dye was separated by a neutral alumina chromatography column, and an orange fraction was collected using a mixed solvent of dichloromethane and methanol as an eluent, yield 55%. MS (TOF LD+) C ₃₆ H ₄₁ BrClN ₃ m/z : 525.2 [M-Br]+

Example 5

Preparation of Compound C:

Add 1 mmol of quinoline quaternary ammonium salt and 1 mmol of benzoxazole quaternary ammonium salt to 100 ml round bottom flask containing 20 ml of chloroform Medium. Under a nitrogen atmosphere, 10 mmol of triethylamine was slowly added dropwise and stirred at room temperature for 12 h to stop the reaction. The resulting reaction solution was poured into diethyl ether to precipitate an orange-yellow solid dye, which was filtered and dried. The dye was separated by a neutral alumina column, and an orange component was collected using a mixed solvent of dichloromethane and methanol as an eluent, yield 19%. MS (TOF LD ⁺ ) C ₂₄ H ₂₈ C1N ₃ 0 ₂ m/z: 472.7 [M-C1] ⁺

Example 6

Preparation of Compound D:

1 mmol of quinoline quaternary ammonium salt and 1 mmol of benzoselenazole quaternary ammonium salt were added to 100 ml of a round bottom flask containing 20 ml of dichloromethane. Under nitrogen protection, 10 mmol of triethylamine was slowly added dropwise and stirred at room temperature for 10 h to stop the reaction. The resulting reaction solution was poured into diethyl ether to precipitate an orange-yellow solid dye, which was filtered and dried. The dye was dissolved in 3 ml of DMF, and after adding 2 mmol of NaC10 _{4 , the} reaction was stirred at 110 ° C for 30 min. The resulting reaction solution was poured into diethyl ether to precipitate an orange-yellow solid dye, which was filtered and dried. The dye was separated by a neutral alumina chromatography column, and an orange fraction was collected using a mixed solvent of dichloromethane and methanol as an eluent, yield 11%. MS (TOF LD+) C ₃₆ H ₄₃ ClN ₄ 0 ₄ Se m/z: 606.1 [M-C10 ₄ ]+.

Example 7

Determination of fluorescence emission spectra and relative fluorescence intensity before and after compound A and commercial dye ethidium bromide (EB) were combined with calf thymus DNA:

A solution of Compound A in DMSO (dimethyl sulfoxide) at a concentration of 1 mM and an aqueous solution of ethidium bromide (EB) were taken, respectively, and 1.5 L was added, followed by pH 7.4, 10 mM tris(hydroxymethyl)aminomethane hydrochloride. The salt buffer was diluted to 3 mL and placed in a cuvette to determine the fluorescence intensity. An aqueous solution of a certain concentration of calf thymus DNA was prepared, and the absorbance at 260 nm was measured by an ultraviolet absorption spectrophotometer, and the concentration was determined to be 1.8 mM. Another 1 mM solution of Compound A in DMSO (dimethyl sulfoxide) and 1.5 ml of ethidium bromide (EB) in a cuvette were added to the calf thymus DNA at a concentration of 1.8 mM. The solution was 85 μm, and finally diluted with a pH 7.4, 10 mM tris (hydroxymethyl) aminomethane hydrochloride buffer to 3 mL, and the fluorescence intensity was measured. Under the same conditions (same substrate concentration and DNA concentration), the commercial dye ethidium bromide (EB), combined with calf thymus DNA, increased the relative fluorescence intensity by 19 (I/I _Q = 19.8/1.06 = 19) times; When Compound A is combined with calf thymus DNA, the relative fluorescence intensity can be increased by 304 (1/1. = 332.1/1.09 = 304) times. The instrument used was an ultraviolet-visible spectrophotometer, model: Hp8453; fluorescence spectrophotometer, model: FP-6500.

Example 8

The change of fluorescence intensity of compound B with the increase of DNA concentration in calf thymus and the linear relationship between the intensity of maximum fluorescence emission peak and the DNA concentration of calf thymus:

A certain concentration of aqueous solution of calf thymus DNA was prepared, and the absorbance at 260 nm was measured by ultraviolet absorption spectrophotometer. The photometric value was calibrated to a concentration of 1.8 mM. 100 L of calf thymus DNA, which has been calibrated to 1.8 mM, was diluted with 0.5 mM of calf thymus DNA aqueous solution after addition of 290 water. Configure a concentration of 1 mM Compound B in DMSO (dimethyl sulfoxide) solution, take 1.5 L, add pH 7.4, 10 mM tris(hydroxymethyl)aminomethane hydrochloride buffer to 3 mL, place In the cuvette, the fluorescence intensity was measured. Subsequently, 0.6 0.5 mM of calf thymus DNA aqueous solution was taken in a cuvette each time, and the buffer was evenly stirred, and then left to stand at 37 ° C for 3 min, and then the fluorescence intensity was measured. Finally, the concentration of calf thymus DNA in the cuvette is 1 μΜ. The intensity of the maximum fluorescence emission peak (530 nm) of each calf thymus DNA concentration was taken as a linear relationship between fluorescence intensity and calf thymus DNA concentration (R=0.998). The instrument used was an ultraviolet-visible spectrophotometer, model: Hp8453; fluorescence spectrophotometer, model: FP-6500.

Example 9

Determination of the fluorescence quantum yield of Compound A and the commercial dye thiazole orange (TO) before and after binding to calf thymus DNA: Take a certain amount of compound A at a concentration of 1 mM and a commercial dye thiazole orange (TO) solution, and add to pH. 7.4. In a buffer solution of 10 mM tris(hydroxymethyl)aminomethane hydrochloride, the maximum absorption value determined by the ultraviolet-visible spectrophotometer is between 0.06 and 0.08. The excitation wavelength was selected to determine the fluorescence intensity. The measurement was performed three times in parallel, and the fluorescence quantum yield was calculated and the average value was taken. Using fluorescein as a standard (Φ _F = 0.95, 0.1 MN _a OH aqueous solution, 15 ° C), the fluorescence quantum yields of thiazole orange (TO) and compound A were all less than 0.01 in the buffer solution, which was low. Fluorescent background. The fluorescence quantum yield of the compound Α after binding to the same concentration of calf thymus (150 μΜ) (3⁄4 = 0.33; the fluorescence quantum yield of thiazole orange (TO) Φ _Ρ = 0.23;

Example 10

Determination of the fluorescence intensity of the compound hydrazine and the commercial dye thiazole orange (TO) before and after binding to calf thymus DNA and bovine serum albumin (BSA):

Take 3 DMSO solution of I mM compound A and commercial dye thiazole orange (TO), dilute to 3 mL of tris(hydroxymethyl)aminomethane hydrochloride buffer with BpH 7.4 and 10 mM. In the color dish, the fluorescence intensity was measured. Take 3 concentrations of I mM Compound A and commercial dye thiazole orange (TO) in DMSO solution in two cuvettes, then add 4 L of BSA solution at a concentration of 30 mg/mL, force BpH 7.4, The fluorescence intensity was measured by diluting to 10 mL of 10 mM tris(hydroxymethyl)aminomethane hydrochloride buffer. In addition, 3 concentrations of 1 mM compound hydrazine and commercial dye thiazole orange (TO) in DMSO were placed in two cuvettes, and then 600 μg/mL of calf thymus DNA solution was added thereto. ^, Finally, pH 7.4, 10 mM tris(hydroxymethyl)aminomethane hydrochloride buffer was diluted to 3 mL, and the fluorescence intensity was measured. Under the same conditions (same substrate concentration, BSA concentration and DNA concentration), the relative fluorescence intensity of the commercial dye thiazole orange (TO) combined with calf thymus DNA was increased by 173 times; combined with bovine serum albumin (BSA) The relative fluorescence intensity increased by 5.7 times. The relative fluorescence intensity of Compound A combined with calf thymus DNA increased by 304 times; the relative fluorescence intensity increased by 1.7 times after binding to bovine serum albumin (BSA). It can be seen that Compound A has a good specific binding to DNA. The instrument used is an ultraviolet-visible spectrophotometer, model: Hp8453 _; fluorescence spectrophotometer, model: FP-6500 o

Example 11

The staining of HeLa living cells by Compound A was observed under confocal laser scanning microscopy:

Compound A, PBS buffer 10 with a concentration of 1 mM was added to a six-well plate in which HeLa cells were cultured, and incubated in a 37 ° C, 5% CO ₂ incubator for 30 min. Then, the cells were washed with PBS for 3 times, and then cell culture medium was added, and the cell morphology was observed by a confocal laser scanning microscope. Select representative regions, 488 nm channel excitation, using oil mirror (100><) Inspect, repeat three times. Figure 5A is a white field photomicrograph of Compound A staining HeLa living cells, and 5B is a fluorescence micrograph of Compound A staining HeLa living cells. As shown in the figure, Compound A was clearly stained for HeLa cell nuclei. The instrument used was a confocal laser scanning microscope, model: FV1000IX81, Japan. Excitation channel: 488 nm.

Example 12

The staining of MCF-7 living cells by Compound B was observed under confocal laser scanning microscopy:

Compound B, PBS buffer 10 with a concentration of 1 mM was added to a six-well plate in which MCF-7 cells were cultured, and incubated in a 37 ° C, 5% CO ₂ cell incubator for 30 min. Then, the PBS buffer was shaken and washed 3 times, and then the cell culture medium was added, and the cell morphology was observed by a confocal laser scanning microscope. Representative regions were selected, challenged on a 488 nm channel, and observed three times with an oil mirror (100><). Figure 6A is a white field photomicrograph of Compound B staining for MCF-7 live cells, and 6B is a fluorescence micrograph of Compound B staining for MCF-7 live cells. As shown in the figure, Compound B was clearly stained for MCF-7 cell nuclei. The instrument used was a confocal laser scanning microscope, model: FV1000IX81, Japan. Excitation channel: 488 nm.

Example 13

The staining of HeLa living cells by Compound C was observed under confocal laser scanning microscopy:

Add a compound C, 1 mM PBS buffer 10 to HeLa cells in a six-well plate, and incubate in a 37 ° C, 5% CO ₂ incubator for 30 min. Then, PBS was shaken and washed 3 times, and then cell culture medium was added, and the cell morphology was observed by a confocal laser scanning microscope. Representative regions were selected and challenged on a 488 nm channel, observed with an oil mirror (100x) and repeated three times. Figure 7A is a white field photomicrograph of Compound C staining HeLa living cells, and 7B is a fluorescence micrograph of Compound C staining HeLa living cells. As shown in the figure, Compound C was clearly stained for HeLa cell nuclei. The instrument used was a confocal laser scanning microscope, model: FV1000IX81, Japan. Excitation channel: 488 nm.

Example 14

The staining of MCF-7 living cells by Compound D was observed under confocal laser scanning microscopy:

Compound DD, 1 mM PBS buffer 10 was added to a well-cultured MCF-7 cell in a six-well plate and incubated for 60 min at 37 ° C in a 5% CO ₂ incubator. Then, PBS was shaken and washed 3 times, and then cell culture medium was added, and the cell morphology was observed by a confocal laser scanning microscope. Representative regions were selected and challenged on a 488 nm channel, observed with an oil mirror (100x) and repeated three times. Figure 8A is a white field photomicrograph of Compound D staining MCF-7 live cells, and 8B is a fluorescence micrograph of Compound D staining MCF-7 live cells. As shown in the figure, Compound D was clearly stained for MCF-7 cell nuclei. The instrument used was a confocal laser scanning microscope, model: FV1000IX81, Japan. Excitation channel: 488 nm.

The above is a further detailed description of the present invention in connection with the specific preferred embodiments, and the specific embodiments of the present invention are not limited to the description. It will be apparent to those skilled in the art that the present invention may be practiced without departing from the spirit and scope of the invention. As a fluorescent dye is a use of the novel compounds of the present invention, it is not believed that the compounds of the present invention are used only for fluorescent dyes, and the same effect of the compounds of the present invention as fluorescent dyes is known to those of ordinary skill in the art to which the present invention pertains. Under the consideration of the mechanism, a number of simple inferences can also be made, and other application uses of the compounds of the present invention are considered to be within the scope of the present invention.

Claims

Claim

1. A class of green photochromic fluorescent dyes having the following structural formula I:

Formula I

X is C(CH ₃ ) ₂ , 0, S or Se;

Ri and R ₂ are each independently selected from gH, d- ₁₈ alkyl, 0, d- ₆ alkyl or halogen;

R ₃ is specific ₅ ) (R ₇ );

R ₅ and R ₇ are each independently selected from H or d- ₁₈ alkyl;

R6 is H or _ ₁₈ alkyl;

Y" is a negative ion.

2. A fluorescent dye according to claim 1, wherein said and are each independently selected from H, d_ ₁₈ alkyl, OR ₆ and halogen.

The fluorescent dye according to claim 2, wherein the compound is 11 or Cl, and is 11 or a methoxy group.

4. The fluorescent dye according to claim 1, wherein R _{4 is} selected from the group consisting of d- ₁₈ alkyl, benzyl and substituted benzyl, and the substituted benzyl is optionally substituted by the following groups: COOH, NH ₂ , OH, D_ ₆ alkoxy or halogen.

5. A fluorescent dye according to claim 4, characterized in that said R4 is selected from the group consisting of d- ₆ alkyl, benzyl and halogen substituted benzyl.

6. The fluorescent dye according to claim 1, wherein each of R ₅ and R _{7 is} independently selected from H or d- ₆ alkyl.

The fluorescent dye according to claim 1, wherein the Y- is a halogen ion, C1 (V, PF ₆ ", BF ₄ -, CH ₃ COO - or OTs".

8. The method for preparing a green photochromic fluorescent dye according to claim 1, comprising the steps of:

1) The compound of the formula Ila or lib is reacted with R ₄ Z to prepare a first quaternary ammonium salt intermediate Ilia or nib, wherein Z is a halogen or OTs, and Z- is a halogen anion or OTs- formed by the reaction, which is halogen:

The reaction temperature is 60-140 ° C, the reaction time is 10 minutes to 2 hours, the reaction solvent is DMF, DMSO or a mixed solvent thereof, and the molar ratio of the sodium salt or potassium salt containing Y- to the compound of the formula VII is 1: 1- 10:1 ο

9. The use of the green photochromic fluorescent dye of claim 1 for staining biological samples.

A method of dyeing a biological sample, comprising the step of contacting a compound according to any one of claims 1 to 6 with a biological sample.