CN117466834A - Cyanine dyes, preparation methods and uses thereof - Google Patents

Abstract

The present application relates to fluorescent dyes, in particular cyanine dyes, a process for their preparation and their use. Specifically, the present application provides a compound having a structure as shown in formula I or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, wherein R ₁ 、R ₂ 、R ₃ As defined in the specification. The cyanine dye provided by the application has the advantages of good stability, good biological penetrability and the like.

Description

Cyanine dye, its preparation method and use

Technical Field

The application relates to a fluorescent dye, in particular to a cyanine dye, a preparation method and application thereof. The application also relates to conjugates comprising said cyanine dyes and to compositions for staining biological samples.

Background

DNA (DeoxyriboNucleic Acid ) is a class of biological macromolecules with genetic information. Normal cells of an organism typically have a relatively stable DNA diploid content and when diseased, undergo abnormal changes; and the DNA content of different species of organisms generally varies. Therefore, the method has great significance for specific recognition and accurate measurement of DNA, especially detection in living cells. Qualitative or quantitative analysis of DNA by using fluorescent dye has the advantages of high sensitivity, quick response and the like, and is interesting to a large number of scientific researchers.

At present, commercial dyes mainly comprise phenanthridines, acridines, imidazoles, cyanine families and the like. However, there are certain limitations to the application of these dyes. Firstly, a larger part of dye is combined with DNA to present fluorescence quenching, so that the dye has low practical value in the visualization application of fluorescence imaging and the like. Secondly, most dyes at present have poor specificity and cannot be specifically combined with DNA, so that the application potential of the dyes is greatly limited. Thirdly, most dyes at present have great toxicity and carcinogenicity per se, and biological samples can be marked by increasing the permeability of cell membranes. However, such permeability treatments cause damage to cells and biological tissues, limiting their use in living cells.

Among the fluorescent dyes, cyanine fluorescent dyes have been widely used as biomolecular fluorescent probes due to their advantages of wide wavelength range, large molar extinction coefficient, moderate fluorescence quantum yield, and the like. Although some of the cyanine dyes have been commercialized, most of these dyes have large molecules, complex structures, and low living cell permeability. In addition, the stokes shift of most dyes is small, so that serious crosstalk between an excitation spectrum and an emission spectrum is caused, background interference and fluorescence self-quenching phenomena are caused, and the application of the dyes is limited.

Disclosure of Invention

In order to solve the problems, the inventor of the application obtains novel benzothiazole cyanine dye through molecular modification, the thermal stability of the cyanine dye is obviously improved, and compared with the prior benzothiazole cyanine dye, the cyanine dye has high biological penetrability, and is more suitable for cell dye and imaging.

Compounds of formula (I)

In a first aspect, the present application provides a compound having a structure as shown in formula I or a hydrate, solvate, stereoisomer, tautomer or crystalline form thereof,

wherein,

R ₁ and R is ₂ Identical or different, and independently selected from C _1-18 Straight-chain or branched alkyl, C _1-18 A linear or branched alkylene group-M selected from the group consisting of sulfonic acid groups, phenyl groups, carboxyl groups, mercapto groups, and amino groups;

R ₃ selected from hydrogen, sulfonic acid group, halogen, cyano group, C _1-6 Alkyl, hydroxy, C _1-6 Alkoxy, halo C _1-6 An alkyl group;

y is absent or a counter anion;

and defines:

when R is ₃ When hydrogen, R ₁ And R is ₂ Not both methyl and R ₁ And R is ₂ Not both benzyl.

In the compounds of the invention, R ₁ And R is ₂ May be the same or different. In some embodiments, R ₁ And R is ₂ Independently selected from C _1-6 Straight chain alkyl, C _1-6 The linear alkylene group M is selected from sulfonic acid group, phenyl group, carboxyl group, mercapto group and amino group.

In some embodiments, R ₁ And R is ₂ At least one of which is C _1-18 Linear or branched alkylene-sulfonic acid groups.

In some embodiments, R ₁ And R is ₂ Is different and independently selected from C _1-6 Straight chain alkyl, benzyl, C _1-6 Straight chain alkylene-carboxyl, C _1-6 Straight chain alkylene-sulfonic acid group, C _1-6 Straight chain alkylene-mercapto, C _1-6 Linear alkylene-amino groups.

In some embodiments, R ₁ And R is ₂ Identical and selected from C _1-6 Straight-chain alkaneRadical, C _1-6 Straight chain alkylene-sulfonic acid group, C _1-6 Linear alkylene-carboxyl groups.

In some embodiments, R ₃ Selected from hydrogen, sulfonic acid group, halogen, cyano group, C _1-6 An alkyl group.

In some embodiments, Y in formula I is a counter anion, in which case Y may be selected from the group consisting of halide ions (e.g., F ^- 、Cl ^- 、Br ^- 、I ^- )、ClO ₄ ^- 、PF ₆ ^- 、CF ₃ SO ₃ ^- 、BF ₄ ^- Acetate, methanesulfonate or p-toluenesulfonate.

In some embodiments, Y in formula I is absent, in which case the compound may be an internal salt. The "inner salt" is also referred to in the art as a "zwitterionic". In some embodiments, the compounds of the present invention may contain both an acidic group (e.g., a sulfonic acid group or a carboxyl group) and a basic group (e.g., an amino group or a thiazole ring) within the molecule, which are neutralized with each other to form an inner salt.

It will be appreciated that when a compound contains a plurality of acidic groups and/or a plurality of basic groups in the molecule, each acidic group and/or each basic group may act as a salt-forming group. Salts of the compounds formed by various salt-forming means are included within the scope of the present invention.

In some embodiments, R ₁ And R is ₂ At least one of which is selected from C _1-18 Straight-chain or branched alkylene-sulfonic acid groups, C _1-18 Straight-chain or branched alkylene-carboxyl groups.

The compounds of the present invention may have any of the following structures:

in some embodiments, the compounds of the present invention are compounds represented by structural formulas 5, 6, or 9 above.

Conjugate and composition for staining biological samples

The compounds of the present invention and their hydrates, solvates, stereoisomers, tautomers or crystal forms may be used directly for staining biological samples, and may also be used in the form of derivatives, including but not limited to conjugates.

As used herein, "conjugate" refers to a compound of the invention, a hydrate, solvate, stereoisomer, tautomer, or crystal form thereof, that is formed by covalent linkage to other molecules. The molecules that may be conjugated to the compounds of the invention, hydrates, solvates, stereoisomers, tautomers or crystal forms thereof, may be molecules that specifically bind to a cell or cell 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 cell components in the test sample, which may also be referred to as staining. The staining procedure may be performed multiple times in sequence, or multiple stains may be performed simultaneously with multiple conjugates. After the staining is completed, the sample is analyzed in an analytical instrument comprising an excitation light source that excites the fluorescent dye of the invention in the conjugate and an assay device that measures the emitted light generated by the excited fluorescent dye.

The present application also provides a composition for staining a biological sample, wherein the composition comprises a compound of the invention, a hydrate, solvate, stereoisomer, tautomer or crystal thereof, or a conjugate of the invention. In some embodiments, the biological sample is a nucleic acid. In some embodiments, the biological sample is deoxyribonucleic acid.

Use of the same

The present application also provides the use of a compound of the invention, a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, or a conjugate of the invention, or a composition of the invention, for staining a biological sample. In some embodiments, the biological sample is a nucleic acid. In some embodiments, the biological sample is deoxyribonucleic acid.

The present application also provides the use of a compound of the invention or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, or a conjugate of the invention, or a composition of the invention, for identifying parasites in a blood sample to be tested using flow cytometry. In some embodiments, the parasite is selected from the group consisting of: roundworm, hookworm, tapeworm, trichomonas vaginalis, liver fluke, paragonium wegener, toxoplasma gondii, cysticercus suis, trichina, amoeba, leishmania donovani, plasmodium, schistosome, filarial, artemia, scabies, follicular mites, lice, fleas.

The application also provides the use of the compound of the invention or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, or the conjugate of the invention, or the composition of the invention, for identifying microorganisms in a blood sample or a body fluid sample to be tested by flow cytometry.

In some embodiments, the microorganism is selected from the group consisting of:

bacteria (e.g., staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, bacillus dysenteriae, bacillus pertussis, bacillus diphtheriae, neisseria meningitidis, mycobacterium tuberculosis, clostridium tetani, bacillus leptospiriformis, group A hemolytic streptococcus, brucella, bacillus cholerae, typhoid bacillus, bacillus anthracis, neisseria gonorrhoeae, vibrio cholerae, pseudomonas klebsiella, and paratyphoid A, B or C.propiolatus),

viruses (e.g., influenza virus, mumps virus, rubella virus, japanese encephalitis virus, dengue virus, epidemic hemorrhagic fever virus, rabies virus, human papilloma virus, polio virus, measles virus, varicella zoster virus, hepatitis virus, novel enterovirus type 70, coxsackie virus type A24 variant, human immunodeficiency virus, poxvirus (e.g., monkey poxvirus)),

Fungi (e.g., candida albicans, trichophyton rubrum, and epizoon floccosum),

mycoplasma (such as mycoplasma pneumoniae, ureaplasma urealyticum, mycoplasma hominis, and mycoplasma genitalium),

chlamydia (e.g., chlamydia trachomatis, chlamydia pneumoniae, chlamydia psittaci, and chlamydia of livestock),

rickettsia (e.g., rickettsia praecox, rickettsia moellendorffimbriae, rickettsia tsutsugamushi) and,

actinomycetes (e.g., actinomycetes israeli),

spirochetes (e.g., leptospira, treponema pallidum).

In some embodiments, blood or body fluid refers to blood or body fluid from a mammal, particularly a human. Among them, body fluids (body fluids) can be classified into urine, sweat, cerebrospinal fluid (cerebrospinal fluid), serosal fluid (serous cavity fluid), joint synovial fluid (synovial fluid) and the like according to the location. The body fluids are all present in small amounts in normal humans. The effusion in serosal cavities includes pleural effusion (pleuroperitoneal effusion), peritoneal effusion (ascites) and pericardial effusion (pericardial effusion), and the effusion in articular cavities is synovial fluid (joint effusion).

General synthetic method

The compounds of the present invention may be synthesized by methods common in the art. Illustratively, the benzothiazole compounds of the present invention may be synthesized by the following methods: starting first with unsubstituted or substituted methylbenzothiazole, this is reacted with R ₂ X (X is F, cl, br or I) is heated for reflux reaction, and the intermediate I in the form of quaternary ammonium salt is obtained. And then, carrying out heating reflux reaction on the connecting molecule 4-hydroxy isophthalaldehyde and the intermediate I (the molar ratio of the intermediate I to the 4-hydroxy isophthalaldehyde is more than or equal to 2) so as to enable the intermediate I to be condensed with the connecting molecule, thereby obtaining the benzothiazole compound.

When R is ₂ Is C _1-18 In the case of straight-chain or branched alkylene-sulphonic acid groups, intermediate I may also be synthesised by the following method: from nothingStarting from substituted or substituted methylbenzothiazoles and the like, intermediate I is obtained by ring-opening reaction of the substituted or substituted methylbenzothiazoles and the like with proper sultones.

The above method is suitable for R ₁ And R is ₂ The same is true.

When R is ₁ And R is ₂ When not identical, the benzothiazole compounds of the present invention may be synthesized by the following exemplary methods: r is R ₁ Or R is ₂ The substituted methylbenzothiazole reacts with the connecting molecule 4-hydroxy isophthalaldehyde by heating and refluxing (4-hydroxy isophthalaldehyde and R) ₁ Or R is ₂ The molar ratio of substituted methylbenzothiazole is about 2), giving the formyl-containing intermediate I'; allowing the resulting intermediate I' to react with R ₂ Or R is ₁ The substituted methylbenzothiazole is subjected to condensation reaction to obtain the benzothiazole compound.

In the above process, each intermediate or product may be recovered by isolation and purification techniques well known in the art to achieve the desired purity.

The various starting materials used in the above-described process are commercially available or may be prepared from the starting materials known in the art by methods known to those skilled in the art or methods disclosed in the prior art.

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the experimental procedures used herein are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "C _1-18 By straight or branched alkyl "is meant a radical obtained by removing one hydrogen atom from a straight or branched alkane containing from 1 to 18 carbon atoms, specific examples of which include, but are not limited to: methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, t-butyl, isobutyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, N-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, and n-octadecyl.

As used herein, "C _1-6 Alkyl "refers to a group obtained by removing one hydrogen atom from a straight or branched alkane containing 1 to 6 carbon atoms, specific examples of which include, but are not limited to: methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, t-butyl, isobutyl, and the like.

As used herein, "C _1-6 By straight chain alkyl "is meant a group derived from a straight chain alkane containing 1 to 6 carbon atoms with one hydrogen atom removed, specific examples of which include, but are not limited to: methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl.

C _1-18 The straight-chain or branched alkylene group means a group obtained by removing two hydrogen atoms from a straight-chain or branched alkane having 1 to 18 carbon atoms, and specific examples thereof include, but are not limited to, methylene, ethylene, propylene, butylene and the like. The term "C _1-6 The straight chain alkylene group means a group obtained by removing two hydrogen atoms from a straight chain alkane having 1 to 6 carbon atoms.

As used herein, the term "halogen" includes fluorine, chlorine, bromine and iodine.

As used herein, the term "halo" refers to the substitution of a hydrogen on a group or compound with one or more halogen atoms, including perhalo and partially halo.

As used herein, the term "C _1-6 Alkoxy "means, in C _1-6 alkyl-O-formed radicals.

As used herein, the term "solvate" (or "solvate") refers to a substance formed by molecular association of a compound with an organic solvent (e.g., methanol, ethanol, propanol, acetonitrile, etc.).

As used herein, the term "hydrate" refers to a substance formed by association of a compound with water molecules.

As used herein, the term "crystalline form" refers to the crystalline structure of a substance. The material is affected by various factors during crystallization, so that the bonding mode of molecules or molecules is changed, and molecules or atoms are arranged differently in lattice space, so that different crystal structures are formed. The compounds of the present invention may exist in one crystal structure or in a plurality of crystal structures, i.e. have a "polymorphic form". The compounds of the present invention may exist in different crystalline forms.

As used herein, the term "stereoisomers" includes conformational isomers and configurational isomers, wherein the configurational isomers include predominantly cis-trans isomers and optical isomers. The compounds of the present invention may exist in stereoisomeric forms and thus encompass all possible stereoisomeric forms, as well as any combination or any mixture thereof. For example, a single enantiomer, a single diastereomer, or a mixture thereof. When the compounds of the present invention contain olefinic double bonds, they include cis-isomers and trans-isomers, and any combination thereof, unless specified otherwise.

The compounds described herein may exist in tautomeric forms having different points of attachment of hydrogen through displacement of one or more double bonds. For example, the ketone and its enol form are keto-enol tautomers. It is to be understood that the present invention encompasses keto-enol tautomers of all compounds. Each tautomer and mixtures thereof are included within the scope of the present invention.

Advantageous effects of the invention

Compared with the prior art, the fluorescent dye has one or more of the following beneficial effects:

1. compared with the existing dye, the dye has obviously improved thermal stability;

2. the dye needs higher cell penetrating power for cell staining, and compared with the existing dye, the dye has high biological penetrating power, and is more suitable for cell staining and imaging;

3. the dye can be specifically combined with DNA, and is favorable for specific recognition and accurate measurement of the DNA;

4. the dye has good living cell permeability, can enter cells to dye nucleic acid under the condition of not damaging cell membranes, and has low toxicity and low carcinogenicity;

5. the excitation light of the dye is blue-green light with smaller wavelength, so that tiny particles can be identified, and the detection capability of the tiny particles is improved;

6. The dye can use a common green semiconductor laser as a light source, so that the use cost is greatly reduced;

7. the dye has the advantages of simple structure, easily available raw materials for preparing the dye, high synthesis yield and easy realization of industrialization.

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Drawings

FIG. 1 shows the change in fluorescence spectrum as the concentration of DNA increases with dye in example 7.

FIG. 2 shows the change in fluorescence spectrum as the concentration of RNA increases for the dye of example 7.

FIG. 3 is a graph showing the linear relationship between fluorescence intensity and calf thymus DNA and RNA concentrations in example 7.

FIG. 4 shows the result of observing the staining of HepG2 living cells by the compound II under the laser microscope in example 8.

FIG. 5 shows the result of observing the staining of HepG2 living cells by the compound III under the laser microscope in example 9.

FIG. 6 shows the evaluation results of the stability of the dye in example 10.

FIG. 7 shows the evaluation results of dye cell penetrating power in example 11.

Fig. 8 is a scatter diagram of the results of simultaneously obtaining the identification of microorganisms and the classification of white blood cells in one test of the same blood sample according to example 12 of the present invention.

Fig. 9 is a scattergram obtained by testing a body fluid sample according to example 13 of the present invention.

Fig. 10 is a scatter diagram of the results of simultaneously obtaining the identification of microorganisms and the classification of white blood cells in one test of the same blood sample according to example 14 of the present invention.

Fig. 11 is a scattergram obtained by testing a body fluid sample according to example 15 of the present invention.

Fig. 12 is a scatter diagram of the results of simultaneously obtaining the identification of microorganisms and the classification of white blood cells in one test of the same blood sample according to example 16 of the present invention.

FIG. 13 is a scatter plot of a body fluid sample tested in accordance with example 17 of the present invention.

FIG. 14 is a scatter plot of results of both microbiological identification and nucleated red blood cell identification obtained in one test of the same blood sample, according to example 18 of the present invention.

FIG. 15 is a scatter plot of a body fluid sample tested in accordance with example 19 of the present invention.

Fig. 16 is a scatter diagram of the results of both the identification of microorganisms and the identification of nucleated red blood cells obtained in one test of the same blood sample according to embodiment 20 of the present invention.

Fig. 17 is a scattergram obtained by testing a body fluid sample according to example 21 of the present invention.

Fig. 18 is a scatter plot of both parasite identification results and nucleated red blood cell identification results obtained in one test of the same blood sample, in accordance with example 22 of the present invention.

Fig. 19 is a scatter plot of both parasite identification results and nucleated red blood cell identification results obtained in one test of the same blood sample, in accordance with example 23 of the present invention.

Fig. 20 is a scatter plot of both parasite identification results and nucleated red blood cell identification results obtained in one test of the same blood sample, in accordance with example 24 of the present invention.

Fig. 21 is a scatter plot of parasite identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample in accordance with example 25 of the present invention.

Fig. 22 is a scatter plot of parasite identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample in accordance with example 26 of the present invention.

Fig. 23 is a scatter plot of parasite identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample in accordance with example 27 of the present invention.

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it. The experiments and methods described in the examples were performed substantially in accordance with conventional methods well known in the art and described in various references unless specifically indicated. In addition, the specific conditions are not specified in the examples, and the process is carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

EXAMPLE 1 Synthesis of Compound I

The structure of compound I can be represented by structural formula 1.

In the first step, the compound (E) -4-2- (5-formyl-2-hydroxystyryl) benzothiazole-3-butyl-1-sulfonic acid inner salt (structure shown on the right side of formula I) is prepared according to formula I below.

Into the reaction flask were added 5mL of methanol, and 1.33mmol of 4-hydroxy-isophthalaldehyde (Shanghai Bi Co., ltd.) and 0.67mmol of (E) -4- (2- (5-formyl-2-hydroxystyryl) benzothiazol-3-yl) -butyl-1-sulfonate and 2.66mmol of pyridine (Shanghai Bi Co., ltd.) were sequentially added. The reaction was stirred at 80℃for 43 hours.

The reaction mixture was suction-filtered, the filter cake was washed 3 times with 10mL of ethanol, and the filter cake was collected and dried under reduced pressure to give 0.44mmol of a yellow solid powder, which was (E) -4- (2- (5-formyl-2-hydroxystyryl) benzothiazol-3-yl) -butyl-1-sulfonic acid inner salt. The yield of this reaction of formula I was about 33%.

The compounds were subjected to nuclear magnetic resonance testing and the results are as follows.

1H NMR(400MHz,DMSO-d6)δ＝12.04(s,1H),9.95(s,1H),8.78(d,J＝2.0Hz,1H),8.42-8.40(m,2H),8.34-8.24(m,2H),7.94-7.86(m,2H),7.80(t,J＝7.6Hz,1H),7.18-7.12(m,1H),5.00-4.88(m,2H),2.59–2.56(m,2H),2.08-2.10(m,2H),1.89-1.79(m,2H)。

The second step was to prepare an inner salt of 4- (2- ((E) -3- ((Z)) -2- (3-methylbenzothiazole-2 (3H) -enyl) vinyl) -6-oxo-1, 4-cyclohexadien-1-yl) vinyl) benzothiazol-3-yl) butanesulfonic acid according to the following reaction formula II (compound I).

To the reaction flask was added 5mL of acetic acid, 0.44mmol of the inner salt of (E) -4- (2- (5-formyl-2-hydroxystyryl) benzothiazol-3-yl) -butyl-1-sulfonic acid (structure shown in the left side of reaction formula II) obtained in the second step, 0.72mmol of 2, 3-dimethylbenzothiazole salt (Shanghai Haihong biomedical science and technology Co., ltd.) and 1.58mmol of sodium acetate were sequentially added to the reaction flask, and reacted at 110℃for 12 hours.

The reaction mixture was filtered and the filter cake was purified with 5mL acetonitrile/water=1: 1 for 3 times to obtain a crude product.

The above crude product was purified by preparative HPLC to give about 0.01mmol of a red solid powder which was 4- (2- ((E) -3- ((Z)) -2- (3-methylbenzothiazole-2 (3H) -enyl) vinyl) -6-oxo-1, 4-cyclohexadien-1-yl) vinyl) benzothiazol-3-yl) butanesulfonic acid inner salt (compound I). The yield of reaction formula II was about 20%.

The product was subjected to nuclear magnetic resonance testing and the results were as follows:

1H NMR(400MHz,DMSO-d6)δ＝8.35-8.14(m,4H),8.09-7.94(m,3H),7.91-7.82(m,2H),7.77-7.74(m,1H),7.70-7.63(m,2H),7.59-7.55(m,1H),7.44-7.29(m,1H),6.39(d,J＝9.2Hz,1H),4.68-4.66(m,2H),4.15-4.13(m,3H),2.62-2.58(m,2H),2.01-1.92(m,2H),1.85-1.81(m,2H)。

the test result is verified to be in accordance with the structure of the structural formula 1.

EXAMPLE 2 Synthesis of Compound II

The structure of compound II can be represented by structural formula 2.

In the first step, 2-methyl-3- (butylsulphonic acid) benzothiazole salt is prepared according to the following reaction scheme I (structure is shown on the right side of reaction scheme I).

50mL of toluene was weighed into a vessel, and 34mmol of benzothiazole (left side of the reaction formula I) and 40mmol of 1, 4-butansultone (Shanghai Honghai Biomedicine Co., ltd.) were charged into the vessel. Reflux and stir under nitrogen for 24 hours before stopping the reaction.

The mixture after the reaction was suction filtered, and then the filter cake was washed 3 times with 50mL of ethyl acetate to obtain a yellow solid product, namely 2-methyl-3- (butylsulfonic acid) benzothiazole salt. The yield of this reaction of formula I was about 62%.

In a second step, compound II (4- (2- ((E) -6-oxo-3- ((Z) -2- (3- (4-sulfobutyl) benzo [ d ] thiazol-2-ylidene) ethylidene) cyclohexyl-1, 4-dien-1-yl) vinyl) benzo [ d ] thiazol-3-yl) butanesulfonic acid is prepared according to reaction formula II below.

30mL of acetic acid was weighed into a vessel, and 1.7mmol of the 2-methyl-3- (butylsulfonic acid) benzothiazole salt obtained in the first reaction (structure shown above arrow of reaction formula II), 0.7mmol of 4-hydroxy isophthalaldehyde, and 2.2mmol of sodium acetate were added into the vessel. The reaction was stirred under nitrogen at 80℃for 24 hours.

The reacted mixture was poured into 50mL of a petroleum ether:ethyl acetate=5:1 solution prepared in advance, and then the supernatant was poured out to obtain a crude product. The crude product was slurried with 10mL acetonitrile/water 1:1 to give about 0.34mmol of a brown solid powder, compound II, in about 52% yield.

The product was subjected to nuclear magnetic resonance testing and the results are as follows.

¹ H NMR(400MHz,DMSO-d6，TMS)δ＝8.87(bs,1H),8.42-8.34(m,4H),8.30-8.22(m,2H),8.21-8.07(m,3H),7.88-7.73(m,4H),7.09(d,J＝8.0Hz,1H),5.00-4.95(m,4H),2.68-2.65(m,4H),2.11-2.03(m,4H),1.90-1.84(m,4H).

The test result is verified to be in accordance with the structure of the structural formula 2.

EXAMPLE 3 Synthesis of Compound VI

The structure of compound VI can be represented by formula 6, wherein Y ^- Is iodide ion.

In a first step, a quaternary ammonium iodide of 2, 3-dimethyl-5-chlorobenzothiazole is prepared according to the following reaction formula I

To the reaction flask was added 20mL of methyl iodide followed by 10.9mmol of 5-chloro-2-methylbenzothiazole (Shanghai Bi. Medical technologies Co., ltd.), and the reaction was heated to 80℃and stirred at this temperature for 16 hours.

The reaction mixture was filtered and the filter cake was washed three times with 10mL of ethyl acetate. The filter cake was collected and dried under reduced pressure to give 7.35mmol of a white powder solid. The white powder solid was 2, 3-dimethyl-5-chlorobenzothiazole quaternary ammonium iodide (right side of formula I). The yield of this reaction of formula I was about 67%.

The product was subjected to nuclear magnetic resonance testing and the results are as follows.

1H NMR(400MHz,DMSO-d6)δ＝8.53(d,J＝12.0Hz,1H),8.45(d,J＝8.8Hz,1H),7.88(dd,J＝2.0,8.8Hz,1H),4.18(s,3H),3.17(s,3H)

In a second step, a 5-chloro-2- ((E) -2- ((E) -3- ((Z) -2- (5-chloro-3-methylbenzothiazole-2 (3H) -ylidene) vinyl) -6-oxocyclohex-1, 4-dien-1-yl) vinyl-3-methylbenzothiazole salt (Compound VI) is prepared according to the following reaction scheme II

6mL of acetic acid was weighed into a vessel, and 1.67mmol of the quaternary ammonium salt of 5-chloro-2, 3-dimethylbenzothiazole (structure shown in the left side of the formula II) obtained in the first reaction was added to a mixture of 0.67mmol of 4-hydroxy-isophthalaldehyde (Shanghai Honghai biomedical science and technology Co., ltd.) and 2.2mmol of sodium acetate. The reaction was stirred at 110℃for 12 hours.

And after the reaction is finished, filtering the mixture after the reaction, and collecting a filter cake to obtain a crude product.

The above crude product was added to 20mL of acetonitrile and stirred at 90℃for 2 hours. The solid was collected by filtration to give about 0.46mmol of a tan solid which was a 5-chloro-2- ((E) -2- ((E) -3- ((Z) -2- (5-chloro-3-methylbenzothiazole-2 (3H) -ylidene) vinyl) -6-oxocyclohex-1, 4-dien-1-yl) vinyl-3-methylbenzothiazole salt (compound VI) in about 70% yield of formula II.

The product was subjected to nuclear magnetic resonance testing and the results are as follows.

1H NMR(400MHz,DMSO-d6)δ＝8.73(d,J＝1.2Hz,1H),8.53(d,J＝2.0Hz,1H),8.47-8.41(m,3H),8.26-8.12(m,4H),7.95(d,J＝16.4Hz,1H),7.90-7.84(m,2H),7.16(d,J＝8.8Hz,1H),4.36(d,J＝6.8Hz,6H).

The test result is verified to be in accordance with the structure of the structural formula 6.

EXAMPLE 4 Synthesis of Compound VII

The structure of compound VII may be represented by structural formula 7.

In the first step, 3- (5-carboxypentyl) -2-methylbenzothiazole bromide salt is prepared according to the following reaction formula I (structure is shown on the right side of the reaction formula I).

67mmol of benzothiazole (Shanghai Haohong Biomedicine technologies Co., ltd.) with the structure shown on the left side of reaction formula I was added to the vessel. Then 73.72mmol of 6-bromohexanoic acid was added, and after the reaction system was warmed to 140℃it was stirred for 12 hours. After that, the reaction was stopped and cooled to room temperature.

The mixture after the reaction was filtered and the filter cake was washed three times with 30mL petroleum ether. About 45mmol of a yellow solid, i.e. 3- (5-carboxypentyl) -2-methylbenzothiazole bromide salt, is obtained in a yield of about 78% for reaction scheme I.

The product was subjected to nuclear magnetic resonance testing and the results are as follows.

1H NMR(400MHz,DMSO-d6)δ＝8.47(d,J＝8.0Hz,1H),8.35(d,J＝8.4Hz,1H),7.91-7.87(m,1H),7.82-7.78(m,1H),4.73-4.70(m,2H),3.22(s,3H),2.22(t,J＝7.2Hz,2H),1.89-1.78(m,2H),1.60-1.53(m,2H),1.50-1.41(m,2H).

In a second step, 2- ((trans) -6-oxo-3- ((Z) -2- (5-carboxypentyl) benzothiazol-2 (3H) -yl) vinyl) cyclohexa-1, 4-dienyl) vinyl-3- (5-carboxypentyl) benzothiazole salt (compound VII) is prepared according to the following reaction formula II.

1.7mmol of 3- (5-carboxypentyl) -2-methylbenzothiazole bromide salt obtained in the first reaction step and 0.7mmol of 4-hydroxy-isophthalaldehyde were added to 10mL of acetic acid, followed by addition of 2.2mmol of sodium acetate. The reaction was stirred at 110℃for 12 hours. After the reaction was completed, the mixture was cooled to room temperature and allowed to stand for 16 hours. A large amount of solid is separated out, and the crude product is obtained by filtration.

After stirring the above crude product over 10mL of acetonitrile for 1 hour, it was filtered again, the filter cake was washed three times with 5mL of acetonitrile, the solid was collected and dried under reduced pressure to give about 0.43mmol of a brown solid powder which was 2- ((trans) -6-oxo-3- ((Z) -2- (5-carboxypentyl) benzothiazol-2 (3H) -yl) vinyl) cyclohexa-1, 4-dienyl) vinyl-3- (5-carboxypentyl) benzothiazole salt (compound VII), yield of reaction formula II was about 64%.

The product was subjected to nuclear magnetic resonance testing and the results are as follows.

1H NMR(400MHz,DMSO-d6)δ＝12.09-11.94(m,2H),8.76(bs,1H),8.43-8.38(m,3H),8.30(d,J＝8.4Hz,1H),8.24-8.20(m,2H),8.16-8.10(m,2H),7.99-7.95(m,1H),7.89-7.73(m,4H),7.02(d,J＝8.8Hz,1H),4.96-4.95(m,4H),2.23-2.18(m,4H),1.92-1.83(m,4H),1.62-1.47(m,8H).

The test result is verified to be in accordance with the structure of structural formula 7.

EXAMPLE 5 Synthesis of Compound VIII

The structure of compound VIII can be represented by formula 8, wherein Y ^- Is iodide ion.

In the first step, 2, 3-dimethyl-5-fluorobenzothiazole quaternary ammonium iodide was prepared according to the following reaction scheme I (structure shown on the right side of the reaction scheme I).

To the reaction flask was added 10mL of methyl iodide followed by 9.0mmol of 5-fluoro-2-methylbenzothiazole (Shanghai Hahong Biotechnology Co., ltd.), and the reaction was heated to 80℃and stirred at this temperature for 12 hours.

The reaction mixture was filtered and the filter cake was washed three times with 10mL of ethyl acetate. The filter cake was collected and dried under reduced pressure to give 7.1mmol of white powder solid B. The white powder solid is the quaternary ammonium iodide of 2, 3-dimethyl-5-fluorobenzothiazole (the structure is shown on the right side of the reaction formula I). The yield of this reaction of formula I was about 79%.

The product was subjected to nuclear magnetic resonance testing and the results are as follows.

H NMR(400MHz,DMSO-d6)δ＝8.50(dd,J＝5.2,9.2Hz,1H),8.34(dd,J＝2.4,8.4Hz,1H),7.74(dt,J＝2.0,8.8Hz,1H),4.18(s,3H),3.18(s,3H).

The test result is verified to be in accordance with the structure of the product in the reaction formula I.

Second step, 5-fluoro-2- ((E) -2- ((E) -3- ((Z) -2- (5-fluoro-3-methylbenzothiazole-2 (3H) -ylidene) vinyl) -6-oxocyclohex-1, 4-dien-1-yl) vinyl-3-methylbenzothiazole salt (Compound VIII) is prepared according to the following reaction formula II

2mmol of the quaternary ammonium salt of 2, 3-dimethyl-5-fluorobenzothiazole (structure shown in the left side of the formula II) obtained in the first reaction and 0.67mmol of 4-hydroxy-m-xylylene-dicarboxaldehyde were added to 10mL of acetic acid, followed by addition of 2.2mmol of sodium acetate. The reaction was stirred at 120℃for 12 hours.

After the reaction was completed, the mixture after the reaction was filtered, and the filter cake was washed 3 times with 10mL of acetonitrile, and the solid was collected to give about 0.46mmol of a tan solid, which was 5-fluoro-2- ((E) -2- ((E) -3- ((Z) -2- (5-fluoro-3-methylbenzothiazole-2 (3H) -ylidene) vinyl) -6-oxocyclohex-1, 4-dien-1-yl) vinyl-3-methylbenzothiazole salt (Compound VIII), the yield of reaction formula II was about 95%.

The product was subjected to nuclear magnetic resonance testing and the results are as follows.

1H NMR(400MHz,DMSO-d6)δ＝8.72(bs,1H),8.48-8.18(m,8H),7.97-7.94(m,1H),7.73(bs,2H),7.15(bs,1H),4.35(bs,6H).

The test result is verified to be in accordance with the structure of structural formula 8.

EXAMPLE 6 Synthesis of Compound IX

The structure of compound IX may be represented by formula 9, wherein Y ^- Is iodide ion.

In the first step, 2-methyl-5-cyanobenzothiazole was prepared according to the following reaction scheme I (structure shown on the right side of reaction scheme I).

To the reaction flask was added 55mL of deionized water, 0.29mmol of potassium acetate was added under nitrogen atmosphere and dissolved, followed by 55mL of dioxane, 5.7mmol of 5-bromo-2-methylbenzothiazole (Shanghai, available from medical technologies Co., ltd.), 2.85mmol of potassium ferricyanate trihydrate and 0.57mmol of XPhos-Pd-G3. After all the raw materials and reagents are added, the mixture is replaced by nitrogen atmosphere. The reaction mixture was reacted at 100℃for 1 hour.

After the reaction was completed, the mixture was diluted with 60mL of water and extracted with 200mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate for 30min, filtered, and the filtrate was concentrated under reduced pressure to give the crude product as a yellow oil. After purification of the crude product by silica gel column chromatography (petroleum ether/ethyl acetate=10:1 to 5:1), 5.72mmol of a grey powder solid is obtained, which is 2-methyl-5-cyanobenzothiazole (structure shown on the right side of the reaction formula I).

The product was subjected to nuclear magnetic resonance testing and the results are as follows.

1H NMR(400MHz,DMSO-d6)δ＝8.44(d,J＝1.2Hz,1H),8.28(d,J＝8.6Hz,1H),7.79(dd,J＝1.6,8.4Hz,1H),2.85(s,3H).

The test result is verified to be in accordance with the structure of the product in the reaction formula I.

In the second step, 2, 3-dimethyl-5-cyanobenzothiazole quaternary ammonium iodide was prepared according to the following reaction scheme II (structure shown on the right side of the reaction scheme II).

To the reaction flask was added 20mL of methyl iodide followed by 5.7mmol of 5-cyano-2-methylbenzothiazole (Chemicals, inc. of Legendoresst technology development Co., ltd.), and the reaction was heated to 80℃and stirred at this temperature for 16 hours.

The reaction mixture was filtered and the filter cake was washed three times with 10mL of ethyl acetate. The filter cake was collected and dried under reduced pressure to give 2.18mmol of a yellow powdery solid which was 2, 3-dimethyl-5-cyanobenzothiazole quaternary ammonium iodide (structure shown on the right side of formula II). The yield of this reaction of formula II was about 38%.

The product was subjected to nuclear magnetic resonance testing and the results are as follows.

1H NMR(400MHz,DMSO-d6)δ＝8.98(s,1H),8.63(d,J＝8.8Hz,1H),8.22(dd,J＝2.0,8.8Hz,1H),4.22(s,3H),3.21(s,3H).

The test results are verified to be in accordance with the structure of the product in the reaction formula II.

Third step, 2- ((trans) -6-oxo-3- ((Z) -2- (5-cyanobenzothiazol-2 (3H) -yl) vinyl) cyclohex-1, 4-dienyl) vinyl-3- (5-cyano) benzothiazole salt (Compound IX) was prepared according to the following reaction formula III

30mL of acetic acid was measured and placed in a vessel, and 2mmol of 5-cyano-2, 3-dimethylbenzothiazole quaternary ammonium salt (structure shown in the left side of the reaction formula III) obtained in the first step, 0.67mmol of 4-hydroxy isophthalaldehyde, and 2.2mmol of sodium acetate were added to the vessel. The reaction was stirred for 12 hours under nitrogen at 120 ℃.

The mixture after the reaction was filtered, and the cake was washed with acetonitrile, and the cake was collected to obtain a crude product.

The above crude product was repeatedly slurried three times with 10mL of acetonitrile to give about 0.37mmol of a brown black solid as a powder of 2- ((trans) -6-oxo-3- ((Z) -2- (5-cyanobenzothiazol-2 (3H) -yl) vinyl) cyclohex-1, 4-dienyl) vinyl-3- (5-cyano) benzothiazole salt (compound IX) in about 52% yield of formula III.

The product was subjected to nuclear magnetic resonance testing and the results are as follows.

1H NMR(400MHz,DMSO-d6)δ＝8.95-8.87(m,2H),8.66-8.60(m,3H),8.30-8.15(m,6H),7.93-7.89(m,1H),7.11(d,J＝8.0Hz,1H),4.36-4.34(m,6H).

The test result is verified to be in accordance with the structure of structural formula 9.

Example 7 evaluation of the specificity of the dye

And (3) measuring a fluorescent intensity change graph of the dye compound II along with the increase of calf thymus DNA and RNA concentration and a linear relation graph of the maximum fluorescent emission peak intensity and calf thymus DNA and RNA concentration to evaluate the specificity of the dye.

An aqueous solution of calf thymus DNA having a certain concentration was prepared, and the absorbance at 260nm was measured by an ultraviolet absorption spectrophotometer to give a concentration of 1.8mM. 100. Mu.L of calf thymus DNA, which had been designated as 1.8mM, was taken and added with 290. Mu.L of water to dilute the aqueous solution of calf thymus DNA at 0.5 mM. 1.5. Mu.L of DMSO (dimethyl sulfoxide) solution of Compound B was prepared, and M-60LN hemolytic agent (Michael) was added to 3mL, and the mixture was placed in a cuvette to measure the fluorescence intensity. Subsequently, 0.6. Mu.L of 0.5mM calf thymus DNA aqueous solution was placed in a cuvette each time, and after the buffer was stirred uniformly, the cuvette was left to stand in an environment of 37℃for 3 minutes, and then the fluorescence intensity was measured. Finally, the calf thymus DNA concentration in the cuvette was 1. Mu.M. Taking the intensity of the maximum fluorescence emission peak of each calf thymus DNA concentration as a linear relation graph of the fluorescence intensity and the calf thymus DNA concentration. Experiments on the linear relationship between the concentration of RNA and the fluorescence intensity were also performed according to the above procedure. The used instrument is an ultraviolet-visible spectrophotometer, and the model is as follows: hp8453; fluorescence spectrophotometer, model: FP-6500.

FIG. 1 shows the change in fluorescence spectrum of Compound II with increasing DNA concentration. FIG. 2 shows the change in fluorescence spectrum of Compound II with increasing RNA concentration. FIG. 3 is a graph showing the linear relationship between fluorescence intensity and calf thymus DNA and RNA concentrations.

As can be seen from the figure, compound II has a concentration dependence on DNA, but not on RNA, indicating that compound II can specifically bind to DNA.

Example 8 staining of HepG2 living cells by Compound II under a laser microscope

10. Mu.L of PBS buffer at 1mM concentration, which was added to a six-well plate in which HepG2 cells were cultured, was added at 37℃with 5% CO ₂ Is incubated for 30min in a cell incubator. Then PBS was washed 3 times with shaking, and then cell culture medium was added thereto, followed by confocal laser scanning microscopy to observe cell morphology. The type of the used instrument is as follows: FV1000IX81, japan.

Fig. 4 shows the observation results. The middle panel is a white field micrograph of compound II staining HepG2 living cells, the left panel is a fluorescent micrograph of compound II staining HepG2 living cells, and the right panel is a superposition of the bright field and fluorescent images. As can be seen from the figure, the compound II can be used for clearly staining HepG2 cell nuclei, which shows that the dye has good permeability and strong nucleic acid staining capability.

Example 9 staining of HepG2 living cells by Compound III under a laser microscope

10. Mu.L of PBS buffer at a concentration of 1mM in compound III was added to a six-well plate in which HepG2 cells were cultured, and incubated at 37℃in a 5% CO2 cell incubator for 30min. Then PBS was washed 3 times with shaking, and then cell culture medium was added thereto, followed by confocal laser scanning microscopy to observe cell morphology. The type of the used instrument is as follows: FV1000IX81, japan.

Fig. 5 shows the observation results. The middle panel is a white field micrograph of compound III staining HepG2 living cells, the left panel is a fluorescent micrograph of compound III staining HepG2 living cells, and the right panel is a superposition of the bright field and fluorescent images. As can be seen from the figure, the compound III can be used for clearly staining HepG2 cell nuclei, which shows that the dye has good permeability and strong nucleic acid staining capability.

Example 10 evaluation of dye stability

The dye is used for commercialized cell dye agents, needs to have certain high-temperature stability, and the existing dye is limited in practical commercialized application due to poor high-temperature stability. In order to improve the situation, the invention designs from the aspect of molecular structure, and develops various novel dyes so as to improve the stability of the dyes.

To evaluate the high temperature stability, 50mg/L of different dye/glycol solutions were placed in a 50℃incubator, 0.5ml of the solutions were measured for dye concentration at different times, and degradation curves were drawn according to the dye concentration at different time points. The dyes tested were: dyes QCy-DT, dye 5 (compound V), dye 6 (compound VI), dye 9 (compound IX) reported in literature (Nucleic Acids Research,2015,Vol.43,No.18 8651-8663). The used instrument is a UV-VIS ultraviolet visible spectrophotometer, model: TCC-240A, SHIMADZU, japan.

Fig. 6 shows degradation curves, four curves corresponding to dye 5, dye 6, dye 9, dye QCy-DT in order from top to bottom. The graph shows that the degradation degree of the dye QCY-DT is the most serious, and the degradation conditions of the dye 5, the dye 6 and the dye 9 are slight, which indicates that the introduction of the electron withdrawing group at the compound R3 can improve the stability of the compound to a certain extent, and is beneficial to the commercial application of the compound such as blood cell staining.

Example 11 evaluation of dye cell penetration

The dye is used for commercialized cell dye agents, and has better cell penetration, and the existing dye has weaker penetration capability on living cells due to smaller molecular structure log P value (logarithmic value of distribution coefficient ratio of a compound in n-octanol and water) and is limited in practical commercialized application. In order to improve the situation, the invention designs from the angle of molecular structure, introduces various chemical groups, and improves the penetration of dye molecules to cells while improving the stability of the dye.

To verify cell penetration, different dye compounds were prepared as 1mM PBS buffer, 10. Mu.L, respectively, and added to 12-well plates of cultured HepG2 cells at 37℃and 5% CO ₂ Is incubated in a cell incubator. Then, at different time points, PBS was used for washing 3 times with shaking, then cell culture medium was added, and fluorescence of each channel was measured by a multifunctional enzyme-labeled instrument (Thermo, USA).

Fig. 7 shows the test results, four curves corresponding to dye 6, dye 9, dye 5, dye QCy-DT in order from top to bottom. As shown in the figure, the dye 5, 6, 9 has better penetration to cells than the dye QCY-DT.

EXAMPLE 12 use of the cyanine dye of the invention for identifying microorganisms of a blood sample to be tested by flow cytometry

Dye reagent A1 and hemolytic agent B1 were first prepared according to the following formulation.

Wherein the first dye includes a compound having the above structural formula 1 and is used for staining microorganisms, and the second dye includes a compound having the following chemical formula and is used for staining cells:

then, 20 microliter of dye reagent A1, 1 milliliter of hemolytic agent B1 and 20 microliter of anticoagulated blood sample to be tested containing pseudomonas klebsiella are mixed, and the mixture is incubated for 30 seconds at 42 ℃ to form a sample liquid to be tested for measurement; next, a flow analyzer having a blue laser with an excitation wavelength of about 450nm is used to test the sample liquid to be tested to obtain forward scattered light intensity FSC, side scattered light intensity SSC, first fluorescence intensity FL1 and second fluorescence intensity FL2; generating a first scatter plot as shown in fig. 8A from the forward scattered light intensity information and the first fluorescence intensity, and generating a second scatter plot as shown in fig. 8B from the side scattered light intensity information and the second fluorescence intensity; the presence of microorganisms in the blood sample to be tested can be identified based on the first scattergram shown in fig. 8A, and the white blood cells in the blood sample to be tested can be four-classified based on the second scattergram shown in fig. 8B, resulting in the white blood cell classification results shown in table 1.

The same blood sample to be tested was tested on an existing blood analyzer (michaelk, model BC-6800) using a DIFF channel (using the michaelk BC-6800 DIFF kit) to obtain the white blood cell classification results shown in table 1.

TABLE 1 results of leukocyte classification

Parameters (parameters)	BC-6800	The invention is that
			Percent lymphocyte (%)	31.5	31.3
Percentage of monocytes (%)	4.3	4.4
			Percent neutrophil (%)	58.1	58.2
Percentage of eosinophils (%)	3.3	3.2

As can be seen from Table 1, the results of the classification of white blood cells obtained according to the present invention are substantially identical to those obtained according to the prior BC-6800. Therefore, the embodiment of the invention can simultaneously realize the microorganism detection and the leucocyte detection in blood by detecting the same blood sample to be detected at one time.

EXAMPLE 13 use of the cyanine dye of the invention for identifying microorganisms of a sample of simulated body fluid to be tested by flow cytometry

Adding a certain amount of pseudomonas klebsiella into physiological saline so as to obtain a simulated body fluid sample to be tested; testing the simulated body fluid sample to be tested by using the reagent and the method of the embodiment 12 to obtain forward scattered light intensity FSC and first fluorescence intensity FL1; generating a first scatter plot as shown in FIG. 9 from the forward scattered light intensity information and the first fluorescence intensity; the presence of microorganisms in the simulated body fluid sample can be identified based on the first scattergram shown in fig. 9.

EXAMPLE 14 use of the cyanine dye of the invention for identifying microorganisms of a blood sample to be tested by flow cytometry

Dye reagent A2 and hemolytic agent B2 were first prepared according to the following formulation.

Wherein the first dye comprises a compound having the above structural formula 2 and is used for staining microorganisms, and the second dye comprises a compound having the following chemical formula and is used for staining cells:

then, 20 microliter of dye reagent A2 and 1 milliliter of hemolytic agent B2 are mixed with 20 microliter of anticoagulated blood sample to be tested containing pseudomonas klebsiella, and the mixture is incubated for 30 seconds at 42 ℃ to form a sample liquid to be tested for measurement; next, a flow analyzer having a blue laser with an excitation wavelength of about 450nm is used to test the sample liquid to be tested to obtain forward scattered light intensity FSC, side scattered light intensity SSC, first fluorescence intensity FL1 and second fluorescence intensity FL2; generating a first scatter plot as shown in fig. 10A from the forward scattered light intensity information and the first fluorescence intensity, and generating a second scatter plot as shown in fig. 10B from the side scattered light intensity information and the second fluorescence intensity; the presence of microorganisms in the blood sample to be tested can be identified based on the first scattergram shown in fig. 10A, and the white blood cells in the blood sample to be tested can be four-classified based on the second scattergram shown in fig. 10B, resulting in the white blood cell classification results shown in table 2.

The same blood sample to be tested was tested on an existing blood analyzer (michaelk model BC-6800) using a DIFF channel (using michaelk BC-6800 DIFF kit) to obtain the white blood cell classification results shown in table 2.

TABLE 2 results of leukocyte classification

Parameters (parameters)	BC-6800	The invention is that
			Percent lymphocyte (%)	42.6	42.3
Percentage of monocytes (%)	3.5	3.3
			Percent neutrophil (%)	51.8	51.6
Percentage of eosinophils (%)	2.1	2.3

As can be seen from Table 2, the results of the classification of white blood cells obtained according to the present invention are substantially identical to those obtained according to the prior BC-6800. Therefore, the embodiment of the invention can simultaneously realize the microorganism detection and the leucocyte detection in blood by detecting the same blood sample to be detected at one time.

EXAMPLE 15 use of the cyanine dyes of the present invention for identifying microorganisms of a simulated body fluid sample to be tested using flow cytometry

Adding a certain amount of pseudomonas klebsiella into physiological saline so as to obtain a simulated body fluid sample to be tested; testing the simulated body fluid sample to be tested by using the reagent and the method in the embodiment 14 to obtain forward scattered light intensity FSC and first fluorescence intensity FL1; generating a first scatter plot as shown in FIG. 11 from the forward scattered light intensity information and the first fluorescence intensity; the presence of microorganisms in the simulated body fluid sample can be identified based on the first scattergram shown in fig. 11.

EXAMPLE 16 use of the cyanine dye of the invention for identifying microorganisms of a blood sample to be tested by flow cytometry

Dye reagent A3 and hemolytic agent B3 were first prepared according to the following formulation.

Wherein the first dye comprises a compound having the above structural formula 3 and is used for staining microorganisms, and the second dye comprises a compound having the following chemical formula and is used for staining cells:

then, 20 microliter of dye reagent A3 and 1 milliliter of hemolytic agent B3 are mixed with 20 microliter of anticoagulated blood sample to be tested containing escherichia coli, and the mixture is incubated for 30 seconds at 42 ℃ to form a sample liquid to be tested for measurement; next, a flow analyzer having a blue laser with an excitation wavelength of about 450nm is used to test the sample liquid to be tested to obtain forward scattered light intensity FSC, side scattered light intensity SSC, first fluorescence intensity FL1 and second fluorescence intensity FL2; generating a first scatter plot as shown in fig. 12A from the forward scattered light intensity information and the first fluorescence intensity, and generating a second scatter plot as shown in fig. 12B from the side scattered light intensity information and the second fluorescence intensity; the presence of microorganisms in the blood sample to be tested can be recognized based on the first scattergram shown in fig. 12A, and the white blood cells in the blood sample to be tested can be four-classified based on the second scattergram shown in fig. 12B, resulting in the white blood cell classification result shown in table 3.

The same blood sample to be tested was tested on an existing blood analyzer (michaelk model BC-6800) using a DIFF channel (using michaelk BC-6800 DIFF kit) to obtain the white blood cell classification results shown in table 3.

TABLE 3 results of leukocyte classification

Parameters (parameters)	BC-6800	The invention is that
			Percent lymphocyte (%)	33.5	33.8
Percentage of monocytes (%)	12.9	12.8
			Percent neutrophil (%)	52.5	52.2
Percentage of eosinophils (%)	0.8	0.8

As can be seen from Table 3, the results of the classification of white blood cells obtained according to the present invention are substantially identical to those obtained according to the prior BC-6800. Therefore, the embodiment of the invention can simultaneously realize the microorganism detection and the leucocyte detection in blood by detecting the same blood sample to be detected at one time.

EXAMPLE 17 use of the cyanine dye of the invention for identifying microorganisms of a simulated body fluid sample to be tested by flow cytometry

Adding a certain amount of escherichia coli into physiological saline so as to obtain a simulated body fluid sample to be tested; testing the simulated body fluid sample to be tested by using the reagent and the method of the embodiment 16 to obtain forward scattered light intensity FSC and first fluorescence intensity FL1; generating a first scatter plot as shown in FIG. 13 from the forward scattered light intensity information and the first fluorescence intensity; the presence of microorganisms in the simulated body fluid sample can be identified based on the first scattergram shown in fig. 13.

EXAMPLE 18 use of the cyanine dye of the invention for identifying microorganisms of a blood sample to be tested by flow cytometry

Dye reagent A4 and hemolytic agent B4 were first prepared according to the following formulation.

Wherein the first dye includes a compound having the above structural formula 1 and is used for staining microorganisms, and the second dye includes a compound having the following chemical formula and is used for staining cells:

then, 20 microliter of dye reagent A4 and 1 milliliter of hemolytic agent B4 are mixed with 20 microliter of anticoagulated blood sample to be tested containing pseudomonas klebsiella, and the mixture is incubated for 30 seconds at 42 ℃ to form a sample liquid to be tested for measurement; next, a flow analyzer having a blue laser with an excitation wavelength of about 450nm is used to test the sample liquid to be tested to obtain forward scattered light intensity FSC, first fluorescence intensity FL1, and second fluorescence intensity FL2; generating a first scatter plot as shown in fig. 14A from the forward scattered light intensity information and the first fluorescence intensity, and generating a second scatter plot as shown in fig. 14B from the forward scattered light intensity information and the second fluorescence intensity; the presence of microorganisms in the blood sample to be tested can be identified based on the first scattergram shown in fig. 14A, and white blood cells, nucleated red blood cells, and basophils in the blood sample to be tested can be identified and counted based on the second scattergram shown in fig. 14B.

Therefore, the embodiment of the invention can simultaneously realize the microorganism detection and the nucleated red blood cell detection in blood by detecting the same blood sample to be detected once.

EXAMPLE 19 use of the cyanine dye of the invention for identifying microorganisms of a sample of simulated body fluid to be tested by flow cytometry

Adding a certain amount of pseudomonas klebsiella into normal saline so as to obtain a simulated body fluid sample to be tested; testing the simulated body fluid sample to be tested by using the reagent and the method in the embodiment 18 to obtain forward scattered light intensity FSC and first fluorescence intensity FL1; generating a first scatter plot as shown in FIG. 15 from the forward scattered light intensity information and the first fluorescence intensity; the presence of microorganisms in the simulated body fluid sample can be identified based on the first scattergram shown in fig. 15.

EXAMPLE 20 use of the cyanine dye of the invention for identifying microorganisms of a blood sample to be tested by flow cytometry

Dye reagent A5 and hemolytic agent B5 were first prepared according to the following formulation.

Wherein the first dye comprises a compound having the above structural formula 3 and is used for staining microorganisms, and the second dye comprises a compound having the following chemical formula and is used for staining cells:

Then, 20 microliter of dye reagent A5 and 1 milliliter of hemolytic agent B5 are mixed with 20 microliter of anticoagulated blood sample to be tested containing pseudomonas klebsiella, and the mixture is incubated for 30 seconds at 42 ℃ to form a sample liquid to be tested for measurement; next, a flow analyzer having a blue laser with an excitation wavelength of about 450nm is used to test the sample liquid to be tested to obtain forward scattered light intensity FSC, first fluorescence intensity FL1, and second fluorescence intensity FL2; generating a first scatter plot as shown in fig. 16A from the forward scattered light intensity information and the first fluorescence intensity, and generating a second scatter plot as shown in fig. 16B from the forward scattered light intensity information and the second fluorescence intensity; the presence of microorganisms in the blood sample to be tested can be identified based on the first scattergram shown in fig. 16A, and white blood cells, nucleated red blood cells, and basophils in the blood sample to be tested can be identified and counted based on the second scattergram shown in fig. 16B.

Therefore, the embodiment of the invention can simultaneously realize the microorganism detection and the nucleated red blood cell detection in blood by detecting the same blood sample to be detected once.

EXAMPLE 21 use of the cyanine dye of the invention for identifying microorganisms of a simulated body fluid sample to be tested by flow cytometry

Adding a certain amount of pseudomonas klebsiella into physiological saline so as to obtain a simulated body fluid sample to be tested; testing the simulated body fluid sample to be tested by using the reagent and the method in the embodiment 20 to obtain forward scattered light intensity FSC and first fluorescence intensity FL1; generating a first scatter plot as shown in FIG. 17 from the forward scattered light intensity information and the first fluorescence intensity; the presence of microorganisms in the simulated body fluid sample can be identified based on the first scattergram shown in fig. 17.

EXAMPLE 22 use of the cyanine dyes of the invention for identifying parasites in blood samples to be tested by flow cytometry

Mixing 20 microliters of dye reagent A4 (example 18), 1 milliliter of hemolysis agent B4 (example 18) with 20 microliters of anticoagulated blood sample to be tested of malaria-infected patients, and incubating the mixture at 42 ℃ for 30 seconds to form a test sample liquid for measurement; next, a flow analyzer having a blue laser with an excitation wavelength of about 450nm is used to test the sample liquid to be tested to obtain forward scattered light intensity FSC, first fluorescence intensity FL1, and second fluorescence intensity FL2; generating a first scatter plot as shown in fig. 18A from the forward scattered light intensity information and the first fluorescence intensity, and generating a second scatter plot as shown in fig. 18B from the forward scattered light intensity information and the second fluorescence intensity; the presence of malaria parasites in the blood sample to be tested can be identified based on the first scattergram shown in fig. 18A, and white blood cells, nucleated red blood cells, and basophils in the blood sample to be tested can be identified and counted based on the second scattergram shown in fig. 18B.

Therefore, the embodiment of the invention can simultaneously realize the parasite detection and the nucleated red blood cell detection in blood by one-time detection of the same blood sample to be detected.

EXAMPLE 23 use of the cyanine dyes of the invention for identifying parasites in blood samples to be tested by flow cytometry

Dye reagent A6 and hemolytic agent B6 were first prepared according to the following formulation.

Wherein the first dye comprises a compound having the above formula 2 and is used to stain parasites and the second dye comprises a compound having the following formula and is used to stain cells:

then, 20 microliter of dye reagent A6 and 1 milliliter of hemolytic agent B6 are mixed with 20 microliter of anticoagulated blood sample to be tested of a patient infected with malaria, and the mixture is incubated for 30 seconds at 42 ℃ to form a sample liquid to be tested for measurement; next, a flow analyzer having a blue laser with an excitation wavelength of about 450nm is used to test the sample liquid to be tested to obtain forward scattered light intensity FSC, first fluorescence intensity FL1, and second fluorescence intensity FL2; generating a first scatter plot as shown in fig. 19A from the forward scattered light intensity information and the first fluorescence intensity, and generating a second scatter plot as shown in fig. 19B from the forward scattered light intensity information and the second fluorescence intensity; the presence of malaria parasites in the blood sample to be tested can be identified based on the first scattergram shown in fig. 19A, and white blood cells, nucleated red blood cells, and basophils in the blood sample to be tested can be identified and counted based on the second scattergram shown in fig. 19B.

Therefore, the embodiment of the invention can simultaneously realize the parasite detection and the nucleated red blood cell detection in blood by one-time detection of the same blood sample to be detected.

EXAMPLE 24 use of the cyanine dyes of the invention for identifying parasites in blood samples to be tested by flow cytometry

Mixing 20 microliters of dye reagent A5 (example 20), 1 milliliter of hemolysis agent B5 (example 20) and 20 microliters of anticoagulated blood sample to be tested of malaria-infected patients, and incubating the mixture at 42 ℃ for 30 seconds to form a test sample liquid for measurement; next, a flow analyzer having a blue laser with an excitation wavelength of about 450nm is used to test the sample liquid to be tested to obtain forward scattered light intensity FSC, first fluorescence intensity FL1, and second fluorescence intensity FL2; generating a first scatter plot as shown in fig. 20A from the forward scattered light intensity information and the first fluorescence intensity, and generating a second scatter plot as shown in fig. 20B from the forward scattered light intensity information and the second fluorescence intensity; the presence of malaria parasites in the blood sample to be tested can be identified based on the first scattergram shown in fig. 20A, and white blood cells, nucleated red blood cells, and basophils in the blood sample to be tested can be identified and counted based on the second scattergram shown in fig. 20B.

Therefore, the embodiment of the invention can simultaneously realize the parasite detection and the nucleated red blood cell detection in blood by one-time detection of the same blood sample to be detected.

EXAMPLE 25 use of the cyanine dyes of the invention for identifying parasites in blood samples to be tested by flow cytometry

Mixing 20 microliters of dye reagent A2 (example 14), 1 milliliter of hemolysis agent B2 (example 14) with 20 microliters of anticoagulated blood sample to be tested of malaria-infected patients, and incubating the mixture at 42 ℃ for 30 seconds to form a test sample liquid for measurement; next, a flow analyzer having a blue laser with an excitation wavelength of about 450nm is used to test the sample liquid to be tested to obtain forward scattered light intensity FSC, side scattered light intensity SSC, first fluorescence intensity FL1 and second fluorescence intensity FL2; generating a first scatter plot as shown in fig. 21A from the forward scattered light intensity information and the first fluorescence intensity, and generating a second scatter plot as shown in fig. 21B from the side scattered light intensity information and the second fluorescence intensity; the presence of malaria parasites in the blood sample to be tested can be identified based on the first scattergram shown in fig. 21A, and the white blood cells in the blood sample to be tested can be four-classified based on the second scattergram shown in fig. 21B, resulting in the white blood cell classification results shown in table 4.

The same blood sample to be tested was tested on an existing blood analyzer (michaelk, model BC-6800) using a DIFF channel (using the michaelk BC-6800 DIFF kit) to obtain the white blood cell classification results shown in table 4.

TABLE 4 results of leukocyte classification

Parameters (parameters)	BC-6800	The invention is that
			Percent lymphocyte (%)	41.7	41.3
Percentage of monocytes (%)	4.2	4.3
			Percent neutrophil (%)	52.9	52.6
Percentage of eosinophils (%)	1.2	1.3

As can be seen from Table 4, the results of the classification of white blood cells obtained according to the present invention are substantially identical to those obtained according to the prior BC-6800. Therefore, the embodiment of the invention can simultaneously realize the parasite detection and the leucocyte detection in blood by one-time detection of the same blood sample to be detected.

EXAMPLE 26 use of the cyanine dyes of the present invention for identifying parasites in blood samples to be tested by flow cytometry

Dye reagent A7 and hemolytic agent B7 were first prepared according to the following formulation.

Wherein the first dye comprises a compound having the structural formula 3 in table 1 above and is used to stain parasites and the second dye comprises a compound having the formula:

then, 20 microliter of dye reagent A7 and 1 milliliter of hemolytic agent B7 are mixed with 20 microliter of anticoagulated blood sample to be tested of a patient infected with malaria, and the mixture is incubated for 30 seconds at 42 ℃ to form a sample liquid to be tested for measurement; next, a flow analyzer having a blue laser with an excitation wavelength of about 450nm is used to test the sample liquid to be tested to obtain forward scattered light intensity FSC, side scattered light intensity SSC, first fluorescence intensity FL1 and second fluorescence intensity FL2; generating a first scatter plot as shown in fig. 22A from the forward scattered light intensity information and the first fluorescence intensity, and generating a second scatter plot as shown in fig. 22B from the side scattered light intensity information and the second fluorescence intensity; the presence of malaria parasites in the blood sample to be tested can be identified based on the first scattergram shown in fig. 22A, and the white blood cells in the blood sample to be tested can be four-classified based on the second scattergram shown in fig. 22B, resulting in the white blood cell classification results shown in table 5.

The same blood sample to be tested was tested on an existing blood analyzer (michaelk model BC-6800) using a DIFF channel (using michaelk BC-6800 DIFF kit) to obtain the white blood cell classification results shown in table 5.

TABLE 5 results of leukocyte classification

Parameters (parameters)	BC-6800	The invention is that
			Percent lymphocyte (%)	30.6	30.3
Percentage of monocytes (%)	5.3	5.4
			Percent neutrophil (%)	59.1	59.2
Percentage of eosinophils (%)	2.3	2.2

As can be seen from Table 5, the results of the classification of white blood cells obtained according to the present invention are substantially identical to those obtained according to the prior BC-6800. Therefore, the embodiment of the invention can simultaneously realize the parasite detection and the leucocyte detection in blood by one-time detection of the same blood sample to be detected.

EXAMPLE 27 use of the cyanine dye of the invention for identifying parasites in blood samples to be tested by flow cytometry

Dye reagent A8 and hemolytic agent B8 were first prepared according to the following formulation.

Wherein the first dye comprises a compound having the above formula 4 and is used to stain parasites and the second dye comprises a compound having the following formula and is used to stain cells:

then, 20 microliters of dye reagent A8 and 1 milliliter of hemolytic agent B8 are mixed with 20 microliters of anticoagulated blood sample to be tested of a patient infected with malaria, and the mixture is incubated for 30 seconds at 42 ℃ to form a sample liquid to be tested for measurement; next, a flow analyzer having a blue laser with an excitation wavelength of about 450nm is used to test the sample liquid to be tested to obtain forward scattered light intensity FSC, side scattered light intensity SSC, first fluorescence intensity FL1 and second fluorescence intensity FL2; generating a first scatter plot as shown in fig. 23A from the forward scattered light intensity information and the first fluorescence intensity, and generating a second scatter plot as shown in fig. 23B from the side scattered light intensity information and the second fluorescence intensity; the presence of malaria parasites in the blood sample to be tested can be identified based on the first scattergram shown in fig. 23A, and the white blood cells in the blood sample to be tested can be four-classified based on the second scattergram shown in fig. 23B, resulting in the white blood cell classification results shown in table 6.

The same blood sample to be tested was tested on an existing blood analyzer (michaelk, model BC-6800) using a DIFF channel (using the michaelk BC-6800 DIFF kit) to obtain the white blood cell classification results shown in table 6.

TABLE 6 results of leukocyte classification

Parameters (parameters)	BC-6800	The invention is that
			Percent lymphocyte (%)	30.5	30.3
Percentage of monocytes (%)	10.0	10.2
			Percent neutrophil (%)	54.5	54.2
Percentage of eosinophils (%)	0.9	1.0

As can be seen from Table 6, the results of the classification of white blood cells obtained according to the present invention are substantially identical to those obtained according to the prior BC-6800. Therefore, the embodiment of the invention can simultaneously realize the parasite detection and the leucocyte detection in blood by one-time detection of the same blood sample to be detected.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims (18)

1. A compound with a structure shown as a general formula I or a hydrate, a solvate, a stereoisomer, a tautomer or a crystal form thereof,

Wherein,

R ₁ and R is ₂ Identical or different, and independently selected from C _1-18 Straight-chain or branched alkyl, C _1-18 A linear or branched alkylene group-M selected from the group consisting of sulfonic acid groups, phenyl groups, carboxyl groups, mercapto groups, and amino groups;

R ₃ selected from hydrogen, sulfonic acid group, halogen, cyano group, C _1-6 Alkyl, hydroxy, C _1-6 Alkoxy, halo C _1-6 An alkyl group;

y is absent or a counter anion;

and defines:

when R is ₃ When hydrogen, R ₁ And R is ₂ Not both methyl and R ₁ And R is ₂ Not both benzyl.

2. The compound of claim 1, or a hydrate, solvate, stereoisomer, tautomer, or crystal form thereof, wherein R ₁ And R is ₂ Identical or different, and independently selected from C ₁ - ₆ Straight chain alkyl, C _1-6 The linear alkylene group M is selected from sulfonic acid group, phenyl group, carboxyl group, mercapto group and amino group.

3. The compound of claim 1 or 2, or a hydrate, solvate, stereoisomer, tautomer, or crystal form thereof, wherein R ₁ And R is ₂ At least one of which is C _1-18 Linear or branched alkylene-sulfonic acid groups.

4. A method as claimed in any one of claims 1 to 3A compound or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, wherein R ₁ And R is ₂ Is different and independently selected from C ₁ - ₆ Straight chain alkyl, benzyl, C _1-6 Straight chain alkylene-carboxyl, C _1-6 Straight chain alkylene-sulfonic acid group, C _1-6 Straight chain alkylene-mercapto, C _1-6 Linear alkylene-amino groups.

5. A compound according to any one of claims 1 to 3, or a hydrate, solvate, stereoisomer, tautomer or crystalline form thereof, wherein R ₁ And R is ₂ Identical and selected from C _1-6 Straight chain alkyl, C _1-6 Straight chain alkylene-sulfonic acid group, C _1-6 Linear alkylene-carboxyl groups.

6. The compound of any one of claims 1-5, or a hydrate, solvate, stereoisomer, tautomer, or crystal thereof, wherein R ₃ Selected from hydrogen, sulfonic acid group, halogen, cyano group, C _1-6 An alkyl group.

7. The compound of any one of claims 1-6, or a hydrate, solvate, stereoisomer, tautomer, or crystal thereof, wherein when Y is a counter anion, Y is selected from the group consisting of a halide (e.g., F-, cl-, br-, I-, clO) ₄ -、PF ₆ -、CF ₃ SO ₃ -、BF ₄ -, acetate, methanesulfonate or p-toluenesulfonate.

8. The compound of any one of claims 1-7, or a hydrate, solvate, stereoisomer, tautomer, or crystal thereof, wherein when Y is absent, the compound is an inner salt.

9. The compound of claim 8, or a hydrate, solvate, stereoisomer, tautomer, or crystal form thereof, wherein R ₁ And R is ₂ At least one of which is selected from C _1-18 Straight-chain or branched alkylene-sulfonic acid groups、C _1-18 Straight-chain or branched alkylene-carboxyl groups.

10. The compound of any one of claims 1-9, a hydrate, solvate, stereoisomer, tautomer, or crystalline form thereof, having any one of the following structures:

11. the compound of any one of claims 1-9, or a hydrate, solvate, stereoisomer, tautomer, or crystalline form thereof, having any one of the following structures:

12. a conjugate comprising a compound according to any one of claims 1-11, or a hydrate, solvate, stereoisomer, tautomer, or crystalline form thereof.

13. A composition for staining a biological sample, wherein the composition comprises a compound of any one of claims 1-11 or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof or a conjugate of claim 12.

14. The composition of claim 13, wherein the biological sample is a nucleic acid, preferably a deoxyribonucleic acid.

15. Use of a compound according to any one of claims 1 to 11, or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, a conjugate according to claim 12 or a composition according to claim 13 for staining a biological sample.

16. Use according to claim 15, wherein the biological sample is a nucleic acid, preferably a deoxyribonucleic acid.

17. Use of a compound according to any one of claims 1 to 11, or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, a conjugate according to claim 12 or a composition according to claim 13 for identifying parasites in a blood sample to be tested using flow cytometry.

18. Use of a compound according to any one of claims 1 to 11, or a hydrate, solvate, stereoisomer, tautomer or crystal form thereof, a conjugate according to claim 12 or a composition according to claim 13 for identifying microorganisms in a blood sample or a body fluid sample to be tested using flow cytometry.