BG67403B1 - Preparation of three-cationic asymmetric mono- and- tri-methycine cyanide dyes - Google Patents

Abstract

The invention relates to novel fluorogenic cyanine derivatives which can dye dead or dying cells or detect DNA in solution or agarose gel by excitation fluorescence at a certain wavelength. The present invention provides the preparation of novel asymmetric mono- and trimethycine cyanide dyes with three positive charges having a high affinity for nucleic acids and rapid binding kinetics. The derivatives, according to the invention, are prepared by reaction of benzothiazole or benzoxazole derivatives with pyridine or quinoline derivatives in the presence of a basic agent and then the resulting compound is quaternized on one side with methyl triethylenediamine iodide.

Description

(54) PREPARATION OF TRICATION UNSYMMETRIC MONO- AND TRIMETHENCYANINE DYES

Field of technique

The invention relates to fluorogenic cyanine derivatives which can stain dead or dying cells or detect DNA in solution or agarose gel by fluorescence upon excitation at a specific wavelength.

Prior art

Fluorescent dyes applied for biological labeling are generally divided into two main classes with respect to the coupling modes between the fluorescent dye and the labeled bioagent. The first class are fluorescent dyes that bind to the bioagent through suitable active groups, such as rhodamine dyes, fluoresceins, naphthalimides, stilbenes, acridine, and fluorenes. The other class of fluorescent dyes have an affinity for DNA and RNA and are incorporated into their structures. These are cyanine dyes, such as thiazole orange (TO), oxazole yellow (YO) and their dimers (TOTO and YOYO) and phenanthridine dyes, of which the most used dye is propidium iodide [1].

The second class of dyes have many advantages over the first class of dyes, such as no fluorescence before the dye enters the nucleic acid structure and no background interference. When the dye molecule is inserted into the DNA molecule, especially the DNA double helix, the fluorescence intensity can increase more than 1000 times, and in RNA more than 3000 times [1, 2]. These dyes have moderate to very high affinity for nucleic acids depending on their structure and their charge, with little or no staining of other biopolymers.

In physical binding for fluorescence labeling purposes, the asymmetric cyanine dyes are mainly used [3]. They bind to nucleic acids by electrostatic bonding and base pair intercalation and depend on the structure and charge of the dye and the concentration ratio of the dyes to the nucleic acids. The first method for the preparation of unsymmetrical monomethinecyanine dyes was developed by Brooker and co-workers [4], in which 2-methylthiobenzazolium quaternary salts reacted with quinoline and pyridine quaternary salts possessing an active methyl group.

Usually, 1-alkyl-4methylquinoline salt and 2-methylthio-3-methylbenzothiazolium salt or 2-methylthio-3methylbenzoxazolium salt [5] are used for the synthesis of the monomethinecyanine dyes TO and YO, respectively, in the presence of the methylating agent iodomethane. This reaction produces as a by-product CH3SH, which is highly poisonous. Other monocyanine dyes PO and BO have been synthesized using benzoxazole and benzothiazole salts with pyridine derivatives, respectively, in the presence of a basic agent [5], but they are less commonly used. The indicated four dyes are single-charged, intercalate in the DNA molecule, have a lower affinity for DNA, and are not permeable to living cells.

There are also known methods for obtaining monomethinecyanine dyes, in which the use of highly toxic products is minimized, such as methylmercaptan [6-13] involving the condensation of equimolar amounts of quaternary 2-methylbenzazoles, 2-sulfobenzazoles, or 2iminobenzazoles and quinoline and pyridine derivatives. .

The derivatives TO-PRO, YO-PRO, PO-PRO and BO-PRO were synthesized, which contain the corresponding cyanine compound and a cationic side chain, which ensures the preparation of a compound with two positive charges and one intercalating unit [14]. Due to the greater charge, these derivatives have a greater affinity for DNA compared to the parent dyes. These monomeric cyanine dyes are also impermeable to cells.

Patent publications US 005321130 A [15], US 5410030 A [16] are known, in which quinoline-containing cyanine dye-dimers, for example TOTO (thiazole orange dimer) and YOYO (oxazole orange dimer), are essentially symmetrical cyanine dyes. fluorescent dyes with a higher number of carbon atoms, with four positive charges, having a high affinity for nucleic acids. It is known that higher charge of dyes provides higher affinity for nucleic acids. This type of dyes do not show fluorescence in solution, but only after binding to nucleic acids, thus reducing background interference. The spectra of TOTO, YOYO and their derivatives lie in the visible light region (490-530 nm), where biological samples have strong absorption and fluorescence emission, thereby significantly reducing the efficiency of the fluorescent sample.

In patent US 5410030 A [16] cyanine dye dimers containing pyridine - POPO and BOVO are described. They were found to have a lower affinity than the quinoline-containing dimers, but their rate of binding to DNA was much greater than the former.

Cyanine dyes with a larger number of carbon atoms in the conjugate chain have been developed, whose absorption and emission spectra are shifted to the near-infrared region (670-1000 nm). Such dyes as TOTAV, TOTIN, TO-PRO-3, PO-PRO-2 and BO-PRO-2 are known by K. M. SovenyhaZy, J. A. Bordelon, J. T. Petty. Nucleic Acids Res, 2003, 31, 2561 [17]. Typically, such structures show a strong tendency to form aggregates within polynucleotide binding. Their favorable spectral properties, such as long-wavelength absorption, position of their respective fluorescence maxima, high molar absorbance, and fluorescence quantum yield make them ideal markers for various types of biomolecules [18-20]. The disadvantages of these dyes are the slower reaching of equilibrium and the tendency of the dyes to aggregate.

The described dyes with a larger number of carbon atoms have a larger molecular mass, easily aggregate, a complex structure and many stages for synthesis, which in advance limits the possibilities for their commercialization and wider application.

In recent years, there has been a growing interest in the synthesis and application of unsymmetrical mono- and trimethincyanine dyes with a larger number of positive charges. The reason for this is that they are characterized by high sensitivity, high speed of binding to DNA, as well as intense fluorescence recorded in the presence of various biomolecules. The developed new unsymmetrical mono- and tri-methinecyanine dyes have different structure, higher charge, high sensitivity to nucleic acids and fast interaction kinetics.

Technical essence of the invention

Taking into account the currently known mono- and tri-methinecyanine dyes, there is a need to propose fluorogenic cyanine dyes - tri-cationic, asymmetric mono- and tri-methinecyanine dyes, with three positive charges (charge) in their molecule, which are characterized by high affinity to nucleic acids and fast interaction kinetics.

The problem to be solved is a cyanine dye characterized by the following formula,

where X is O or S, η = 0 or 1, Ri is C1-C4 alkyl, R ₂ and R ₃ together with the carbon atoms to which they are attached form a fused benzene ring or individually hydrogen, and Y is a biocompatible anion, such as halogen, tosylate, hexafluorophosphate, methosulfate, acetate, perchlorate, nitrate, tetrafluoroborate, trifluoroacetate, etc.

According to the invention, the cyanine dye is a compound where X is S, R ₂ and R ₃ are each hydrogen.

It is convenient to use a compound where X is O. R ₂ and R ₃ are each hydrogen.

According to a preferred embodiment, the compound is as described above, where η = 0.

A compound according to claim 1, wherein η = 1.

A compound according to claim 1, wherein R 1 is C 1 -C 4 alkyl.

Cyanine dye, characterized in that benzthiazole or benzoxazole derivatives of the general formula

where R is SCH ₃ or CH = CH-X(COCH ₃ )CbH 5 a X and Y have the meanings indicated above, react in the presence of a basic agent with pyridine or quinoline derivatives of the general formula

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wherein R ₂ R ₃ and Y are as defined above, whereby a dye of general formula is obtained

where X, Y and R ₂ and R ₃ and n have the above meanings, then the resulting dye is quaternized on one side by triethylenediamine (TED) with a C1-C4 alkyl of the general formula

R 1' / Y where R 1 and Y are as defined above, giving a cyanine dye of the general formula

BG 67403 Bl where X is O or S, η = 0 or 1, Ri is C1-C4 alkyl, R ₂ and R ₃ together with the carbon atoms to which they are attached form a fused benzene ring or individually are hydrogen, and Y is biocompatible anion such as halogen, tosylate, hexafluorophosphate, methosulfate, acetate, perchlorate, nitrate, tetrafluoroborate, trifluoroacetate, etc.

The proposed new fluorogenic cyanine derivatives allow the preparation of tricationic asymmetric mono- and tri-methinecyanine dyes, distinguished by three positive charges in their molecule, which allows these dyes to be applied to DNA analysis (detection of DNA in solution, in gel electrophoresis and you.). Another important application of monomethine and trimethine fluorescent dyes is for staining dead cells.

Explanation of the attached figures

In order to clarify the features of the invention in more detail, the description is accompanied by drawings, where:

Figure 1 - presents DNA titration of PO-TEDM 1 (A) and Propidium iodide (♦).

Figure 2 - presents kinetics of interaction of the fluorescent dyes PO-TEDM-1 (A) and Propidium iodide (♦) with DNA, carried out at the optimal concentration of the components.

Figure 3 - presents microscopic pictures of yeast cells in a sample taken from a fermenter of a local brewery stained with PO-TEDM 1: A) total number of cells in the sample, B) dead cells in the starting sample.

Example of implementation of the invention

The fluorogenic cyanine derivatives described so far allow the preparation of fluorescent asymmetric dyes using standard methods, i.e. the dyes can be prepared in one reaction vessel, without isolation of the intermediate dye. The derivatives according to the invention are obtained by reacting benzothiazole or benzoxazole derivatives with pyridine or quinoline derivatives, in the presence of a basic agent, and then the resulting compound is quaternized on one side by methyl triethylenediamine iodide, providing a greater number of positive charges on the final dye ( three charges).

The invention is illustrated by the following examples.

Example 1. Preparation of dye RO-TEDM 1

2-Methylmercapto-3-methylbenzoxazolium methosulfate - 7.7 g is placed in a 250 ml flat-bottomed, two (3) necked flask; 1-(3-Iodopropyl)-4-methylpyridinium iodide - 10.2 g and 57 ml of acetic anhydride under magnetic stirring at room temperature.

4.6 ml of N-ethyldiisopropylamine was added dropwise to the reaction mixture with continuous stirring. After 30 min, the instillation is repeated. A light green dye is obtained. The reaction mixture was stirred for another 2 h at room temperature and diluted with 50 ml of diethyl ether. The precipitate is filtered through a Buchner. The precipitate was washed with 3 x 20 ml of diethyl ether and dried to constant weight under vacuum. The melting point of the crude product is 186 - 188°C. Recrystallized from CH3OH.

a Q[ι no

Ϊ CH ₃ SO 4

CHj 12

291.34 389.04

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-CH ₃ SH

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2.6 g of PO-PrI and 3.9 g of methyl TED iodide were placed in a three-necked flask and 23 ml of methoxyethanol was added. The mixture is heated on the magnetic stirrer with gentle stirring at a temperature of ~ 120°C until they are completely dissolved. Heating and stirring continued for 4 h. During heating, carefully observe whether a precipitate will fall. After cooling to 70-80°C, the flask is left in the refrigerator chamber to fully crystallize the precipitate ~ 1 hour. This is followed by filtration through a Buchner under vacuum. At the end of the filtration, wash with 30 ml of CH3OH. Dry at t = 60°C under vacuum.

Example 2.

It works analogously to example 1. VO-TEDM 1 dye is obtained with the following formula:

Example 3. Preparation of dye TO-TEDM 1

2-methylmercapto-3-methylbenzothiazolium methosulfate - 8.89 g; quinoline - 13.27 g and 66 ml of acetic anhydride while stirring with a magnetic stirrer at room temperature. 5.3 ml of N-ethyldiisopropylamine was added dropwise to the reaction mixture while stirring was continued. After 30 min, the instillation is repeated.

A yellow-orange dye is immediately obtained. The reaction mixture was stirred for another 2 h at room temperature and diluted with 50 ml of diethyl ether. The precipitate is filtered through a Buchner. Wash with 3 x 20 ml of diethyl ether on the Buchner. The TO-PrI precipitate was dried to constant weight in a vacuum drier.

In a 250 ml round-bottomed flask, dissolve 5 g of TO-PrI in 150 ml of diethyl ether. Mix by hand until dissolved. Place in an aluminum container with water and ice and cool to a temperature of 10-15°C. Place on a magnetic stirrer. 50 ml of diethyl ether and 4.22 ml of methyl TED iodide were added dropwise through a separatory funnel. A white precipitate immediately begins to fall. After adding the contents of the separatory funnel, stirring was continued for 30 min at room temperature. The precipitate is filtered under vacuum and immediately dried in a desiccator because it is hygroscopic.

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Example 4. Preparation of dye YO-TEDM 1

It works analogously to example 3. Dye YO-TEDM 1 with the following formula is obtained:

Example 5. Preparation of dye TO-TEDM 3

To 2-(2-anilidovinyl)-3-methylbenzothiazolium iodide was added 4 mmol (1.75 g) of 1-methyl-4-quinoline quaternary ammonium salt (obtained by milking 1-methyl quinoline) and 10 ml of pyridine and the mixture was heated with stirring at 90°C (1.5 h) in an oil bath. The reaction mixture was poured into ethyl ether to precipitate small, dark purple dye particles, which was then filtered and dried. The dye was purified by silica gel column using dichloromethane/methanol = 100:5 as eluent, the blue fraction was collected to give TO-Pri-3 (cyanine) in 70% yield.

In a two-necked flask of 50 ml (the volume of the flask depends on the amount of substance to be processed) we put 1 g of TO-Pri-3 (cyanine) and 1.35 g of methyl TED iodide, add 8 ml of methoxyethanol. The mixture is heated on the magnetic stirrer with stirring at about 400 rpm at a temperature of ~ 85°C until they are completely dissolved. Heating and stirring continue for 60 min. The solution was then cooled and the flask was left in the refrigerator chamber for 1 h for complete crystallization of the resulting compound.

This is followed by filtration under vacuum. At the end of the filtration, wash with CH2OH - 30 ml. The dye is dried at t = 60°C under vacuum.

Example 6. Preparation of dye YO-TEDM-Z

It works analogously to example 5. Dye YO-TEDM 3 with the following formula is obtained:

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Example 7. Preparation of VO-TEDM 3 dye

To 0.65 g of 2-(2-anilidovinyl)-3-methylbenzothiazolium iodide was added 0.57 g of 1-(3-iodopropyl)-4methylpyridinium iodide and 14 ml of pyridine and the mixture was heated with stirring at 90°C (1, 5 h) in an oil bath. The reaction mixture was poured into ethyl ether and a red precipitate of BO-PrI was obtained which was filtered and dried.

0.9 g of BO-PrI and 1.2 g of methyl EDA are placed in a 50 ml two-necked flask, 8 ml of methoxyethanol are added. The mixture is heated on the magnetic stirrer with stirring at about 400 rpm at a temperature of ~ 85°C until they are completely dissolved. Heating and stirring continue for 60 min. The solution was then cooled and the flask was left in the refrigerator chamber for 1 h for complete crystallization of the resulting compound.

This is followed by filtration under vacuum. At the end of the filtration, wash with CH2OH - 30 ml. The dye is dried at t = 60°C under vacuum.

Example 8. Preparation of dye RO-TEDM 3

It works analogously to example 7. RO-TEDM 3 dye is obtained with the following formula:

Example 9. Optical properties of the obtained asymmetric methcyanine dyes

Further in the description, we present in tabular form the optical properties of the asymmetric methcyanine dyes described in examples 1-8.

Table 1. Optical properties of the obtained asymmetric methcyanine dyes

Number Bshtrilo DMF Buffer pH 7.4 + DNA Absorption maximum Emission maximum Unelichscis 1. PO-TEDM 1 434 456 X52 2. BO-TEDM 1 462 481 X45 3. TOUTED M ] 514 533 X 78 4.. YO-TtDM 1 491 509 X80 5. TO-TEDM 3 642 660 X 53 6. ΥΟ-Ί EDM 3 612 631 X 48 7. PO-TEDM 3 534 572 X45 8. BO-TEDM 3 570 604 HZO

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Table 1 shows the absorption and emission maxima of the obtained dyes. For mono-methinecyanine dyes, the emission maxima lie in the range from 456 to 533 nm, and for trimethine cyanine dyes - in the range from 572 nm to 660 nm. For some of the dyes, a very large increase in fluorescence was obtained upon binding of the dye to DNA relative to fluorescence without DNA - 78-fold for TO-TEDM 1, 80-fold for YO-TEDM 1, and so on.

Example 10. DNA titration of PO-TEDM 1 dye

The synthesized fluorescent dyes, object of the invention are suitable and used for DNA analysis. Laboratory analyzes of DNA were performed using the fluorescent dye POTEDM 1. Experiments were performed at different concentrations of DNA: 0, 5, 10, 25, 40, 50 and 75 pg/ml. For comparison, experiments were made with the well-known and widely used fluorescent dye for dead cells Propidium iodide and the same concentrations of DNA. The concentrations of both dyes were 50 pg/ml and they were mixed with the DNA solutions in a ratio of 1:12.5.

Figure 1 presents a comparative analysis of the interaction of the fluorescent dye POTEDM 1, subject of the invention, with DNA and of the interaction of a known fluorescent dye Propidium iodide with DNA. From the graphical presentation of the results, it can be seen that the fluorescence intensity of the related new, tri-charged dye with different concentrations of DNA is significantly stronger (about 50 times) compared to the intensity of the known dye "Propidium iodide" with the same concentrations of DNA. These results are also valid for the other newly synthesized tri-charged fluorescent dyes.

Additionally, the kinetics of interaction of the obtained fluorescent dye PO-TEDM 1 with DNA was tracked and analyzed. The studies were carried out at the optimal concentration of the components, namely - the concentration of PO-TEDM 1 was 50 μg/ml and of DNA was 50 μg/ml, mixed in a ratio of 1: 12.5. At the same time, the studies with the known dye were carried out at a concentration of PI 50 μg/ml and DNA 500 μg/ml, mixed in a ratio of 1:1. The comparative analysis of the results shows that the newly synthesized fluorescent dyes have unexpectedly very fast kinetics of binding the dye to DNA, reaching equilibrium in only a short period of 3 s. With the well-known fluorescent dye propidium iodide, the time for its binding to DNA is 30 s.

Example 11. Quantitative fluorescence determination of dead cells using ROTEDM 1

Another important application of monomethine and trimethine fluorescent dyes is for staining dead cells. Figure 3 shows two microscopic photographs of yeast cells in a sample taken from a local brewery fermenter stained with PO-TEDM 1. The concentration of PO-TEDM 1 was 5 μg/ml. Photomicrograph A shows the total number of yeast cells in a sample taken from a local brewery fermenter, and photomicrograph B shows the dead cells in the same sample. The sample for determining the total number of cells was pre-treated for 15 min at 80°C in order to kill all cells. The total number of yeast cells in the sample taken from a local brewery fermenter was found to be (9 x 10 ⁶ ) and the number of dead cells in the source sample was (4 x 10 ⁵ ).

Claims (3)

Cyanine dye of general formula: where X is O or S, n = 0 or 1, R 1 is C 1 -C 4 alkyl, R 2 and R 3 together with the carbon atoms to which they are attached form a fused benzene nucleus or individually represent hydrogen, and Y is a biocompatible anion, such as halogen, tosylate, hexafluorophosphate, methosulfate, acetate, perchlorate, nitrate, tetrafluoroborate, trifluoroacetate, etc. Use of a cyanine dye according to claim 1 for analysis of DNA in solution, wherein the dye: DNA solution ratio is 1: 12.5 A process for preparing a fluorogenic cyanine dye according to claim 1 comprises mixing benzothiazole or benzoxazole derivatives of the general formula wherein R is SCH3 or CH = CH-N (COCH3) C6H5; and X is S and Y have the meanings given above, with pyridine derivatives of the general formula: wherein R2 and R3 are each hydrogen, and Y has the meanings given above, characterized in that the dye obtained is quaternized on one side by triethylenediamine with C1-C4 alkyl of general formula: wherein R1 and Y have the meanings given above, to give cyanine dyes according to claim 1