BG112900A - Preparation of three-cationic asymmetric mono- and trimethycine 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

FIELD OF THE INVENTION

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, is a general formula

BACKGROUND OF THE INVENTION

Fluorescent dyes used for biological labeling are generally divided into two main classes with respect to the ways of binding between the fluorescent dye and the labeled bioagent. The first class are fluorescent dyes which 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 commonly used dye is propidium iodide [1].

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

Asymmetric cyanine dyes are mainly used for physical bonding for fluorescent labeling purposes [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 ratio of dye concentration to nucleic acids. The first method for the preparation of asymmetric monomethicyanine dyes was developed by Brooker et al. [4], in which 2-methylthiobenzazole quaternary salts are reacted with quaternary salts of quinoline and pyridine having an active methyl group.

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

Also known are methods for producing monomethincyanine dyes which minimize the use of highly toxic products such as methyl mercaptan [6-13] involving the condensation of equimolar amounts of quaternary 2-methylbenzazoles, 2-sulfobenzazoles, or 2-iminobenzazoles and hyaline derivatives. and pyridine.

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 provides the preparation of a compound with two positive charges and one intercalating unit [14]. Due to the higher charge, these derivatives have a higher affinity for DNA compared to the original dyes. These monomeric cyanine dyes are also impermeable to cells.

Patent publications US3 & 5321130A [15], US5410030A [16] are known, which describe cyanine dyes-dimers containing quinoline, for example TOTO (thiazole orange dimer) and YOYO (oxazole orange dimer), which are essentially symmetric symmetric cyresines. dye is a larger number of carbon atoms, with four positive charges, having a high affinity for nucleic acids. It is known that the higher charge of the dyes provides a higher affinity for nucleic acids. This type of dye does not show fluorescence in solution, but only after binding is the nucleic acids and thus reduces the interfering influence of the background. The spectra of TOTO, YOYO and their derivatives lie in the visible light range (490-530 nm), where the biological samples have strong absorption and fluorescent emission, which significantly reduces the efficiency of the fluorescent sample.

U.S. Pat. No. 5,441,030A [16] describes cyanine dyes containing pyridine-POPO and BOBO dimers. They have been found to have lower affinity than quinoline-containing dimers, but their DNA binding rate is much higher than the former.

Cyanine dyes with a larger number of carbon atoms in the conjugate chain have been developed, whose absorption and emission spectra have been shifted to the near infrared region (670-1000 nm). Such dyes as TOTAV, TOTIN, TO-PRO-3, PO-PRO-2 and B0-PRO-2 are known from 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 fluorescent maxima, high molar absorbency, and fluorescent quantum yield, make them ideal markers for different types of biomolecules [18-20]. The disadvantages of these dyes are the slower balance and the tendency to aggregate the dyes.

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

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

SUMMARY OF THE INVENTION

Taking into account the currently known mono- and tri-methiancin dyes, it is necessary to propose fluorogenic cyanine dyes tri-cationic, asymmetric mono- and tri-methionine dyes, with three positive charges in their molecule. , which are characterized by high affinity for nucleic acids and fast kinetics of interaction.

The problem is solved with a novel cyanine dye characterized by the following formula, wherein X is O or S, η = 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.

with

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

It is suitable to use a compound wherein X is O. R 2 and R ₃ each represent hydrogen.

According to a preferred embodiment, the compound is as described above, wherein n = 0.

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

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

Cyanine dye characterized in that benzothiazole or benzoxazole derivatives of general formula

wherein R is SCH ₃ or CH = CH-X (COCH ₃ ) C 6 H 5 and X and Y have the meanings given above, reacting in the presence of a basic agent with pyridine or quinoline derivatives of general formula

wherein R 2, R ₃ and Y have the meanings given above to give a dye of general formula

wherein X, Y and R 2 and R ₃ and η have the meanings given above, after which the resulting dye is quaternized on one side by triethylenediamine (TED) with C 1 -C 4 alkyl of general formula

wherein R 1 and Y are as defined above to give a cyanine dye of general formula

where X is O or S, η = 0 or 1, R 1 is C 1 -C 4 alkyl, R 2 and R 3 together are 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.

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

DESCRIPTION OF THE DRAWINGS

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

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

Figure 2. - presents the 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.

Fig.Z. - presents Microscopic images of yeast cells in a sample taken by a local brewer's fermenter stained with RO-TEDM 1: A) total number of cells in the sample, B) dead cells in the original sample.

EXAMPLE EMBODIMENT OF THE INVENTION

The fluorogenic cyanine derivatives described herein allow the preparation of fluorescent asymmetric dyes using standard methods, i. the dyes can be prepared in a reaction vessel without isolating the intermediate dye. The derivatives according to the invention are prepared 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 with methyl triethylenediamine iodide providing a larger number of positive charges of the final dye. three charges).

The invention is illustrated by the following examples.

Example 1. Preparation of dye RO-TEDM 1

In a 250 ml flat-bottomed, two (3) neck flask was placed 2-methylmercapto-Zmethylbenzoxazolium methosulfate - 7.7 g; 1- (3-iodopropyl) -4-methylpyridinium iodide 5

10.2 g, and 57 ml of acetic anhydride with stirring with a magnetic stirrer at room temperature. 4.6 ml of N-ethyldiisopropylamine are added dropwise to the reaction mixture with constant stirring. After 30 minutes, the drip is repeated. A light green dye is obtained. The reaction mixture was stirred for a further 2 hours at room temperature and diluted with 50 ml of diethyl ether. The precipitate is filtered through a beaker. The precipitate is washed with 3x20 ml of diethyl ether and dried to constant weight under vacuum. T.t. of the crude product is 186188 ° C. Recrystallizes to CH3OH.

291.34

389.04

520.18

Place 2.6 g of PO-PrI and 3.9 g of methyl TED iodide in a three-necked flask and add 23 ml of methoxyethanol. The mixture was heated on a magnetic stirrer with gentle stirring at a temperature of ~ 120 ° C until completely dissolved. Heating and stirring are continued for 4 hours. When heating, carefully monitor whether sediment will fall.

After cooling to 70-80 ° C, the flask was left in the refrigerator compartment to completely crystallize the precipitate for ~ 1 hour. This is followed by filtration through a vacuum cleaner. At the end of the filtration, wash with 30 ml of CH3OH. Dry at t = 60 ° C under vacuum

Example 2.

Proceed analogously to Example 1. A dye BO-TEDM 1 of the following formula is obtained:

Example 3. Preparation of TO-TEDM dye 1

Place 2-methylmercapto-Zmethylbenzothiazole methosulfate in 250 ml of a flat-bottomed, two (3) neck flask - 8.89 g; quinoline -13.27 g and 66 ml of acetic anhydride with stirring with a magnetic stirrer at room temperature. 5.3 ml of N-ethyldiisopropylamine are added dropwise to the reaction mixture, stirring is continued. After 30 minutes, the drip is repeated. A yellow-orange dye is obtained immediately. The reaction mixture was stirred for a further 2 hours at room temperature and diluted with 50 ml of diethyl ether. The precipitate is filtered through a purifier. Wash with 3x20 ml diethyl ether on the burner. The sludge from

ΊΌ-PrI was dried to constant weight in a vacuum oven.

291.34 439.07 ⁵⁷⁰²¹

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

Example 4. Preparation of YO-TEDM dye 1

The procedure is analogous to Example 3. A YO-TEDM 1 dye is obtained with the following formula:

Example 5. Preparation of TO-TEDM dye 3

To 2- (2-anylidovinyl) -3-methylbenzothiazole iodide were 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 (1.5h) in an oil bath. The reaction mixture was poured into ethyl ether to precipitate small, dark purple dye particles, 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 50 ml two-necked flask (the volume of the flask depends on the amount of substance to be treated) place 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 a magnetic stirrer with stirring at about 400 rpm at a temperature of ~ 85 ° C until completely dissolved. Heating and stirring are continued for 60 minutes. The solution was then cooled and the flask was left in the refrigerator for 1 hour for complete crystallization of the resulting compound.

This is followed by filtration under vacuum. At the end of the filtration, wash with CH ₃ OH - 30 ml. The dye was dried at t = 60 ° C under vacuum.

Example 6. Preparation of dye YO-TEDM-H

Proceed analogously to Example 5. A YO-TEDM 3 dye was prepared with the following formula:

Example 7. Preparation of VO-TEDM dye 3

To 0.65 g of 2- (2-anylidovinyl) -3-methylbenzothiazole iodide were added 0.57 g of 1 (3-iodopropyl) -4-methylpyridinium iodide and 14 ml of pyridine, and the mixture was heated with stirring at 90 °. C (1.5h) in an oil bath. The reaction mixture was poured into ethyl ether to give a red precipitate of BO-PrI, which was filtered off and dried.

Place 0.9 g of BO-PrI and 1.2 g of methyl EDA in a 50 ml two-necked flask, add 8 ml of methoxyethanol. The mixture is heated on a magnetic stirrer with stirring at about 400 rpm at a temperature of ~ 85 ° C until completely dissolved. Heating and stirring are continued for 60 minutes. The solution was then cooled and the flask was left in the refrigerator for 1 hour for complete crystallization of the resulting compound. This is followed by filtration under vacuum. At the end of the filtration, wash with CH3OH - 30 ml. The dye was dried at t = 60 ° C under vacuum.

Example 8. Preparation of dye RO-TEDM 3

The procedure is analogous to Example 7. A RO-TEDM 3 dye of the following formula is obtained:

Example 9. Optical properties of the obtained asymmetric methiancin dyes Further in the description we present in tabular form the optical properties of the asymmetric methiancin dyes described in examples 1-8.

Table 1. Optical properties of the obtained asymmetric methcyanine dyes

number Dye DMF Buffer pH 7.4 + DNA Absorption maximum Emission maximum Raise 1. PO-TEDM 1 434 456 X52 2. BO-TEDM 1 462 481 X45 3. TO-TEDM 1 514 533 X78 4. YO-TEDM 1 491 509 X80 5. TO-TEDM 3 642 660 X53 6. YO-TEDM 3 612 631 X48 7. PO-TEDM 3 534 572 X45 8. BO-TEDM 3 570 604 HZO

Table 1 shows the absorption and emission maxima of the dyes obtained. For mono-methizianin dyes the emission maxima lie in the range from 456 to 533 nm, and for three-methizian dyes - in the range from 572 nm to 660 nm. In some of the dyes a very large increase in the fluorescence was obtained when the dye bound to DNA compared to the fluorescence without DNA - 78 times in TOTEDM 1, 80 times in YO-TEDM 1, etc.

Example 10. DNA titration of PO-TEDM dye 1

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

Figure 1 shows a comparative analysis of the interaction of the fluorescent dye PO-TEDM 1, object of the invention, with DNA and of the interaction of a known fluorescent dye Propidium iodide with DNA. The graphical presentation of the results shows that the fluorescence intensity of the bound new, three-charge dye with different DNA concentrations is significantly stronger (about 50 times) compared to the intensity of the known dye "Propidium iodide" with the same DNA concentrations. These results are also valid for the other newly synthesized three-charge fluorescent dyes.

The kinetics of the interaction of the resulting fluorescent dye PO-TEDM 1 with DNA was further monitored and analyzed. The studies were performed at the optimal concentration of the components, namely - the concentration of PO-TEDM 1 is 50 pg / ml and DNA is 50 pg / ml, mixed in a ratio of 1: 12.5. At the same time, studies with the known dye were performed at a concentration of PI 50pg / ml and DNA 500pg / ml mixed in a ratio of 1: 1. Comparative analysis of the results shows that the newly synthesized fluorescent dyes have an unexpectedly very fast kinetics of dye binding to DNA, with equilibrium being achieved in just a short period of 3 sec. In the known fluorescent dye Propidium iodide, the time to bind to DNA is 30 sec.

Example 11. Quantitative fluorescence determination of dead cells using PO-TEDM 1

Another important application of monomethine and trimethine fluorescent dyes is for staining dead cells. Figure 3 shows two microscopic images of yeast cells in a sample taken from a local brewer's fermenter stained with PO-TEDM 1. The concentration of PO-TEDM 1 is 5 pg / ml. Microscopic image A shows the total number of yeast cells in a sample taken by a fermenter at a local brewery, and microscopic image B shows the dead cells in the same sample. The total cell count was pre-treated for 15 minutes at 80 ° C to kill all cells. It was found that the total number of yeast cells in the sample taken by a fermenter at a local brewery was (9x10 ⁶ ) and the number of dead cells in the original sample was (4x10 ⁵ ).

Claims (12)

PATENT CLAIMS Novel cyanine dyes characterized by the general formula, wherein X is O or S, η = 0 or 1, R 1 is C 1 -C 4 alkyl, R 2 and R 3 together are the carbon atoms to which they are attached, form a fused benzene nucleus or individually denoting hydrogen, and Y is a biocompatible anion such as, halogen, tosylate, hexafluorophosphate, methosulfate, acetate, perchlorate, nitrate, tetrafluoroborate, trifluoroacetate, etc. A compound according to claim 1, wherein X is S, and R _{3 is} independently hydrogen. A compound according to claim 1, wherein X is O. R 2 and R 3 are each hydrogen A compound according to claim 1, wherein n = 0. A compound according to claim 1, wherein n = 1. A compound according to claim 1, wherein R 1 is C 1 -C 4 alkyl Cyanine dyes according to claim 1, characterized in that they consist of benzothiazole or benzoxazole derivatives of general formula wherein R is SCH ₃ or CH = CH-N (COCH ₃ ) C ₆ H ₅ ; and X is S and Y has the meanings given above, said benzothiazole or benzoxazole derivatives reacting with pyridine derivatives of general formula H ₃ C — C © N. / - \ ^Y ® r / R3 where R2 and R3 are each hydrogen and Y has the meanings given above to give a dye by reaction is a general formula wherein X, Y and R 2 and R ₃ and η have the meanings given above, wherein the taught dye is quaternized on one side by TED with C 1 -C 4 alkyl of general formula wherein R 1 and Υ have the meanings given above, whereby cyanine dyes according to claim 1 are obtained. A compound comprising a dye polymer attached to a dye according to claim 1. The compound of claim 4, wherein the nucleic acid polymer is DNA. The compound of claim 4, wherein the nucleic acid polymer is RNA. A compound according to claim 4, wherein enhanced fluorescence is obtained with impaired cell membrane integrity. 12. A method of forming a fluorogenic cyanine dye, comprising: (a) mixing benzothiazole or benzoxazole derivatives of general formula wherein R is SCH ₃ or CH = CH-N (COCH ₃ ) C ₆ H ₅ ; a X e S and Y have the meanings given above, with pyridine derivatives of general formula wherein R 2 and R 3 are each hydrogen and a Υ has the meanings given above, characterized in that the resulting dye is quaternized on one side by TED with C 1 -C 4 alkyl of general formula wherein R 1 and Υ have the meanings given above, whereby cyanine dyes according to claim 1 are obtained.