Preparation method of cyanine dye Cy3

Abstract

The invention provides a novel preparation method of a water-soluble cyanine dye Cy3, which comprises the following steps of: adding reaction raw materials including aromatic nitrogen heterocycle, sultone and triethyl (or methyl) promethyl (or propanoate) into a reaction bottle together, carrying out heating reaction, and purifying a product obtained by the heating reaction to finally obtain the cyanine dye Cy3 containing an alkane sulfonic acid group. The target cyanine dye Cy3 is synthesized in one step, the process is simple, the reaction can be carried out under the conditions of no solvent and no basic catalyst, and the preparation and production period is greatly prolonged.

Description

Technical Field

[0001] The present invention relates to the technical field of compound preparation, and particularly to a preparation method of cyanine dye Cy3. Background Art

[0002] As a member of the cyanine dye family, cyanine dye Cy3 has attracted more attention from researchers in recent years due to its specific absorption band, high molar extinction coefficient, fluorescence quantum yield, high sensitivity and other good characteristics. It is widely used in many fields such as biomolecular fluorescence probes, photosensitive materials, and photosensitizers for solar cells. Especially water-soluble cyanine dye Cy3, especially the structure with substituents introduced on the central carbon atom of the trimethine chain, such as Figure 1 shown, is more widely used as a fluorescence probe material.

[0003] Up to now, the literature (CN 111039897, EP 1408 366A2, US 02503776) shows that the cyanine dye Cy3 with sulfonic acid groups is synthesized by a two-step reaction process to obtain the target product: First, an aromatic heterocyclic nitrogen-containing compound reacts with sulfonic acid lactone to introduce an alkylsulfonic acid group to form an inner amine salt. Then, further, under the condition of using a base as a catalyst, it reacts with various triethyl orthoacids to obtain cyanine dyes Cy3 with different structures. The two-step reaction route is as Figure 2 shown. In the above two-step route, the intermediate needs to be separated, and then further, under the catalysis of bases such as triethylamine and pyridine, an addition and elimination reaction occurs in a solvent to form a trimethine chain. Currently, all Figure 1 shown structural compounds are prepared according to this route to obtain cyanine dye Cy3, and this technology is a general method. Summary of the Invention

[0004] In view of this, the present invention provides a preparation method of cyanine dye Cy3 containing an alkylsulfonic acid group to simplify the preparation process flow of cyanine dye Cy3 and improve the yield.

[0005] The technical solution of the present invention includes: A preparation method of water-soluble cyanine dye Cy3, adding the reaction raw materials aromatic nitrogen heterocycle, sulfonic acid lactone, and triethyl (or methyl) orthoformate (or propionate) into a reaction flask, heating and reacting, and then purifying the product obtained from the heating reaction to finally obtain cyanine dye Cy3, whose structural formula is:

[0006]

[0007] Among them, R 1 is C 1-4 alkyl, phenyl, or -substituted phenyl;

[0008] R 2 is C 1-4Alkyl, alkoxy, halogen, sulfonic acid group, carboxyl group, etc.;

[0009] X is C(CH 3 ) 2 , O, S;

[0010] π is phenyl, naphthyl;

[0011] n is 1 or 2.

[0012] Preferably, the heating reaction conditions are: under N 2 protection, 120 - 150 °C, react for 3 - 10 h.

[0013] Preferably, the purification includes:

[0014] The product obtained from the heating reaction is eluted with acetone solvent, and then the eluted product is dissolved in a mixed solution of anhydrous sodium acetate and methanol. Ethyl acetate is added to the mixed solution to precipitate an orange - yellow solid. The obtained solid is recrystallized with absolute ethanol to finally obtain the cyanine dye Cy3.

[0015] The present invention provides a preparation method of a cyanine dye Cy3 containing an alkylsulfonic acid group. By a one - pot method, three raw materials, namely aromatic nitrogen heterocycle, sultone, and triethyl (or methyl) orthoformate (or propionate), are mixed. Without solvent and catalyst, only by heating for a period of time, the target product can be obtained. The synthesis of the target cyanine dye Cy3 in the present invention has a simple process and can react without solvent and alkaline catalyst, greatly shortening the preparation production cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a schematic diagram of the chemical structure of the cyanine dye Cy3 described in the background art of the present invention;

[0017] Figure 2 is a schematic diagram of the reaction route for synthesizing the cyanine dye Cy3 through a two - step reaction process described in the background art of the present invention;

[0018] Figure 3 is a schematic diagram of the ultraviolet absorption test result of compound I obtained in Example 1 of the present invention;

[0019] Figure 4 is a schematic diagram of the ultraviolet absorption test result of compound II obtained in Example 2 of the present invention;

[0020] Figure 5 is a schematic diagram of the ultraviolet absorption test result of compound III obtained in Example 3 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0021] In the prior art, the preparation of cyanine dye Cy3 adopts a two-step route, in which the intermediate needs to be separated, and then further addition and elimination reactions occur in a solvent under the catalysis of a base such as triethylamine, pyridine, etc. to form a trimethine chain. A variety of organic solvents are used in this method. After the intermediate of quaternary ammonium alkyl sulfonate is purified, further addition and elimination reactions with various triethyl esters in an alcohol solvent are carried out under the condition of a base as a catalyst to obtain the target cyanine dye Cy3.

[0022] In order to simplify the reaction conditions, be more conducive to the large-scale preparation of cyanine dye Cy3, and improve the yield, the present invention adopts a "one-pot" reaction route to prepare the target cyanine dye Cy3 with the following structure:

[0023]

[0024] wherein, R 1 is C 1-4 alkyl, phenyl or -substituted phenyl;

[0025] R 2 is C 1-4 alkyl, alkoxy, halogen, sulfonic acid group, carboxyl group, etc.;

[0026] X is C(CH 3 ) 2 , O, S;

[0027] π is phenyl, naphthyl;

[0028] n is 1 or 2;

[0029] The preparation method of the semi-cyanine dye with the above-described structure: Add three reaction raw materials, namely aromatic nitrogen heterocycle, propyl (or butyl) sulfonic acid lactone, and trimethyl (or propyl) orthoformate, to the reaction flask according to a certain reaction ratio, and react at 120 - 150 °C for 3 - 10 h. After combining with subsequent separation and purification, the target cyanine dye Cy3 with the corresponding structure can be obtained, and the yield of the product is comparable to that of the existing two-step route. The steps of separation and purification are as follows: The reaction mixture obtained by heating the reaction is washed with acetone, and the intermediate cyanine dye Cy3 sulfonic acid inner amine salt with satisfactory purity can be obtained, which can be used for the next salt conversion process without further purification. Further add a methanol solution of anhydrous sodium acetate, dissolve all the intermediate Cy3 sulfonic acid inner amine salt to obtain the sodium salt, then add ethyl acetate, and an orange-yellow solid will precipitate. The obtained solid is recrystallized with absolute ethanol, and finally the sodium salt of the orange solid cyanine dye Cy3 is obtained.

[0030]

[0031] wherein R 1 , R 2 , n, X and π are the same as described above.

[0032] This process realizes the one-pot preparation of water-soluble cyanine dye Cy3 containing an alkylsulfonic acid group from three-component raw materials without solvent and catalyst. The three components are sulfonic acid lactone, triethyl (or methyl) orthoformate (or propionate), and an aromatic azacyclic ring with an active methyl group. The reaction mechanism underlying the present invention is as follows: Under high temperature and without solvent, triethyl (or methyl) orthoformate (or propionate) hardly reacts with sulfonic acid lactone and the active methyl group on the azacyclic ring, thus greatly reducing the complexity of the products during the three-component reaction and providing the possibility of a one-pot reaction. Sulfonic acid lactone and the aromatic azacyclic ring can form a sulfonic acid inner amine salt at high temperature. And under high temperature conditions, a hydrogen atom of the active methyl group on the azacyclic ring of the sulfonic acid inner amine salt intermediate migrates to the N atom, and isomerization can occur to form a methylene group with stronger nucleophilicity. Moreover, the reaction activity of the methylene group is stronger, attacking the active reaction center carbon atom of triethyl (or methyl) orthoformate (or propionate), thereby accelerating the progress of the condensation reaction and finally forming cyanine dye Cy3. It is precisely based on the isomerization that the sulfonic acid inner amine salt intermediate can undergo at the reaction temperature, so that the addition and elimination reactions can occur without an alkaline catalyst, and finally the target product cyanine dye Cy3 is successfully obtained. The reaction process of the speculated "one-pot reaction" is as follows

[0033]

[0034] This reaction is based on the high activity and high selectivity of sulfonic acid lactone when reacting with a nitrogen-containing heterocyclic ring to form a quaternary ammonium salt, and is completed by a one-step one-pot method; it overcomes the limitation that the condensation reaction in the existing two-step method technology must be carried out under alkaline conditions and in an ethanol solvent to form the trimethine conjugate chain of cyanine dye. The reaction conditions are mild, the operation is simple, and the product yield is high.

[0035] The existing two-step method mostly uses one-step process route, and an organic base is used as a catalyst during the condensation reaction process. While the present invention directly adopts a one-pot method, which shortens the reaction process, saves reaction time, reduces the use of some unnecessary reaction raw materials and solvents, the post-treatment operation is simple, and qualified products are also obtained. The yield of the one-step method is comparable to that of the background technology. The new process route is more environmentally friendly, easy to operate, and greatly reduces the emission of "three wastes", saves energy and raw materials, meeting the requirements of green chemistry.

[0036] The present invention synthesizes the target cyanine dye Cy3 in one step, with a simple process, and can react without solvent and alkaline catalyst, greatly shortening the preparation production cycle.

[0037] The following combines specific examples to further verify the preparation method provided by this implementation plan.

[0038] Example 1:

[0039] The synthesis process and structure of Compound I are as follows:

[0040] Take 0.849 g (5.06 mmol) of 2-methyl-5-chlorobenzoxazole, 0.676 g (5.53 mmol) of 1,3-propane sultone, and 0.298 g (2.01 mmol) of triethyl orthoformate in a two-necked flask. Under N 2 protection, react at 150 °C for 5 h, and then cool to room temperature. After rinsing with acetone, add 201 mg of anhydrous sodium acetate and 15 ml of methanol. After the reaction product is completely dissolved, add ethyl acetate, and orange-yellow solid precipitates. Filter the solid by suction, collect the solid, and wash it with ethyl acetate.

[0041] The obtained solid is recrystallized with 10 ml of absolute ethanol, and finally 553 mg of orange solid is obtained. The yield is 44.0%.

[0042]

[0043] Example 2:

[0044] The synthesis process and structure of Compound II are as follows:

[0045] Take 0.421 g (2.80 mmol) of 2-methylbenzothiazole, 0.376 g (3.08 mmol) of 1,3-propane sultone, and 0.167 g (1.13 mmol) of triethyl orthopropionate in a two-necked flask. Under N 2 protection, react at 150 °C for 5 h, and then cool to room temperature. After rinsing with acetone, add 125 mg of anhydrous sodium acetate and 15 ml of methanol. After the reaction product is completely dissolved, add an appropriate amount of ethyl acetate, and dark purple solid precipitates. Filter the solid by suction, collect 443 mg of the solid, and wash it with ethyl acetate. The obtained solid is recrystallized with 10 ml of absolute ethanol, and finally 405 mg of purple solid is obtained. The yield is 60.9%.

[0046]

[0047] Example 3:

[0048] The synthesis process and structure of Compound III are as follows:

[0049] Take 0.595 g (3.55 mmol) of 2-methyl-5-chlorobenzoxazole, 0.474 g (3.88 mmol) of 1,3-propane sultone, and 0.250 g (1.42 mmol) of triethyl orthopropionate in a two-necked flask. Under N 2After reacting for 5 h at 150 °C under protection and cooling to room temperature, it was rinsed with acetone. After adding 140 mg of anhydrous sodium acetate and 10 ml of methanol, and after all the reaction products were dissolved, ethyl acetate was added, and yellow solid precipitated out. 613 mg of solid was collected by suction filtration and washed with ethyl acetate. The obtained solid was recrystallized with 6 ml of absolute ethanol, and finally 553 mg of yellow solid was obtained, with a yield of 61.0%.

[0050]

[0051] The ultraviolet absorption tests (using methanol as the solvent) were carried out on the three compounds synthesized in Examples 1, 2, and 3. The test results are as Figures 3 - 5 shown. The maximum ultraviolet absorption wavelength of Compound I is 490 nm, the maximum ultraviolet absorption wavelength of Compound II is 560 nm, and the maximum ultraviolet absorption wavelength of Compound III is 498 nm. Through the analysis of ultraviolet absorption, it can be determined that the compounds with the target structure are generated, and at the same time, the structure-activity relationship of the cyanine dye Cy3 can be clarified, that is, the influence of structural differences on ultraviolet absorption.

[0052] Comparative Example 1:

[0053] From the literature Wang Li. Research on Cyanine Dyes for Biological Fluorescent Labeling [D]. Dalian University of Technology, 2010. In the synthesis of symmetrical trimethine cyanine dyes in Section 2.2.3 of Chapter 2, the indole quaternary ammonium salt intermediate was first synthesized and directly used in the second-step reaction without purification. The quaternary ammonium salt intermediate and trimethyl orthoformate were dissolved in acid anhydride in a ratio of 2:1. Under nitrogen protection, after heating for 40 min, it was purified by gradient elution, and the yield was only 26%.

[0054] Comparative Example 2:

[0055] From the literature Shi, Q.Q, Sun, R., Ge, J.F., et al. "A comparative study of symmetrical and unsymmetrical trimethine cyanine dyes bearing benzoxazolyl and benzothiazolyl groups." Dyes and Pigments 93. (2012): 1506-1511. In the process of preparing Compound 3a, the iodide salt intermediate 1a was first prepared. Benzoxazole and iodomethane were dissolved in DMF and heated at 60 °C for 24 h. After post-treatment, the intermediate 1a was obtained with a yield of 80.5%. The intermediate 1a was dissolved in acid anhydride, and during the heating reflux process, triethyl orthoformate was added and reacted for 1 h. After post-treatment, the final product 3a was obtained, and the yield was only 8.7%.

[0056] In summary, when synthesizing trimethine cyanine dyes in the comparative examples, it is necessary to first synthesize the inner salt intermediate and use it for the next reaction. In the second reaction, acid anhydride was used as the solvent, and the intermediate was reacted with triethyl orthoformate under heating and reflux, and the target compound was obtained after post-treatment. The synthesis yield of the trimethine cyanine dye prepared thereby is relatively low.

[0057] Other embodiments of the present invention will be readily apparent to those skilled in the art after considering the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the present invention that follow the general principles of the present invention and include known common knowledge or conventional technical means in the technical field not disclosed by the present invention. The specification and examples are only to be considered exemplary, and the true scope and spirit of the present invention are pointed out by the following claims.

[0058] It should be understood that the present invention is not limited to what has been described above and can be variously modified and changed without departing from its scope. The scope of the present invention is only limited by the appended claims.

Claims

1. A method for preparing a water-soluble cyanine dye Cy3, characterized in that: include: The reaction raw materials, aromatic nitrogen heterocycle, sultone and triethyl orthoformate (or propionate) are added into a reaction bottle together, and heated to react, and then the product obtained by the heating reaction is purified to finally obtain cyanine dye Cy3, whose structural formula is: Where R1 is C 1-4 Alkyl, phenyl or -substituted phenyl; R2 is C 1-4 Alkyl, alkoxy, halogen, sulfonic acid, carboxyl, etc.; X is C(CH3)2, O, S; π is phenyl or naphthyl; n is 1 or 2.

2. The method for preparing cyanine dye Cy3 according to claim 1, characterized in that: The heating reaction conditions are: under N2 protection, 120-150°C, reaction time 3-10h.

3. The method for preparing cyanine dye Cy3 according to claim 1, characterized in that: The purification comprises: The product obtained by the heating reaction was eluted with an acetone solvent, and then the eluted product was dissolved in a mixture of anhydrous sodium acetate and methanol. Ethyl acetate was added to the mixture to precipitate an orange-yellow solid. The obtained solid was recrystallized with anhydrous ethanol to finally obtain the cyanine dye Cy3.

Images