阅读说明：本技术 一种磺基-Cy3羧酸荧光染料及其制备工艺 (sulfo-Cy 3 carboxylic acid fluorescent dye and preparation process thereof ) 是由 张伟 刘德平 吕海平 于 2021-08-23 设计创作，主要内容包括：本发明涉及一种磺基-Cy3羧酸荧光染料及其制备工艺,合成的磺基-Cy3羧酸具有荧光量子产率、高发射高激发波长以及易溶于水物理化学性质稳点的特点,同时,本发明的合成方法通过同时构建苯并吲哚两侧支链,利用吲哚α位甲基活性较高的特点,在最后迈克尔与亚胺加成的时候选择性的生成了我们需要的单缩合目标产物,收率较高,通过沉淀和重结晶就能拿到纯品,而避免了C18柱分离,对后续中试放大具有一定的指导意义。(The invention relates to a sulfo-Cy 3 carboxylic acid fluorescent dye and a preparation process thereof, wherein the synthesized sulfo-Cy 3 carboxylic acid has the characteristics of fluorescence quantum yield, high emission, high excitation wavelength and easy water solubility and stable physical and chemical properties, and meanwhile, the synthetic method of the invention selectively generates a needed mono-condensation target product by simultaneously constructing branched chains at two sides of benzindole and utilizing the characteristic of higher activity of methyl at alpha position of indole during the final addition of Michael and imine, has higher yield, can obtain a pure product by precipitation and recrystallization, avoids C18 column separation, and has certain guiding significance for subsequent pilot scale amplification.)

1. A preparation process of sulfo-Cy 3 carboxylic acid fluorescent dye, which is characterized in that,

(1) dissolving 4-hydrazinobenzenesulfonic acid in glacial acetic acid, and then adding methyl isopropyl ketone for reaction; filtering the solid obtained by the reaction, washing the solid with ethyl acetate, and drying the solid in vacuum to obtain light pink solid; dissolving the obtained solid in methanol, and slowly dropwise adding an isopropanol solution of potassium hydroxide; stirring and filtering to obtain 2,3, 3-trimethyl-3H-indole-5-potassium sulfonate;

(2) dissolving 2,3, 3-trimethyl-3H-indole-5-sulfonic acid potassium and 6-bromohexanoic acid in 1, 2-dichlorobenzene, and heating under the condition of argon for reaction; after the reaction is finished, vacuum concentrating, adding isopropanol, stirring and filtering to obtain a solid, and filtering to obtain a product 1- (5-carboxypentyl) -2,3, 3-trimethyl-3H-indole-1-onium-5-sulfonic acid ester;

(3) adding 2,3, 3-trimethyl-3H-indole-5-potassium sulfonate into iodoethane 1, and heating and refluxing; after the reaction is finished, concentrating the reaction solution, filtering again, washing with acetone for three times, and drying the obtained solid in vacuum to obtain a product 1-ethyl-2, 3, 3-trimethyl-3H-indole-1-onium-5-sulfonate;

(4) dissolving 1-ethyl-2, 3, 3-trimethyl-3H-indol-1-ium-5-sulfonate and 3.3 g of N, N-diphenylformamidine in glacial acetic acid, and heating and refluxing; concentrating to remove the solvent, adding ethyl acetate, stirring, and filtering to obtain a product;

(5) dissolving (E) -1-ethyl-3, 3-dimethyl-2- (2- (phenylamino) vinyl) -3H-indol-1-ium-5-sulfonic acid salt in acetic anhydride and pyridine; adding 1- (5-carboxyl amyl) -2,3, 3-trimethyl-3H-indole-1-onium-5-sulfonate into the reaction solution, and heating for reaction; and adding ether to separate out the product after the reaction is cooled, washing the product with isopropanol for three times, and drying the product in vacuum to obtain the product.

2. The process for preparing sulfo-Cy 3 carboxylic acid fluorescent dye according to claim 1, wherein the process steps are developed according to the following materials and proportions,

(1) dissolving 10 g of 4-hydrazinobenzenesulfonic acid in 35 ml of glacial acetic acid, adding 17 ml of methyl isopropyl ketone, and reacting at 120 ℃ for 4 hours; filtering the solid obtained by the reaction, washing the solid for three times by 50 ml of ethyl acetate, and drying the solid in vacuum to obtain 9.4 g of light pink solid; dissolving the obtained solid in 100 ml of methanol, and slowly dropwise adding 80 ml of isopropanol solution of 2.6 g of potassium hydroxide; stirring for one hour and filtering to obtain 7.5 g of yellow solid;

(2) dissolving 10 g of potassium 2,3, 3-trimethyl-3H-indole-5-sulfonate and 9.8 g of 6-bromohexanoic acid in 100 ml of 1, 2-dichlorobenzene, and heating to 110 ℃ under the condition of argon to react for 12 hours; after the reaction is finished, vacuum concentrating, adding isopropanol, stirring and filtering, and filtering the obtained solid to obtain 9.5 g of a product;

(3) adding 110 ml of iodoethane into 20 g of 2,3, 3-trimethyl-3H-indole-5-potassium sulfonate, and heating to reflux for 24 hours; after the reaction is finished, concentrating the reaction solution, filtering again, washing with 50 ml of acetone for three times, and drying the obtained solid in vacuum to obtain 18 g of a product;

(4) 4 g of 1-ethyl-2, 3, 3-trimethyl-3H-indol-1-ium-5-sulfonate and 3.3 g of N, N-diphenylformamidine are dissolved in 20 ml of glacial acetic acid and heated under reflux for 4 hours; concentrating to remove the solvent, adding 50 ml of ethyl acetate, stirring and filtering to obtain 1.8 g of a product;

(5) 4.7 g of (E) -1-ethyl-3, 3-dimethyl-2- (2- (phenylamino) vinyl) -3H-indol-1-ium-5-sulfonate are dissolved in 25 ml of acetic anhydride and 25 ml of pyridine; then 5.3 g of 1- (5-carboxypentyl) -2,3, 3-trimethyl-3H-indol-1-ium-5-sulfonate is added into the reaction liquid, and the mixture is heated to 110 ℃ for reaction for 4 hours; after the reaction is cooled, diethyl ether is added to separate out the product, the product is washed three times by 10 ml of isopropanol and dried in vacuum to obtain 2.3 g of the product.

3. The sulfo-Cy 3 carboxylic acid fluorescent dye prepared according to the preparation process of claim 1 or 2.

Technical Field

The invention relates to a sulfo-Cy 3 carboxylic acid fluorescent dye and a preparation process thereof, and relates to the technical field of organic chemical industry.

Background

Fluorescent dyes are substances that absorb light of a certain wavelength and emit light of another wavelength that is greater than the light absorbed. Most of them are compounds containing a benzene ring or a heterocycle and having a conjugated double bond. The fluorescent dye can be used independently or combined into a composite fluorescent dye. Wherein, the Cy series belongs to indole cyanine dyes and is formed by connecting indole rings at two ends together through a conjugated methine chain. And the Cy fluorescent probe can effectively weaken the interference from the autofluorescence probe in the organism because the ultraviolet absorption wavelength and the fluorescence emission wavelength of the Cy fluorescent probe are both positioned in the near infrared region.

In the prior art, for example, CN201010523674.1 is a method for preparing and purifying a water-soluble cyanine dye, cy5.5, by using 1, 3-disulfonic acid-6-naphthylhydrazine as a starting material, reacting with methyl isopropyl ketone to generate 1, 1, 2-trimethylbenzindole 1, 3-disulfonate, and then reacting with iodoethane and 6-bromohexanoic acid to generate N-ethyl-2, 3, 3-trimethylbenzindole 5, 7-disulfonate (product 1) and N- (epsilon-carboxypentyl) -2,3, 3-trimethylbenzindole-5, 7-disulfonate (product 2); the product 1 reacts with beta-anilino-acraldehyde aniline hydrochloride and then directly reacts with the product 2 to prepare Cy5.5; and repeatedly purifying and separating by preparative thin-layer chromatography to obtain a pure Cy5.5 product. The method has the advantages of simple operation and high yield, and the product does not need expensive HPLC (C-18 reverse column) separation, thereby having important significance for meeting domestic autonomous production and demand.

In the prior art, sulfo-Cy 3 carboxylic acid is also reported only in English documents at present, almost no patent is reported in China, the synthesis difficulty is high, the purification is difficult, most of English documents are finally purified by using a C18 reverse column, the cost is high, and the sulfo-Cy 3 carboxylic acid is expensive. CN201780051208.8, for example, provides compositions and methods for synthesizing near-infrared, closed-chain, sulfo-cyanine dyes.

As described above, since the near-infrared Cy dye is a kind of fluorescent reagent which has been increasingly emphasized in recent years, the Cy dye technology is continuously being developed.

Disclosure of Invention

The invention aims to provide a novel sulfo-Cy 3 carboxylic acid fluorescent dye and a preparation process thereof, and the preparation method has the advantages of simple conventional method, high yield and low product purification difficulty.

The purpose of the invention is achieved by the following ways:

a preparation process of sulfo-Cy 3 carboxylic acid fluorescent dye, which comprises the following steps,

(1) dissolving 4-hydrazinobenzenesulfonic acid in glacial acetic acid, and then adding methyl isopropyl ketone for reaction; filtering the solid obtained by the reaction, washing the solid with ethyl acetate, and drying the solid in vacuum to obtain light pink solid; dissolving the obtained solid in methanol, and slowly dropwise adding an isopropanol solution of potassium hydroxide; stirring and filtering to obtain 2,3, 3-trimethyl-3H-indole-5-potassium sulfonate;

(2) dissolving 2,3, 3-trimethyl-3H-indole-5-sulfonic acid potassium and 6-bromohexanoic acid in 1, 2-dichlorobenzene, and heating under the condition of argon for reaction; after the reaction is finished, vacuum concentrating, adding isopropanol, stirring and filtering to obtain a solid, and filtering to obtain a product 1- (5-carboxypentyl) -2,3, 3-trimethyl-3H-indole-1-onium-5-sulfonic acid ester;

(3) adding 2,3, 3-trimethyl-3H-indole-5-potassium sulfonate into iodoethane 1, and heating and refluxing; after the reaction is finished, concentrating the reaction solution, filtering again, washing with acetone for three times, and drying the obtained solid in vacuum to obtain a product 1-ethyl-2, 3, 3-trimethyl-3H-indole-1-onium-5-sulfonate;

(4) dissolving 1-ethyl-2, 3, 3-trimethyl-3H-indol-1-ium-5-sulfonate and 3.3 g of N, N-diphenylformamidine in glacial acetic acid, and heating and refluxing; concentrating to remove the solvent, adding ethyl acetate, stirring, and filtering to obtain a product;

(5) dissolving (E) -1-ethyl-3, 3-dimethyl-2- (2- (phenylamino) vinyl) -3H-indol-1-ium-5-sulfonic acid salt in acetic anhydride and pyridine; adding 1- (5-carboxyl amyl) -2,3, 3-trimethyl-3H-indole-1-onium-5-sulfonate into the reaction solution, and heating for reaction; and adding ether to separate out the product after the reaction is cooled, washing the product with isopropanol for three times, and drying the product in vacuum to obtain the product.

Further, the process steps are developed according to the following substances and proportions,

(1) dissolving 10 g of 4-hydrazinobenzenesulfonic acid in 35 ml of glacial acetic acid, adding 17 ml of methyl isopropyl ketone, and reacting at 120 ℃ for 4 hours; filtering the solid obtained by the reaction, washing the solid for three times by 50 ml of ethyl acetate, and drying the solid in vacuum to obtain 9.4 g of light pink solid; dissolving the obtained solid in 100 ml of methanol, and slowly dropwise adding 80 ml of isopropanol solution of 2.6 g of potassium hydroxide; stirring for one hour and filtering to obtain 7.5 g of yellow solid;

(2) dissolving 10 g of potassium 2,3, 3-trimethyl-3H-indole-5-sulfonate and 9.8 g of 6-bromohexanoic acid in 100 ml of 1, 2-dichlorobenzene, and heating to 110 ℃ under the condition of argon to react for 12 hours; after the reaction is finished, vacuum concentrating, adding isopropanol, stirring and filtering, and filtering the obtained solid to obtain 9.5 g of a product;

(3) adding 110 ml of iodoethane into 20 g of 2,3, 3-trimethyl-3H-indole-5-potassium sulfonate, and heating to reflux for 24 hours; after the reaction is finished, concentrating the reaction solution, filtering again, washing with 50 ml of acetone for three times, and drying the obtained solid in vacuum to obtain 18 g of a product;

(4) 4 g of 1-ethyl-2, 3, 3-trimethyl-3H-indol-1-ium-5-sulfonate and 3.3 g of N, N-diphenylformamidine are dissolved in 20 ml of glacial acetic acid and heated under reflux for 4 hours; concentrating to remove the solvent, adding 50 ml of ethyl acetate, stirring and filtering to obtain 1.8 g of a product;

(5) 4.7 g of (E) -1-ethyl-3, 3-dimethyl-2- (2- (phenylamino) vinyl) -3H-indol-1-ium-5-sulfonate are dissolved in 25 ml of acetic anhydride and 25 ml of pyridine; then 5.3 g of 1- (5-carboxypentyl) -2,3, 3-trimethyl-3H-indol-1-ium-5-sulfonate is added into the reaction liquid, and the mixture is heated to 110 ℃ for reaction for 4 hours; after the reaction is cooled, diethyl ether is added to separate out the product, the product is washed three times by 10 ml of isopropanol and dried in vacuum to obtain 2.3 g of the product.

Further, sulfo-Cy 3 carboxylic acid fluorescent dye prepared by the preparation process is provided.

In the prior art CN201780051208.8 (CN 109689626A), 2- (2, 3, 3-trimethyl-3H-indol-1-onium-1-yl) ethane-1-sulfonate and N- ((Z) - ((E) -2-chloro-3- ((phenylamino) methylene) cyclohex-1-en-1-yl) methylene) aniline chloride, which are molecules with quite complex and expensive structures, are used, and are only suitable for laboratory lab laboratory research, but have a vast prospect of process amplification production. Meanwhile, the reaction is easy to produce mono-condensation products and di-condensation products when condensing in alkali and alcohol, the yield is not compatible with the selectivity, the yield is low when the selectivity is good, and the proportion of the di-condensation and the mono-condensation is basically equal when the yield is high, so the condition is very limited.

In contrast, the invention directly uses cheap 4-hydrazino benzene sulfonic acid and methyl isopropyl ketone as starting materials, the production cost is obviously lower, because the two parts of benzindole structures are separately synthesized, and the activity of 1- (5-carboxyl amyl) -2,3, 3-trimethyl-3H-indole-1-onium-5-sulfonic acid ester is stronger because of the methyl group at the 2-position of indole, the methyl group can directly generate Michael addition with conjugated double bonds to remove aniline under the condition of pyridine acetic anhydride in the last step, and the final product basically has no single condensation and self double condensation, so the yield is ensured. Meanwhile, after the reaction is finished, the product is separated out by using ether, and the pure product of the product can be obtained by directly recrystallizing with isopropanol without using C-18 columns in many documents, so that the method has strong guiding significance for experimental amplification.

The sulfo-Cy 3 carboxylic acid synthesized by the method has the characteristics of fluorescence quantum yield, high emission and excitation wavelength and stable physical and chemical properties of being easily dissolved in water, and meanwhile, the synthetic method has the advantages of cheap raw materials, simple and conventional experimental method, high yield and low product purification difficulty, only needs conventional purification methods such as column chromatography, recrystallization, pulping and the like, and has certain guiding significance for subsequent pilot plant amplification.

Drawings

FIG. 1 is a flow chart of a production process according to an embodiment of the present invention.

Detailed Description

The technical solution of the present invention is further described below by way of examples with reference to the accompanying drawings, however, the scope of the present invention is not limited to the following examples.

Example (b): Sulfo-Cy 3 carboxylic acid (Sulfo-Cyanine 3 carboxylic acid):

1. the production process flow is shown in figure 1:

formula 1 is 4-hydrazinylbenzenesulfonic acid;

formula 2 is potassium 2,3, 3-trimethyl-3H-indole-5-sulfonate;

formula 3 is 1- (5-carboxypentyl) -2,3, 3-trimethyl-3H-indol-1-ium-5-sulfonate;

formula 4 is potassium 2,3, 3-trimethyl-3H-indole-5-sulfonate;

formula 5 is 1-ethyl-2, 3, 3-trimethyl-3H-indol-1-ium-5-sulfonate;

formula 6 is (E) -1-ethyl-3, 3-dimethyl-2- (2- (phenylamino) vinyl) -3H-indol-1-ium-5-sulfonate;

formula 7 is 2- ((E) -3- ((E) -1- (5-carboxypentyl) -3, 3-dimethyl-5-sulfonidolin-2-ylidene) prop-1-en-1-yl) -1-ethyl-3, 3-dimethyl-3H-indol-1-ium-5-sulfonate.

The technical process and technical parameters are as follows:

in a 250 ml single-neck flask, 10 g of 4-hydrazinobenzenesulfonic acid was dissolved in 35 ml of glacial acetic acid, and 17 ml of methyl-isopropyl-ketone was added and the mixture was left at 120 ℃ for 4 hours. The solid obtained from the reaction was filtered, washed three times with 50 ml of ethyl acetate and dried in vacuo to give 9.4 g of a pale pink solid. The solid obtained was dissolved in 100 ml of methanol and 80 ml of a 2.6 g solution of potassium hydroxide in isopropanol was slowly added dropwise. Stirring for one hour and filtering to obtain 7.5 g of yellow solid.

In a 250 ml one-neck flask, 10 g of potassium 2,3, 3-trimethyl-3H-indole-5-sulfonate and 9.8 g of 6-bromohexanoic acid were dissolved in 100 ml of 1, 2-dichlorobenzene and heated to 110 ℃ under argon for 12 hours. After the reaction was completed, vacuum concentration was performed, isopropyl alcohol was then added, stirring and filtration were performed, and the obtained solid was filtered to obtain 9.5 g of a product.

In a 250 ml single-neck flask, 20 g of potassium 2,3, 3-trimethyl-3H-indole-5-sulfonate and 110 ml of iodoethane were added, and the mixture was heated to reflux for 24 hours. After the reaction, the reaction solution was concentrated, filtered again, washed three times with 50 ml of acetone, and the obtained solid was dried in vacuum to obtain 18 g of a product.

In a 100 ml single-neck flask, 4 g of 1-ethyl-2, 3, 3-trimethyl-3H-indol-1-ium-5-sulfonate and 3.3 g of N, N-diphenylformamidine were dissolved in 20 ml of glacial acetic acid, and heated under reflux for 4 hours. The solvent was removed by concentration, 50 ml of ethyl acetate was added, the mixture was stirred and filtered to obtain 1.8 g of a product.

In a 250 ml one-neck flask was added 4.7 g of (E) -1-ethyl-3, 3-dimethyl-2- (2- (phenylamino) vinyl) -3H-indol-1-ium-5-sulfonate and dissolved in 25 ml of acetic anhydride and 25 ml of pyridine. 5.3 g of 1- (5-carboxypentyl) -2,3, 3-trimethyl-3H-indol-1-ium-5-sulfonate was added to the reaction mixture, and the mixture was heated to 110 ℃ and reacted for 4 hours. After the reaction is cooled, diethyl ether is added to separate out the product, the product is washed three times by 10 ml of isopropanol and dried in vacuum to obtain 2.3 g of the product.

The raw materials of the embodiment can be amplified or reasonably adjusted according to the proportion, and the method is particularly suitable for guiding industrial production.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.