High-purity D-fluorescein substance as well as preparation method and application thereof

Abstract

The invention discloses a high-purity D-fluorescein substance as well as a preparation method and application thereof, and relates to the technical field of compound preparation. According to the preparation method, a synthetic method for forming a bi-conjugated thiazole ring system by constructing a thiazole-like ring is adopted, and the whole reaction process is carried out in a dark environment; and pulping and purifying to remove impurities in the product, thereby obtaining the high-purity D-fluorescein free acid. And salifying with different metal ion alkalis to realize the synthesis of D-fluorescein series salts. The preparation method is few in working procedures, simple to operate and convenient to industrialize; according to the method, the problem of waste of materials such as solvent and filler through traditional purification means such as column chromatography is avoided, so that the generation amount of waste liquid and waste residues is greatly reduced, environmental protection treatment is facilitated, and the process cost is reduced. The prepared D-fluorescein substance has wide application in the aspects of biological research, medical research, environment monitoring and food safety detection.

Description

Technical Field

[0001] The present invention relates to the technical field of compound preparation, and in particular to a high-purity D-luciferin substance and a preparation method and application thereof. Background Art

[0002] D-luciferin is a substrate of firefly luciferase. In the presence of magnesium ions, luciferase reacts luciferin with ATP, which is oxidized to form a dioxetane structure and emits yellow-green light. At present, the conventional D-luciferin synthesis method has low raw material cost, but due to the harsh synthesis conditions, it is easy to cause the generation of by-products (luciferin with opposite configuration), and the subsequent processing steps are complicated, resulting in the final luciferin, the free acid purity is not high, which in turn reduces the added value of the overall product.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] The purpose of the present invention is to provide a high-purity D-luciferin substance and a preparation method and application thereof to solve the above technical problems.

[0005] The present invention is achieved in that:

[0006] In a first aspect, an embodiment of the present invention provides a method for preparing a high-purity D-luciferin-like substance, comprising the following steps: reacting 2-cyano-6-hydroxybenzothiazole and D-cysteine ​​hydrochloride in a mass ratio of 1:(0.5-1.5) in an organic solvent aqueous solution, adding carbonate when the color of the system is light yellow, and reacting for 5h-18h to obtain D-luciferin free acid; the entire reaction is carried out in a light-proof environment.

[0007] In a second aspect, an embodiment of the present invention provides a high-purity D-luciferin free acid prepared by the aforementioned preparation method, wherein the purity of the high-purity D-luciferin free acid is >99.3%.

[0008] In a third aspect, an embodiment of the present invention provides a luciferin metal salt, which is prepared by reacting high-purity D-luciferin free acid prepared by the preparation method as described above with an alkali metal compound in an aqueous solution; wherein the alkali metal compound is selected from any one of sodium hydroxide and potassium hydroxide; and the mass ratio of high-purity D-luciferin free acid to the alkali metal compound is 1:(0.1-0.5).

[0009] In a fourth aspect, an embodiment of the present invention provides an application of high-purity D-luciferin free acid prepared by the aforementioned preparation method, which includes at least one of the following application modes:

[0010] (1) luc marker gene in living cells, tissues or organisms;

[0011] (2) Imaging analysis of luciferase-fusion gene expression in vivo or in vitro;

[0012] (3) Reporter gene analysis;

[0013] (4) Immunoassay;

[0014] (5) ATP fluorescence monitoring analysis.

[0015] The present invention has the following beneficial effects:

[0016] The method for preparing high-purity D-luciferin substances provided in the embodiment of the present invention has fewer steps, simple operation, and is easy to realize industrialization; the method avoids the waste of materials such as solvent fillers by traditional purification means such as column chromatography, thereby greatly reducing the amount of waste liquid and waste residue generated, which is beneficial to environmental protection and reduces process costs.

[0017] High-purity D-luciferin substances are synthesized by constructing a thiazole-like ring to form a diconjugated thiazole ring system, and then pulping and purification are applied to remove impurities in the product to obtain high-purity D-luciferin free acid. The synthesis of D-luciferin series salts is achieved by salt formation with different metal ion bases. The obtained D-luciferin substances are widely used in biological research, medical research, environmental monitoring and food safety testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 During the preparation of high-purity D-luciferin substances in Example 1, the color of the system changed: A was light pink, and B was light yellow. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0021] In a first aspect, an embodiment of the present invention provides a method for preparing a high-purity D-luciferin-like substance, comprising the following steps: reacting 2-cyano-6-hydroxybenzothiazole and D-cysteine ​​hydrochloride in a mass ratio of 1:(0.5-1.5) in an organic solvent aqueous solution, adding carbonate when the color of the system is light yellow, and reacting for 5h-18h to obtain D-luciferin free acid; the entire reaction is carried out in a light-proof environment.

[0022] Specifically, the mass ratio of 2-cyano-6-hydroxybenzothiazole to D-cysteine ​​hydrochloride can be selected from any one of 1:0.5, 1:0.7, 1:0.9, 1:1 and 1:1.5, or other values ​​within the range of 1:(0.5-1.5).

[0023] It should be noted that the reaction raw materials or reaction products will change color, deteriorate, etc. under light, affecting their use effect; using a light-proof environment for reaction can effectively prevent deterioration and ensure that the appearance and properties of the target product remain unchanged; in addition, a light-proof environment can prevent light from participating in the reaction process, ensure the effectiveness of the reaction, reduce the occurrence of side reactions, and improve the purity of the product.

[0024] In an optional embodiment of the present invention, the organic solvent aqueous solution comprises water and an organic solvent in a volume ratio of (1-3):(2-1), wherein the organic solvent is selected from at least one of methanol, ethanol, dichloromethane and acetonitrile.

[0025] It should be noted that the organic solvent used in the embodiment of the present invention has low toxicity and low volatility, and is relatively less harmful to the environment and human body. The addition of an organic solvent will increase the solubility of the raw material, and can provide a suitable reaction environment and conditions so that the catalyst and the reactant can be fully mixed, thereby improving the rate and selectivity of the reaction, and improving the actual yield of the final product; however, the type of organic solvent will affect the formation of the final product. For example, when acetonitrile and water are combined, the cyano group of acetonitrile will participate in the reaction process, a competitive reaction will occur, and by-products will be generated, reducing the yield. And changing the ratio of the solvent, the phenomenon in the preparation process and the purity and total yield of the final product will also be different.

[0026] In an optional embodiment of the present invention, based on the mass of 2-cyano-6-hydroxybenzothiazole, the ratio of the organic solvent to 2-cyano-6-hydroxybenzothiazole is (10-14) mL: 1 g; specifically, it can be selected from any one of 10 mL: 1 g, 11 mL: 1 g, 12 mL: 1 g, 13 mL: 1 g and 14 mL: 1 g, or other values ​​within the range of (10-14) mL: 1 g.

[0027] In an optional embodiment of the present invention, the organic solvent aqueous solution is water and methanol in a volume ratio of (1-3):1; further, the volume ratio of water and methanol is (1.5-2.5):1, which can be selected from any one of 1.5:1, 1.8:1, 2:1 and 2.5:1, or other values ​​within the range of (1.5-2.5):1.

[0028] It should be noted that after adding water and methanol solution in a volume ratio of (1.5-2.5):1 to the reactants 2-cyano-6-hydroxybenzothiazole and D-cysteine ​​hydrochloride, a light pink suspension is formed. After the solution system is stirred and reacted at room temperature in the dark for 1 min-10 min, the color of the system changes to light yellow, and then carbonate is added to continue the reaction. The reaction time is 5 h-18 h, until 2-cyano-6-hydroxybenzothiazole is completely consumed, and a solution system containing D-luciferin free acid is obtained.

[0029] The complete consumption of 2-cyano-6-hydroxybenzothiazole is detected by TLC thin layer chromatography. In addition, the reaction time is also related to the amount of raw materials used.

[0030] Therefore, D-luciferin free acid can be obtained by selecting a suitable type of organic solvent and a mixed solution of an organic solvent and an aqueous solution in a suitable ratio for reaction.

[0031] In an optional embodiment of the present invention, the carbonate is selected from any one of potassium carbonate and sodium carbonate; preferably potassium carbonate.

[0032] It should be noted that carbonates are used as catalysts in the reaction system. Carbonate catalysts are generally low in cost and suitable for large-scale industrial production. In addition, carbonate catalysts have good catalytic effects and fast reaction rates, and exhibit high selectivity and high activity in organic synthesis reactions.

[0033] In an optional embodiment of the present invention, the color of the blended system of 2-cyano-6-hydroxybenzothiazole, D-cysteine ​​hydrochloride and organic solvent aqueous solution changes from light pink to light yellow, and carbonate is added after the color stabilizes.

[0034] It should be noted that in the embodiment of the present invention, the color of the blending system changes from light pink to light yellow, which can be used as a basis for judging whether the reactants are fully activated; if carbonate is added when the reactants are not fully activated, it will affect the color and yield of the product.

[0035] Further, based on the mass of 2-cyano-6-hydroxybenzothiazole, the mass ratio of the mass of carbonate to the mass of 2-cyano-6-hydroxybenzothiazole is (0.5-1): 1. It can be selected from any one of 0.5: 1, 0.65: 1, 0.7: 1 and 1: 1, or other values ​​within the range of (0.5-1): 1.

[0036] In an optional embodiment of the present invention, the preparation method further comprises, after the reaction is completed, adjusting the pH value of the solution system to 2.8-3.2 in a light-proof environment at a temperature of 2°C-10°C, and performing a crystallization treatment.

[0037] Specifically, after the reaction is completed, the temperature of the solution system is reduced to 2°C-10°C in an ice bath environment, and the pH value of the solution system is adjusted to 3 in a light-proof state. At this time, precipitation continues to precipitate in the solution. After standing in an ice bath for 1.5h-2.5h, the product is filtered and recovered. During this period, the filter cake is rinsed with anhydrous ethanol solution several times, such as 3 times, or 5 times, to ensure that the impurities on the surface of the product are cleaned, especially those fat-soluble substances that are difficult to remove with water. This makes the filter cake purer and is conducive to subsequent processing and use. In addition, anhydrous ethanol is highly volatile, and the surface residue will evaporate quickly after cleaning, reducing the impact of the residue on the filter cake. By using anhydrous ethanol to clean the filter cake, the quality and performance of the product can be significantly improved.

[0038] In an optional embodiment of the present invention, the product obtained by filtration is recovered and then placed in a vacuum drying oven for drying. The drying temperature is 40°C-55°C and the drying time is 30min-60min. The specific drying temperature and drying time can be reasonably adjusted according to the actual amount of product to be processed. In addition, the drying form can be selected from other forms according to actual conditions, such as desiccant, convection drying, etc. During the vacuum drying process, anhydrous calcium chloride can be added to the drying oven according to actual needs to absorb ethanol vapor.

[0039] After drying, the product was weighed, the single yield was calculated and its purity was tested.

[0040] The weighing was carried out using a conventional electronic balance; the calculation formula for the single yield was Y%=m(actual product) / m(theoretical)×100%, where Y represents the yield and m represents the mass; the purity was determined by HPLC using Shimadzu HPLC-20.

[0041] In an optional embodiment of the present invention, the crystallization process includes slurrying and purifying the crystals.

[0042] It should be noted that the pulping purification operation is a purification method between washing and recrystallization, which uses the difference in solubility of the product and impurities in a single solvent (or mixed solvent) to purify the product. The solvent is mainly selected according to the properties of the product. The ideal solvent for pulping has a good solubility for impurities and is basically insoluble in the product, so that the impurities can be removed without losing the product. Therefore, in actual operation, pulping is usually performed using a poor solvent for the product and a good solvent for the impurities.

[0043] It should be noted that the beating experimental device and the reaction device in the present invention are the same.

[0044] The present invention adopts pulping purification to purify the product, which has the following characteristics: it avoids traditional purification methods such as column chromatography, avoids the waste of materials such as solvent fillers, thereby greatly reducing the amount of waste liquid and waste residue generated, is beneficial to environmental protection, and reduces process costs.

[0045] Furthermore, the solvent used in the pulping purification process is at least one of anhydrous ethanol, methanol, ethyl acetate, acetonitrile and dichloromethane.

[0046] Specifically, in one embodiment of the present invention, after adding an appropriate amount of anhydrous ethanol to the collected product, the mixture is stirred thoroughly, and then the precipitated product is collected and placed in a vacuum drying oven for drying. The drying temperature is 40°C-55°C, and the drying time is 30min-60min. The specific drying temperature and drying time can be reasonably adjusted according to the actual amount of the product. In addition, the drying form can be selected from other forms according to the actual situation, such as freeze drying, desiccant, convection drying, etc.

[0047] It should be noted that the type and performance of the pulping equipment will also affect the amount of solvent used. Specifically, the amount of solvent used is preferably 3 to 5 times the amount of the product. The amount of anhydrous ethanol used and the specific stirring time are related to the actual amount of product processed, and can be reasonably adjusted according to actual conditions. If the solution is red during pulping purification, it needs to be stirred for at least 2 hours. If the solution is lighter or colorless, the stirring time during the purification process can be reasonably reduced.

[0048] The D-luciferin free acid prepared by the preparation method of the present invention has a purity of 97%-99% after the first pulping and purification treatment as determined by HPLC. If the purity is lower than 99%, the above operation is repeated to finally obtain crystals with a purity greater than 99%.

[0049] It should be noted that the number of times for pulping purification is 2 to 5 times, and the more the better is not necessarily true, because the solvent will dissolve part of the target product, resulting in a decrease in the yield.

[0050] In a second aspect, an embodiment of the present invention provides a high-purity D-luciferin free acid prepared by the aforementioned preparation method, wherein the purity of the high-purity D-luciferin free acid is >99.3%.

[0051] In a third aspect, an embodiment of the present invention provides a luciferin metal salt, which is prepared by reacting high-purity D-luciferin free acid prepared by the preparation method as described above with an alkali metal compound in an aqueous solution; wherein the alkali metal compound is selected from any one of sodium hydroxide and potassium hydroxide; and the mass ratio of high-purity D-luciferin free acid to the alkali metal compound is 1:(0.1-0.5).

[0052] In an optional embodiment of the present invention, the preparation of the luciferin metal salt is to use the high-purity D-luciferin free acid prepared above as a raw material, then add an alkali metal compound, react in an aqueous solution, stir continuously during the reaction, and react at a temperature of 22° C.-26° C. for 1 h-12 h to obtain the D-luciferin metal salt. For example, when preparing D-luciferin potassium salt, potassium hydroxide aqueous solution is used to react with high-purity D-luciferin free acid.

[0053] In a fourth aspect, an embodiment of the present invention provides an application of high-purity D-luciferin free acid prepared by the aforementioned preparation method, which includes at least one of the following application modes:

[0054] (1) luc marker gene in living cells, tissues or organisms;

[0055] (2) Imaging analysis of luciferase-fusion gene expression in vivo or in vitro;

[0056] (3) Reporter gene analysis;

[0057] (4) Immunoassay;

[0058] (5) ATP fluorescence monitoring analysis.

[0059] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0060] Example 1

[0061] This embodiment provides a method for preparing high-purity D-luciferin-based substances, which comprises the following steps:

[0062] (1) Addition reaction:

[0063] Take 10g of 2-cyano-6-hydroxybenzothiazole and 10g of D-cysteine ​​hydrochloride and add them to a 1L light-proof reaction bottle, add 1L of methanol-water solution (the volume ratio of water to methanol is 2:1, recorded as H2O2-MeOH1) mixed solvent to react and form a light pink suspension (see Figure 1 A), and the suspension was stirred at room temperature for 8 min, and the solution color turned light yellow (see Figure 1 B), then 7.84 g of potassium carbonate was added, and stirring was continued for 12 h at room temperature in the dark until 2-cyano-6-hydroxybenzothiazole was completely consumed.

[0064] It should be noted that after the reaction process continues for a certain period of time, such as 6 hours, the TLC thin layer chromatography technique is used to detect whether the 2-cyano-6-hydroxybenzothiazole in the raw material is completely consumed.

[0065] (2) Crystallization treatment:

[0066] After the reaction (1) is completed, the temperature of the reaction system is lowered to below 8°C in an ice bath environment, and the pH value of the solution system is adjusted to 3 in a light-proof state. At this time, precipitation continues to precipitate in the solution. After standing in an ice bath for 2 hours, the product is filtered and recovered. During this period, the filter cake is rinsed 3 times with anhydrous ethanol solution.

[0067] All precipitated solids were collected and placed in a vacuum drying oven for drying at a temperature of 45°C and a drying time of 30 min.

[0068] After drying, 15.6 g of crystals were weighed and analyzed to find that the free acid of D-luciferin was 13.6 g with a purity of 87.2%.

[0069] Among them, the instrument and model for purity detection are specifically Shimadzu HPLC-20.

[0070] (3) Pulping and purification:

[0071] Collect the solid in (2), add 300 mL of anhydrous ethanol, the amount of which is 4 times that of D-luciferin free acid, stir for 2 hours, collect all the precipitated solids, and put them into a vacuum drying oven for drying, wherein the drying temperature T = 45 ° C, and the drying time t = 30 min.

[0072] After the first pulping, the purity of the product was determined by HPLC to be 97.52%.

[0073] The purity was lower than 99%. The above operation was repeated, and after beating twice, crystals with a purity greater than 99% were obtained, namely 13.7 g of high-purity D-luciferin free acid was obtained. After analysis, there were 13.6 g of D-luciferin free acid with a purity of 99.36% and a total yield of 84.85%.

[0074] Example 2

[0075] This example provides a method for preparing a high-purity D-luciferin-based substance, which differs from Example 1 only in that the crystallization temperature is changed, as follows:

[0076] (2) Crystallization treatment:

[0077] After the reaction in step (1) is completed, the pH value of the solution is adjusted to 3 at room temperature of 25° C. in a light-proof state.

[0078] After drying, 17.3 g of crystals were weighed and analyzed to find that the free acid of D-luciferin was 12.5 g with a purity of 72.2%.

[0079] (3) Beating and purification:

[0080] After the first pulping, the product purity is generally 93.2% as determined by HPLC.

[0081] The purity was lower than 99%. The above operation was repeated, and after beating for 4 times, crystals with a purity greater than 99% were obtained. After analysis, 12.1 g of D-luciferin free acid was found, with a purity of 99.01% and a total yield of 76.05%.

[0082] Example 3

[0083] This example provides a method for preparing high-purity D-luciferin-based substances, which differs from Example 1 only in that the reaction solvent is changed, as follows:

[0084] (1) Addition reaction:

[0085] The mixed solvent is an acetonitrile aqueous solution, wherein the volume ratio of acetonitrile to water is 2:1.

[0086] (2) Crystallization treatment:

[0087] After drying, 14.2 g of crystals were weighed and analyzed to find that the free acid of D-luciferin was 10.1 g with a purity of 71.13%.

[0088] It should be noted that the crystals precipitated in this process are darker in color, and during the subsequent pulping and purification process, the pulping solution is red.

[0089] (3) Beating and purification:

[0090] After the first pulping, the product purity is generally 91.3% as determined by HPLC.

[0091] The purity was lower than 99%. The above operation was repeated, and after beating for 7 times, crystals with a purity greater than 99% were obtained. After analysis, 9.7 g of D-luciferin free acid was found, with a purity of 99.21% and a total yield of 60.93%.

[0092] It should be noted that when the acetonitrile aqueous solution is used as a mixed solvent for the reaction, the yield is reduced because the cyano group of acetonitrile participates in the reaction process, competes with the reaction, and generates by-products.

[0093] Experimental Example 1

[0094] This experimental example provides a D-luciferin potassium salt, and the preparation process thereof is as follows:

[0095] The high-purity D-luciferin free acid prepared in Example 1 was reacted with potassium hydroxide in an aqueous solution at a mass ratio of 1:1.5, with continuous stirring. The reaction temperature was 25°C. After reacting for 2 hours, the solution system was filtered, and the filtered solid was freeze-dried to obtain D-luciferin potassium salt.

[0096] In summary, the method for preparing high-purity D-luciferin substances provided in the embodiments of the present invention has fewer steps, simple operation, and is easy to realize industrialization; the method avoids the waste of materials such as solvent fillers by traditional purification means such as column chromatography, thereby greatly reducing the amount of waste liquid and waste residue generated, which is beneficial to environmental protection and reduces process costs.

[0097] High-purity D-luciferin substances are synthesized by constructing a thiazole-like ring to form a diconjugated thiazole ring system, and then pulping and purification are applied to remove impurities in the product to obtain high-purity D-luciferin free acid. The synthesis of D-luciferin series salts is achieved by salt formation with different metal ion bases. The obtained D-luciferin substances are widely used in biological research, medical research, environmental monitoring and food safety testing.

[0098] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing high-purity D-luciferin-based substances, characterized in that: The method comprises the following steps: reacting 2-cyano-6-hydroxybenzothiazole and D-cysteine ​​hydrochloride in a mass ratio of 1:(0.5-1.5) in an organic solvent aqueous solution, adding carbonate when the color of the system is light yellow, and reacting for 5h-18h to obtain D-luciferin free acid; the whole reaction is carried out in a light-proof environment.

2. The preparation method according to claim 1, characterized in that: The organic solvent aqueous solution comprises water and an organic solvent in a volume ratio of (1-3):(2-1), wherein the organic solvent is selected from at least one of methanol, ethanol, dichloromethane and acetonitrile; Preferably, based on the mass of 2-cyano-6-hydroxybenzothiazole, the ratio of the organic solvent to the 2-cyano-6-hydroxybenzothiazole is (10-14) mL: 1 g.

3. The preparation method according to claim 2, characterized in that: The organic solvent aqueous solution comprises water and methanol in a volume ratio of (1-3):1; Preferably, the volume ratio of water to methanol is (1.5-2.5):

1.

4. The preparation method according to claim 1, characterized in that: The carbonate is selected from any one of potassium carbonate and sodium carbonate; preferably potassium carbonate; Preferably, based on the mass of 2-cyano-6-hydroxybenzothiazole, the mass ratio of the carbonate to 2-cyano-6-hydroxybenzothiazole is (0.5-1):

1.

5. The preparation method according to claim 1, characterized in that: The preparation method further comprises, after the reaction is completed, adjusting the pH value of the solution system to 2.8-3.2 in a light-proof environment at a temperature of 2° C.-10° C., and then performing a crystallization treatment.

6. The preparation method according to claim 5, characterized in that: The crystallization treatment further includes pulping and purifying the crystals; Preferably, the solvent used in the pulping purification process is at least one of anhydrous ethanol, methanol, ethyl acetate, acetonitrile and dichloromethane.

7. The preparation method according to claim 1, characterized in that: The beating purification is stopped when the crystal purity is greater than 99%, thereby obtaining high-purity D-luciferin free acid.

8. A high-purity D-luciferin free acid prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The purity of the high-purity D-luciferin free acid is >99.3%.

9. A luciferin metal salt, characterized in that: The luciferin metal salt is prepared by reacting the high-purity D-luciferin free acid prepared by the preparation method according to any one of claims 1 to 7 with an alkali metal compound in an aqueous solution; Wherein, the alkali metal compound is selected from any one of sodium hydroxide and potassium hydroxide; The mass ratio of the high-purity D-luciferin free acid to the alkali metal compound is 1:(0.1-0.5).

10. An application of high-purity D-luciferin free acid obtained by the preparation method according to any one of claims 1 to 7, characterized in that: The application method includes at least one of the following: (1) luc marker gene in living cells, tissues or organisms; (2) Imaging analysis of luciferase-fusion gene expression in vivo or in vitro; (3) Reporter gene analysis; (4) Immunoassay; (5) ATP fluorescence monitoring analysis.

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