Preparation method of dithiothreitol

Abstract

The invention provides a preparation method of dithiothreitol, which comprises the following steps: step S1, carrying out epoxidation reaction on 1, 4-dibromo-2-butene and peroxy acid to generate 1, 4-dibromo-2-epoxidized butene; s2, carrying out a substitution reaction on the 1, 4-dibromo-2-epoxidized butylene and thioacetate, so as to generate 1, 4-diacetyl thio-2-epoxidized butylene, and carrying out a reaction on the 1, 4-dibromo-2-epoxidized butylene and thioacetate so as to generate 1, 4-diacetyl thio-2-epoxidized butylene; and S3, carrying out a hydrolysis reaction on the 1, 4-diacetyl thio-2-epoxidized butylene, so as to generate the dithiothreitol. According to the preparation method disclosed by the embodiment of the invention, 1, 4-dibromo-2-butene is used as a starting raw material, the epoxidation reaction is firstly carried out, then thioacetate is used for replacing bromine at the tail end, and finally the hydrolysis reaction is carried out to obtain the target product dithiothreitol, so that the whole synthesis route is short, the raw material is safe and non-toxic, impurities are easy to remove, and the preparation method is suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of compound preparation, and more specifically, relates to a method for preparing dithiothreitol. Background Art

[0002] Dithiothreitol (DTT) is a commonly used thiol reagent with low volatility, less unpleasant odor, and low oxidation potential. The oxidized state of dithiothreitol (DTTox) is a ring structure, and its structure is stable, so dithiothreitol has a high reducibility. In DNA extraction experiments, dithiothreitol is often added to break the disulfide bonds between proteins, so that DNA is fully free and the extraction efficiency of DNA is improved. Dithiothreitol is also often used for the reduction of disulfide bonds in proteins, and can be used to prevent the intramolecular or intermolecular disulfide bonds formed between cysteines in proteins. Therefore, adding dithiothreitol in protein extraction can protect the protein structure from being destroyed.

[0003] There are relevant literature reports on the synthesis of dithiothreitol, and the main synthesis route is as follows.

[0004] Route 1: Document CN101503384A records that cis-1,4-butenediol is used as the starting material, undergoes bromination reaction with bromine, undergoes ring-closing reaction with sodium hydroxide, undergoes ring-opening addition reaction with thioacetic acid, and then undergoes hydrolysis reaction with sodium hydroxide solution to obtain dithiothreitol. Its synthetic route reaction route is:

[0005]

[0006] However, this route is prone to produce isomers during the reaction process, and the use of highly toxic bromine in the bromination reaction and the use of extremely smelly thioacetic acid in the ring-opening addition reaction can also cause harm to the human body and the environment, making it unsuitable for industrial production.

[0007] Route 2: Document CN117510383A records that 1,4-dithiothreitol is prepared by using erythritol as raw material through two steps of bromination with hydrogen bromide and substitution with thiourea. The synthetic route is:

[0008]

[0009] The bromination reaction has poor selectivity, and all four hydroxyl groups are easily brominated, which will generate a large amount of impurities that are difficult to separate using conventional methods and are not suitable for the preparation and synthesis of dithiothreitol.

[0010] Route 3: Document CN115093352A records that 1,4-dibromobutene and pyridine are reacted to form a quaternary ammonium salt, which is oxidized to a diol with potassium permanganate, and the diol is protected to form an ester with acetic anhydride, and reacted with potassium thioacetate to obtain 1,4-diacetylmercapto-2,3-diacetoxybutane, and then sulfuric acid is added to methanol for reflux decomposition reaction to obtain dithiothreitol. The synthetic route reaction route is:

[0011]

[0012] However, the reaction steps are relatively complicated, and it uses pyridine, which is reproductively toxic, and potassium permanganate, a controlled hazardous chemical, which poses a high safety hazard and is not suitable for industrial production. Summary of the invention

[0013] In view of this, the object of the present invention is to provide a method for preparing dithiothreitol which is simple to operate, has a short route and is suitable for industrial production.

[0014] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0015] The method for preparing dithiothreitol according to an embodiment of the present invention comprises the following steps:

[0016] Step S1, 1,4-dibromo-2-butene is subjected to epoxidation reaction with a peroxy acid to generate 1,4-dibromo-2-epoxidized butene;

[0017] Step S2, allowing 1,4-dibromo-2-epoxybutene to undergo a substitution reaction with thioacetate to generate 1,4-diacetylthio-2-epoxybutene;

[0018] Step S3, hydrolyzing 1,4-diacetylthio-2-epoxybutene to generate dithiothreitol.

[0019] In some embodiments of the present invention, the peracid is any one or more of peracetic acid, meta-chloroperbenzoic acid, and trifluoroperacetic acid.

[0020] Furthermore, the epoxidation reaction is carried out in a first solvent, which is selected from one or more of dichloromethane, chloroform, toluene, and 1,2-dichloroethane. After the epoxidation reaction is completed, the reaction solution is washed with water, and the organic phase is dried over anhydrous sodium sulfate and evaporated to obtain the 1,4-dibromo-2-epoxidized butene.

[0021] Furthermore, the molar ratio of the 1,4-dibromo-2-butene to the peroxy acid is 1:(1.0-1.2), the reaction temperature of the epoxidation reaction is 0-60° C., and the reaction time is 2-8 hours.

[0022] In some embodiments of the present invention, the substitution reaction is carried out in the presence of a catalyst, the thioacetate is one or more of potassium thioacetate and sodium thioacetate, and the catalyst is one or more of potassium iodide and sodium iodide.

[0023] Furthermore, the substitution reaction is carried out in a second solvent under weak alkaline conditions, the second solvent is selected from one or more of dichloromethane, chloroform, toluene, and 1,2-dichloroethane, and the pH value of the reaction system is 7-8.

[0024] Furthermore, in the step S2, after the substitution reaction is completed, the reaction solution is washed with water, and the organic phase is dried over anhydrous sodium sulfate and evaporated to dryness to obtain the 1,4-diacetylthio-2-epoxybutene.

[0025] Furthermore, the molar ratio of the 1,4-dibromo-2-epoxybutene to the thioacetate is 1:(1.0-1.2), the reaction temperature of the substitution reaction is 20-50° C., and the reaction time is 6-10 hours.

[0026] In some embodiments of the present invention, in step S3, the hydrolysis reaction is carried out in the presence of a base, the base is selected from one or more of potassium hydroxide and sodium hydroxide, the molar ratio of 1,4-diacetylthio-2-epoxybutene to the base is 1:(0.05-0.1), the reaction temperature of the hydrolysis reaction is 20-80°C, and the reaction time is 4-8 hours.

[0027] Furthermore, the hydrolysis reaction is carried out in an aqueous solution. After the hydrolysis reaction is completed, the reaction product is extracted using an extractant, and after removing water and the extractant, it is recrystallized using dichloromethane / n-heptane to obtain the dithiothreitol; the extractant includes at least one of dichloromethane and chloroform.

[0028] The above technical solution of the present invention has at least one of the following beneficial effects:

[0029] According to the preparation method of the embodiment of the present invention, 1,4-dibromo-2-butene is used as the starting raw material, an epoxidation reaction is first carried out, then the bromine at the terminal is replaced with thioacetate, and finally the target product dithiothreitol is obtained by a hydrolysis reaction. The overall synthesis route is short, the raw materials are safe and non-toxic, and impurities are easy to remove, which is suitable for industrial production. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.

[0031] The following first describes in detail the preparation method of dithiothreitol according to an embodiment of the present invention.

[0032] The method for preparing dithiothreitol according to an embodiment of the present invention comprises the following steps:

[0033] Step S1, 1,4-dibromo-2-butene is subjected to epoxidation reaction with a peroxy acid to generate 1,4-dibromo-2-epoxidized butene;

[0034] Step S2, allowing 1,4-dibromo-2-epoxybutene to undergo a substitution reaction with thioacetate to generate 1,4-diacetylthio-2-epoxybutene;

[0035] Step S3, hydrolyzing 1,4-diacetylthio-2-epoxybutene to generate dithiothreitol.

[0036] Specifically, the synthesis route is shown in the following formula (1):

[0037]

[0038] According to the preparation method of the embodiment of the present invention, 1,4-dibromo-2-butene is used as the starting raw material, an epoxidation reaction is first carried out, then the bromine at the terminal is replaced with thioacetate, and finally the target product dithiothreitol is obtained by a hydrolysis reaction. The overall synthesis route is short, the raw materials are safe and non-toxic, and impurities are easy to remove, which is suitable for industrial production.

[0039] Below, each step is described in detail.

[0040] (i) Step S1, 1,4-dibromo-2-butene is epoxidized with a peracid to generate 1,4-dibromo-2-epoxidized butene.

[0041] That is, first, 1,4-dibromo-2-butene is used as a starting material and subjected to epoxidation reaction to generate 1,4-dibromo-2-epoxybutene.

[0042] Compared with the above-mentioned route 3, which uses reproductively toxic pyridine to react with it to form a quaternary ammonium salt, the first step of the present application uses peroxy acid to cause an epoxidation reaction to generate 1,4-dibromo-2-epoxybutene. The intermediate process is safe and easy to operate.

[0043] In some embodiments of the present invention, the peracid is any one or more of peracetic acid, meta-chloroperbenzoic acid, and trifluoroperacetic acid.

[0044] Furthermore, the epoxidation reaction is carried out in a first solvent, and the first solvent is selected from one or more of dichloromethane, chloroform, toluene, and 1,2-dichloroethane, preferably dichloromethane, in which other reagents have better solubility, and dichloromethane is easily removed later.

[0045] In addition, the step may also include: after the epoxidation reaction is completed, the reaction solution is washed with water, the organic phase is dried with anhydrous sodium sulfate, and evaporated to dryness to obtain the 1,4-dibromo-2-epoxidized butene. In other words, the separation between the intermediate product and the impurities is simple and easy to operate, with less three wastes, and is suitable for industrial production.

[0046] Furthermore, the molar ratio of the 1,4-dibromo-2-butene to the peroxy acid is 1:(1.0-1.2), the reaction temperature of the epoxidation reaction is 0-60° C., and the reaction time is 2-8 hours.

[0047] (ii) Step S2, allowing 1,4-dibromo-2-epoxybutene to undergo a substitution reaction with thioacetate to generate 1,4-diacetylthio-2-epoxybutene.

[0048] That is, after obtaining the intermediate 1,4-dibromo-2-epoxybutene, it is subjected to a substitution reaction with thioacetate to generate 1,4-diacetylthio-2-epoxybutene.

[0049] Compared with thioacetic acid, the odor of thioacetate is much smaller than that of thioacetic acid, and thioacetate is safer, produces less three wastes, and the generated intermediates are easy to separate from impurities.

[0050] Specifically, the thioacetate may be, for example, one or more of potassium thioacetate and sodium thioacetate.

[0051] In some embodiments of the present invention, the substitution reaction is carried out in the presence of a catalyst. The presence of a catalyst is conducive to promoting the substitution reaction and improving the reaction efficiency. When a catalyst is added, the reaction speed is much faster than when no catalyst is added.

[0052] Specifically, the catalyst may be one or more of potassium iodide and sodium iodide.

[0053] Furthermore, the substitution reaction is carried out in a second solvent, and the second solvent is selected from one or more of dichloromethane, chloroform, toluene, and 1,2-dichloroethane.

[0054] Furthermore, the substitution reaction is preferably carried out under weakly alkaline conditions, for example, the pH value of the reaction system is 7-8. An organic base or an inorganic base can be used to adjust the pH value of the reaction system to 7-8. This is beneficial to promote the conversion of thioacetate into a free state, thereby promoting its replacement of the terminal bromine of 1,4-dibromo-2-epoxybutene.

[0055] Furthermore, the molar ratio of the 1,4-dibromo-2-epoxybutene to the thioacetate is 1:(1.0-1.2), the reaction temperature of the substitution reaction is 20-50° C., and the reaction time is 6-10 hours.

[0056] Furthermore, after the substitution reaction is completed, the reaction solution is washed with water, the organic phase is dried over anhydrous sodium sulfate, and evaporated to dryness to obtain the 1,4-diacetylthio-2-epoxybutene. In other words, the intermediate produced in this step can be easily separated from impurities and can be used for the next reaction after simple treatment, with less three wastes, low cost, and suitable for industrial production.

[0057] (iii) Step S3, hydrolyzing 1,4-diacetylthio-2-epoxybutene to generate dithiothreitol.

[0058] That is, after 1,4-diacetylthio-2-epoxybutene is generated by substitution reaction, ene is hydrolyzed to generate the target product dithiothreitol.

[0059] In some embodiments of the present invention, in step S3, the hydrolysis reaction is carried out in the presence of a base, and the base is selected from one or more of potassium hydroxide and sodium hydroxide.

[0060] The amount of base used does not need to be too much. The greater the amount of base, the greater the chance of generating impurities. For example, the molar ratio of 1,4-diacetylthio-2-epoxybutene to the base is 1:(0.05-0.1).

[0061] In addition, the reaction temperature of the hydrolysis reaction can be 20 to 80° C., and the reaction time can be 4 to 8 hours.

[0062] Furthermore, the hydrolysis reaction is carried out in an aqueous solution. After the hydrolysis reaction is completed, the reaction product is extracted using an extractant, and after removing water and the extractant, it is recrystallized using dichloromethane / n-heptane to obtain the dithiothreitol; the extractant includes at least one of dichloromethane and chloroform.

[0063] From the above, it can be seen that the preparation method of the present application has simple post-treatment of each intermediate, less three wastes, and the post-treatment of the target product does not require column treatment, only simple operations such as extraction and recrystallization are required, and the extractant and the reagents used for recrystallization can be recycled after simple treatment.

[0064] The preparation method of dithiothreitol of the present invention is further described in detail below through specific examples.

[0065] Example 1

[0066] (1) Synthesis of 1,4-dibromo-2-epoxybutene

[0067] 1,4-dibromo-2-butene (42.8 g, 0.2 mol) was dissolved in dichloromethane (400 mL), and peracetic acid (15.2 g, 0.2 mol) was added to react. The reaction was stirred at 25°C for 8 hours. After the reaction, the reaction solution was washed twice with water, and the organic phase was dried over anhydrous sodium sulfate. The dichloromethane was removed by rotary evaporation to obtain 36 g of 1,4-dibromo-2-epoxybutene with a yield of 78%.

[0068] The NMR data of the intermediate product are shown below:

[0069] 1H-NMR: (d6-DMSO): δppm=3.22-3.24(m,2H),3.94-3.96(m,4H). The detection results are consistent with the structure of 1,4-dibromo-2-epoxybutene.

[0070] (2) Synthesis of 1,4-diacetylthio-2-epoxybutene

[0071] The compound 2,3-dibromomethyloxirane (23 g, 0.1 mol) obtained in 1.1 and potassium thioacetate (25.1 g, 0.22 mol) were dissolved in 250 mL of dichloromethane, and 0.1 g of potassium iodide was added. The mixture was stirred at 40°C for 6 hours. After the reaction, the reaction solution was washed twice with water, and the organic phase solution was dried over anhydrous sodium sulfate. Then, dichloromethane was removed by rotary evaporation to obtain 18 g of 1,4-diacetylthio-2-epoxybutene with a yield of 82%.

[0072] The NMR data of the intermediate product are shown below:

[0073] 1H-NMR: (d6-DMSO): δppm=2.1(s,6H),3.22-3.25(m,2H),3.93-3.95(m,4H). The detection results are consistent with the structure of 1,4-diacetylthio-2-epoxybutene.

[0074] (3) Synthesis of dithiothreitol

[0075] The intermediate compound 1,4-diacetylthio-2-epoxybutene (17.6 g, 0.08 mol) obtained in 1.2 was dissolved in water (200 mL), and sodium hydroxide (2 g, 0.05 mol) was added. The reaction was carried out at 30°C for 8 hours. After the reaction was completed, it was extracted with dichloromethane (3×200 mL), and the dichloromethane was dried over anhydrous sodium sulfate. The dichloromethane was removed by rotary evaporation to obtain a solid, which was recrystallized from dichloromethane / n-heptane to obtain 10 g of the target product dithiothreitol with a yield of 81%.

[0076] The NMR data of the final product are shown below:

[0077] 1H-NMR: (d6-DMSO): δppm=1.95-2.10(dd,2H),2.66-2.83(dd,2H),4.97(s,2H). The detection results are consistent with the structure of dithiothreitol.

[0078] Example 2

[0079] (1) Synthesis of 1,4-dibromo-2-epoxybutene

[0080] 1,4-dibromo-2-butene (42.8 g, 0.2 mol) was dissolved in dichloromethane (400 mL), and trifluoroperacetic acid (26 g, 0.2 mol) was added to react. The reaction was stirred at 40° C. for 4 hours. After the reaction was completed, the reaction solution was washed twice with water, and the organic phase was dried over anhydrous sodium sulfate. The dichloromethane was removed by rotary evaporation to obtain 38 g of 1,4-dibromo-2-epoxybutene with a yield of 83%.

[0081] (2) Synthesis of 1,4-diacetylthio-2-epoxybutene

[0082] The compound 2,3-dibromomethyloxirane (23 g, 0.1 mol) obtained in 2.1 and potassium thioacetate (26.7 g, 0.24 mol) were dissolved in 250 mL of dichloromethane, 0.1 g of potassium iodide was added, and the mixture was stirred at 50°C for 8 hours. After the reaction, the reaction solution was washed twice with water, and the organic phase solution was dried over anhydrous sodium sulfate. Then, dichloromethane was removed by rotary evaporation to obtain 19 g of 1,4-diacetylthio-2-epoxybutene with a yield of 86%.

[0083] (3) Synthesis of dithiothreitol

[0084] The intermediate compound 1,4-diacetylthio-2-epoxybutene (17.6 g, 0.08 mol) obtained in 2.2 was dissolved in water (200 mL), potassium hydroxide (2.8 g, 0.05 mol) was added, and the reaction was carried out at 40°C for 4 hours. After the reaction was completed, it was extracted with dichloromethane (3×200 mL), and the dichloromethane was dried over anhydrous sodium sulfate. The dichloromethane was removed by rotary evaporation to obtain a crude product, which was recrystallized from dichloromethane / n-heptane to obtain 10.2 g of the target product dithiothreitol with a yield of 83%.

[0085] Example 3

[0086] (1) Synthesis of 1,4-dibromo-2-epoxybutene

[0087] 1,4-dibromo-2-butene (42.8 g, 0.2 mol) was dissolved in dichloromethane (400 mL), and peracetic acid (15.2 g, 0.2 mol) was added to react. The reaction was stirred at 40°C for 3 hours. After the reaction, the reaction solution was washed twice with water, and the organic phase was dried over anhydrous sodium sulfate. The dichloromethane was removed by rotary evaporation to obtain 36 g of 1,4-dibromo-2-epoxybutene with a yield of 79%.

[0088] (2) Synthesis of 1,4-diacetylthio-2-epoxybutene

[0089] The compound 2,3-dibromomethyl oxirane (23 g, 0.1 mol) obtained in 2.1 and sodium thioacetate (27.8 g, 0.24 mol) were dissolved in 250 mL of dichloromethane, 0.1 g of sodium iodide was added, and the mixture was stirred at 50°C for 10 hours. After the reaction, the reaction solution was washed twice with water, and the organic phase solution was dried over anhydrous sodium sulfate. Then, dichloromethane was removed by rotary evaporation to obtain 20 g of 1,4-diacetylthio-2-epoxybutene with a yield of 90%.

[0090] (3) Synthesis of dithiothreitol

[0091] The intermediate compound 1,4-diacetylthio-2-epoxybutene (17.6 g, 0.08 mol) obtained in 2.2 was dissolved in water (200 mL), potassium hydroxide (2.8 g, 0.05 mol) was added, and the reaction was carried out at 50°C for 4 hours. After the reaction was completed, it was extracted with dichloromethane (3×200 mL), and the dichloromethane was dried over anhydrous sodium sulfate. The dichloromethane was removed by rotary evaporation to obtain a crude product, which was recrystallized from dichloromethane / n-heptane to obtain 9.4 g of the target product dithiothreitol with a yield of 76%.

[0092] In summary, the method for preparing dithiothreitol of the present invention has the advantages of short synthesis route, simple operation, low cost and high yield.

[0093] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing dithiothreitol, characterized in that: The steps include: Step S1, 1,4-dibromo-2-butene is subjected to epoxidation reaction with a peroxy acid to generate 1,4-dibromo-2-epoxidized butene; Step S2, allowing 1,4-dibromo-2-epoxybutene to undergo a substitution reaction with thioacetate to generate 1,4-diacetylthio-2-epoxybutene; Step S3, hydrolyzing 1,4-diacetylthio-2-epoxybutene to generate dithiothreitol.

2. The preparation method according to claim 1, characterized in that: The peracid is any one or more of peracetic acid, m-chloroperbenzoic acid, and trifluoroperacetic acid.

3. The preparation method according to claim 2, characterized in that: The epoxidation reaction is carried out in a first solvent, wherein the first solvent is selected from one or more of dichloromethane, chloroform, toluene, and 1,2-dichloroethane. After the epoxidation reaction is completed, the reaction solution is washed with water, and the organic phase is dried over anhydrous sodium sulfate and evaporated to dryness to obtain the 1,4-dibromo-2-epoxidized butene.

4. The preparation method according to claim 2, characterized in that: The molar ratio of the 1,4-dibromo-2-butene to the peroxy acid is 1:(1.0-1.2), the reaction temperature of the epoxidation reaction is 0-60° C., and the reaction time is 2-8 hours.

5. The preparation method according to claim 1, characterized in that: The substitution reaction is carried out in the presence of a catalyst, the thioacetate is one or more of potassium thioacetate and sodium thioacetate, and the catalyst is one or more of potassium iodide and sodium iodide.

6. The preparation method according to claim 5, characterized in that: The substitution reaction is carried out in a second solvent under weak alkaline conditions, the second solvent is selected from one or more of dichloromethane, chloroform, toluene, and 1,2-dichloroethane, and the pH value of the reaction system is 7-8.

7. The preparation method according to claim 6, characterized in that: In the step S2, after the substitution reaction is completed, the reaction solution is washed with water, and the organic phase is dried over anhydrous sodium sulfate and evaporated to dryness to obtain the 1,4-diacetylthio-2-epoxybutene.

8. The preparation method according to claim 5, characterized in that: The molar ratio of the 1,4-dibromo-2-epoxybutene to the thioacetate is 1:(1.0-1.2), the reaction temperature of the substitution reaction is 20-50° C., and the reaction time is 6-10 hours.

9. The preparation method according to claim 1, characterized in that: In step S3, the hydrolysis reaction is carried out in the presence of a base, the base is selected from one or more of potassium hydroxide and sodium hydroxide, the molar ratio of 1,4-diacetylthio-2-epoxybutene to the base is 1:(0.05-0.1), the reaction temperature of the hydrolysis reaction is 20-80° C., and the reaction time is 4-8 hours.

10. The preparation method according to claim 9, characterized in that: The hydrolysis reaction is carried out in an aqueous solution. After the hydrolysis reaction is completed, the reaction product is extracted using an extractant, and after removing water and the extractant, it is recrystallized using dichloromethane / n-heptane to obtain the dithiothreitol; the extractant includes at least one of dichloromethane and chloroform.

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