L-thioglutamic acid is a natural product compound that is converted from glucosinolate-like precursors in grannymonda vegetables such as kale, mustard greens and broccoli. It has a variety of health effects, and is widely used to prevent chronic diseases such as cancer, cardiovascular disease, and diabetes. It is widely found in cruciferous plants, such as kale, cauliflower, and cabbage. It has many pharmacological activities, such as anticancer, anti-inflammatory, antioxidative, neuroprotective, etc. Therefore, the synthetic method of L-thioglutamic acid has attracted much attention.
At present, the methods for synthesizing L-thioglutamic acid can be divided into two categories: biological fermentation and chemical synthesis. These two methods are described in detail below.
L-Sulforaphane biological fermentation method:
The biological fermentation method is a method of synthesizing compounds with microorganisms. Through the screening of bacterial strains and the optimization of culture conditions, the microorganisms can produce L-thioglutamic acid during the fermentation process. Now it has been found that many strains can synthesize L-thioglutamic acid, such as Escherichia coli, Streptococcus thermophilus and so on. Among them, the method for synthesizing L-thioglutamic acid by strains represented by Escherichia coli is the most common. Specific steps are as follows:
1. Isolate L-thioglutamate-producing strains, such as Escherichia coli, from soil or plant tissue;
2. Adopt suitable medium and culture conditions, such as temperature, pH value, culture time, etc., to cultivate Escherichia coli;
3. Harvest the Escherichia coli fermentation broth, carry out certain processing and purification, and obtain L-thioglutamic acid.
L-Sulforaphane chemical synthesis method:
The chemical synthesis method is to use the method of chemical molecular reaction to synthesize L-thioglutamic acid through different synthetic routes. At present, chemical synthesis methods are mainly divided into four types: choline salt method, sulfuryl chloride method, carbonyl reduction method, and agarose sulfate method.
1. Choline salt method:
This method is one of the commonly used synthetic methods. Proceed as follows:
① First, L-methionine and choline are reacted under acidic conditions to generate a rare nitrogen substitute of L-thioglutamic acid.
② Heating and hydrolyzing nitrogen substitutes into L-thioglutamic acid under alkaline conditions.
The advantage of this method is that the reaction is simple and easy to master, but the yield is low.
2. Sulfonyl chloride method:
This method utilizes electrophilic substitution reaction to synthesize L-thioglutamic acid. Proceed as follows:
① Reaction of L-methionine and sulfonyl chloride under alkaline conditions to obtain L-thioglutamic acid sulfinate.
② The sulfinate is hydrolyzed under alkaline conditions to generate L-thioglutamic acid.
The advantage of this method is that the reaction is easy to control and the yield is high, but it needs to use toxic sulfonyl chloride.
3. Carbonyl reduction method:
The method is to utilize reduction reaction to synthesize L-thioglutamic acid. Proceed as follows:
①L-methionine reacts with acetonimine to generate pyridine pyrimidin-2-en-4-one and pyrimidine pyrimidin-2-en-4-one-5-sulfate at the same time
②Pyrimidine pyrimidine-2-en-4-one-5-sulfate is reduced to obtain L-thioglutamic acid.
4. Agarose sulfate method: This method is to react L-methionine and agarose sulfate under acidic conditions to generate L-thioglutamic acid. Due to the good catalytic activity of sulfate, the reaction efficiency and selectivity are improved. Higher, but more cumbersome to operate.
In scientific research, the detection of L-sulforaphane is very important. Among them, the agarose sulfate method is one of the classic detection methods. The agarose sulfate method and its steps will be described in detail below.
Experimental principle
The agarose sulfate method is based on the formation of a yellow complex of L-thiogalactopicrin and barium sulfate, a salt formed under acidic conditions, using agarose as a scaffold to separate L- The method of sulforaphane. The method is based on the fact that under acidic conditions, the yellow complex formed by the reaction of barium sulfate and L-sulforaphane is not easily soluble in water and precipitates in agarose, so as to separate and detect L-sulforaphane.
Experimental steps:
Step 1: Prepare the agarose-barium sulfate suspension
Weigh an appropriate amount of agarose and barium sulfate, add a certain amount of distilled water after mixing and stir thoroughly, heat until dissolved or evenly mixed, filter the suspension with a filter with a pore size of 0.22um, and then boil for 3 to 5 minutes. When the suspension cools down to below 50°C but still does not solidify, pour the solution into a Petri dish and place it at room temperature until it solidifies.
Step 2: Sample preparation and processing:
Weigh the sample or standard to be tested, add enough 0.01 M sodium pyrophosphate solution to dissolve, add corresponding volume of chloroform to extract, centrifuge for 10-15 minutes, remove the upper layer liquid, evaporate the chloroform to collect the residue, add 2 mL of water and 2 mL of concentrated hydrochloric acid, extracted again with chloroform, centrifuged, and the upper layer was taken. Take 24mL of the upper liquid, add 0.2 mL of 2% copper nitrate solution and 0.4 mL of 5% lead nitrate solution, extract once with chloroform, centrifuge, and take the upper liquid, which can be used as an experimental sample for testing.
Step 3: Reanalysis experiment:
The samples were added to the agarose-barium sulfate suspension and heated in a water bath to solidify at 80°C for 2 hours. After the sample is solidified, the agarose-barium sulfate suspension is cooled to room temperature, and the agarose-barium sulfate-sample complex is dissolved under the buffer conditions of citric acid and ammonium salt. Finally, rinse with water sequentially, and visually inspect the color of the complex in the agarose sphere.
Step 4: Result analysis:
Measure the absorbance of the filtrate after reanalysis to construct a standard curve, thereby obtaining the content of L-sulforaphane.
The agarose sulfate method is a classic method for separating and detecting L-sulforaphane. Through a series of steps, L-sulforaphane can be separated and its content detected. The method has the advantages of simplicity, high sensitivity, and accurate results, and is commonly used in the detection of L-sulforaphane in food, cosmetics, and pharmaceuticals.
Summary: With the development of science and technology, the method of synthesizing L-thioglutamic acid is being continuously optimized and improved. At the same time, the biological fermentation method and the chemical synthesis method also have their own advantages and disadvantages, and which method to use depends on the actual situation.