CN117603158A - Preparation method of D-fluorescein and D-fluorescein potassium salt - Google Patents
Preparation method of D-fluorescein and D-fluorescein potassium salt Download PDFInfo
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- CN117603158A CN117603158A CN202311631359.4A CN202311631359A CN117603158A CN 117603158 A CN117603158 A CN 117603158A CN 202311631359 A CN202311631359 A CN 202311631359A CN 117603158 A CN117603158 A CN 117603158A
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- fluorescein
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- mixed solvent
- hydroxybenzothiazole
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- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 title claims abstract description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 126
- 239000012046 mixed solvent Substances 0.000 claims abstract description 49
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 48
- SQAVNBZDECKYOT-UHFFFAOYSA-N 6-hydroxy-1,3-benzothiazole-2-carbonitrile Chemical compound OC1=CC=C2N=C(C#N)SC2=C1 SQAVNBZDECKYOT-UHFFFAOYSA-N 0.000 claims abstract description 38
- QIJRTFXNRTXDIP-YBBRRFGFSA-N (2s)-2-amino-3-sulfanylpropanoic acid;hydrate;hydrochloride Chemical compound O.Cl.SC[C@@H](N)C(O)=O QIJRTFXNRTXDIP-YBBRRFGFSA-N 0.000 claims abstract description 32
- 239000003960 organic solvent Substances 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 21
- 239000002994 raw material Substances 0.000 claims abstract description 15
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 60
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 claims description 31
- 239000002904 solvent Substances 0.000 claims description 9
- 238000004519 manufacturing process Methods 0.000 claims description 8
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 abstract description 24
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 21
- 230000000052 comparative effect Effects 0.000 description 21
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 14
- 239000002024 ethyl acetate extract Substances 0.000 description 14
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 14
- 238000000605 extraction Methods 0.000 description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 12
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 8
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 8
- 238000003756 stirring Methods 0.000 description 8
- 238000001035 drying Methods 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- 229910000027 potassium carbonate Inorganic materials 0.000 description 7
- 239000007787 solid Substances 0.000 description 7
- 239000007788 liquid Substances 0.000 description 6
- 239000007810 chemical reaction solvent Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 108060001084 Luciferase Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
- C07D277/62—Benzothiazoles
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
Abstract
The invention particularly discloses a preparation method of D-fluorescein and D-fluorescein potassium salt. The preparation method of the D-fluorescein takes 2-cyano-6-hydroxybenzothiazole and D-cysteine hydrochloride hydrate as raw materials for reaction preparation, and the reaction is prepared by the reaction in a mixed solvent consisting of a water-soluble organic solvent and water. The invention provides a brand-new preparation method of D-fluorescein; the invention takes 2-cyano-6-hydroxy benzothiazole and D-cysteine hydrochloride hydrate as raw materials to react in a mixed solvent composed of methanol and water to prepare D-fluorescein; the actual yield of the D-fluorescein prepared by the method is more than 80%; compared with the D-fluorescein prepared by taking 2-cyano-6-hydroxybenzothiazole and D-cysteine hydrochloride hydrate as raw materials and reacting in a water-insoluble organic solvent (dichloromethane) described in Chinese patent No. CN102532053A, the actual yield of the D-fluorescein can be remarkably improved.
Description
Technical Field
The invention relates to the technical field of compound preparation, in particular to a preparation method of D-fluorescein and D-fluorescein potassium salt.
Background
D-luciferin is a commonly used substrate for luciferases, and is applied to the biotechnology field, in particular to in vivo imaging technology. Chinese patent No. CN102532053a discloses a method for preparing and purifying high purity D-fluorescein; the method comprises the steps of firstly, reacting 2-cyano-6-hydroxybenzothiazole with D-cysteine hydrochloride hydrate in a non-water-soluble organic solvent (dichloromethane) with certain polarity to prepare a crude product of D-fluorescein, and then, carrying out purification steps such as extraction and the like to obtain high-purity D-fluorescein. The method can obtain D-fluorescein with purity of more than 90 percent; however, the actual yield of the D-fluorescein prepared by the method is only 54.6-79.33 percent (see the description of the 75 th section of the specification in Table 5), and the actual yield does not break through 80 percent; further improvements are needed.
Disclosure of Invention
In order to overcome at least one technical problem existing in the prior art, the invention provides a preparation method of D-fluorescein.
The preparation method of the D-fluorescein takes 2-cyano-6-hydroxybenzothiazole and D-cysteine hydrochloride hydrate as raw materials for reaction, and the reaction is prepared by the reaction in a mixed solvent consisting of a water-soluble organic solvent and water.
Preferably, the water-soluble organic solvent is methanol.
The inventors have surprisingly found in a number of experiments that in the process of the present invention for preparing D-luciferin starting from 2-cyano-6-hydroxybenzothiazole and D-cysteine hydrochloride hydrate; the composition of the reaction solvent has an important influence on the actual yield of D-luciferin. The inventor finds in the research that the actual yield of D-fluorescein can be remarkably improved by taking 2-cyano-6-hydroxybenzothiazole and D-cysteine hydrochloride hydrate as raw materials to react in a mixed solvent consisting of methanol and water compared with the reaction in a water-insoluble organic solvent (dichloromethane) described in Chinese patent No. CN 102532053A.
Further preferably, the mixed solvent consists of methanol and water; wherein the volume ratio of the methanol to the water is 2-3:1.
Preferably, the weight ratio of the 2-cyano-6-hydroxybenzothiazole to the D-cysteine hydrochloride hydrate is 1:1-1.6.
Most preferably, the weight ratio of 2-cyano-6-hydroxybenzothiazole to D-cysteine hydrochloride hydrate is 1:1.05.
Preferably, the dosage ratio of the mixed solvent to the 2-cyano-6-hydroxybenzothiazole is 30-50 mL:1g based on the metering of the 2-cyano-6-hydroxybenzothiazole.
Preferably, the ratio of the mixed solvent to the 2-cyano-6-hydroxybenzothiazole is 36mL to 1g based on the metering of the 2-cyano-6-hydroxybenzothiazole.
Preferably, acetonitrile is also added to the mixed solvent; the mixed solvent consists of methanol, acetonitrile and water.
Preferably, the volume ratio of methanol, acetonitrile and water is 2-3:1-2:1.
Most preferably, the volume ratio of methanol, acetonitrile and water is 2:1:1.
The inventors have surprisingly found in further studies that the actual yield of D-fluorescein can be further significantly improved by using 2-cyano-6-hydroxybenzothiazole and D-cysteine hydrochloride hydrate as starting materials to react in a mixed solvent consisting of methanol, acetonitrile and water, as compared with the mixed solvent consisting of methanol and water.
Preferably, the preparation method of the D-fluorescein specifically comprises the following steps: adding 2-cyano-6-hydroxybenzothiazole and D-cysteine hydrochloride hydrate into a mixed solvent consisting of a water-soluble organic solvent and water, uniformly stirring, then introducing nitrogen, and then adding potassium carbonate for reaction; removing the organic solvent after the reaction is finished, adding water, stirring uniformly, adjusting the pH value to 2-3, and extracting with ethyl acetate to obtain ethyl acetate extract; concentrating and drying the ethyl acetate extract to obtain a solid, namely the D-fluorescein.
The D-cysteine hydrochloride hydrate as referred to in the present invention means D-cysteine hydrochloride monohydrate unless otherwise specified.
The invention also provides a preparation method of the D-fluorescein potassium salt, which comprises the preparation method of the D-fluorescein, and further comprises the following steps:
dissolving D-fluorescein in a solvent, then adding KOH solution for reaction, and obtaining a product to obtain the D-fluorescein potassium salt.
The reaction of the present invention with KOH using D-luciferin as a raw material may be carried out under the conventional reaction conditions of the present invention.
The beneficial effects are that: the invention provides a brand-new preparation method of D-fluorescein; the invention takes 2-cyano-6-hydroxy benzothiazole and D-cysteine hydrochloride hydrate as raw materials to react in a mixed solvent composed of methanol and water to prepare D-fluorescein; the actual yield of the D-fluorescein prepared by the method is more than 80%; compared with the D-fluorescein prepared by taking 2-cyano-6-hydroxybenzothiazole and D-cysteine hydrochloride hydrate as raw materials and reacting in a water-insoluble organic solvent (dichloromethane) described in Chinese patent No. CN102532053A, the actual yield of the D-fluorescein can be remarkably improved.
In addition, the D-fluorescein prepared by taking the 2-cyano-6-hydroxybenzothiazole and the D-cysteine hydrochloride hydrate as raw materials has higher yield; thus, on the basis thereof, the D-potassium fluorescein salt can be further prepared by a conventional method; can ensure that the preparation of the D-potassium fluorescein salt by taking the 2-cyano-6-hydroxybenzothiazole and the D-cysteine hydrochloride hydrate as the initial raw materials has higher yield.
Detailed Description
The present invention is further explained below with reference to specific examples, which are not intended to limit the present invention in any way.
Example 1 preparation of D-luciferin
(1) 1g of 2-cyano-6-hydroxybenzothiazole and 1.05g of D-cysteine hydrochloride hydrate are added into 36mL of mixed solvent consisting of methanol and water and stirred uniformly; wherein the volume ratio of the methanol to the water in the mixed solvent is 2:1;
(2) Then nitrogen is introduced, and 0.8g of potassium carbonate is added to react for 30min at room temperature;
(3) Removing the organic solvent after the reaction is finished, adding 100mL of water, stirring uniformly, adjusting the pH value to 3 by using 1M hydrochloric acid, and extracting by using 100mL of ethyl acetate to obtain ethyl acetate extract; repeating the extraction for 4 times, combining ethyl acetate extract liquid obtained after the extraction for 4 times, concentrating and drying to obtain solid, namely the D-fluorescein.
Example 2 preparation of D-luciferin
(1) 1g of 2-cyano-6-hydroxybenzothiazole and 1.05g of D-cysteine hydrochloride hydrate are added into 36mL of mixed solvent consisting of methanol, acetonitrile and water, and stirred uniformly; wherein the volume ratio of the methanol to the acetonitrile to the water in the mixed solvent is 2:1:1;
(2) Then nitrogen is introduced, and 0.8g of potassium carbonate is added to react for 30min at room temperature;
(3) Removing the organic solvent after the reaction is finished, adding 100mL of water, stirring uniformly, adjusting the pH value to 3 by using 1M hydrochloric acid, and extracting by using 100mL of ethyl acetate to obtain ethyl acetate extract; repeating the extraction for 4 times, combining ethyl acetate extract liquid obtained after the extraction for 4 times, concentrating and drying to obtain solid, namely the D-fluorescein.
Example 2 differs from example 1 in that the solvent used for the reaction in step (1) is different; example 2 a mixed solvent consisting of methanol, acetonitrile and water was used; whereas in example 1, a mixed solvent composed of methanol and water was used.
Process for preparing comparative example 1D-fluorescein
(1) 1g of 2-cyano-6-hydroxybenzothiazole and 1.05g of D-cysteine hydrochloride hydrate are added into 36mL of dichloromethane and stirred uniformly;
(2) Then nitrogen is introduced, and 0.8g of potassium carbonate is added to react for 30min at room temperature;
(3) Removing the organic solvent after the reaction is finished, adding 100mL of water, stirring uniformly, adjusting the pH value to 3 by using 1M hydrochloric acid, and extracting by using 100mL of ethyl acetate to obtain ethyl acetate extract; repeating the extraction for 4 times, combining ethyl acetate extract liquid obtained after the extraction for 4 times, concentrating and drying to obtain solid, namely the D-fluorescein.
Comparative example 1 differs from example 1 in that the solvent used for the reaction in step (1) is different; comparative example 1 using a solvent which is a non-water-soluble organic solvent methylene chloride; whereas in example 1, a mixed solvent composed of methanol and water was used.
Comparative example 2D-fluorescein preparation method
(1) 1g of 2-cyano-6-hydroxybenzothiazole and 1.05g of D-cysteine hydrochloride hydrate are added into 36mL of methanol and stirred uniformly;
(2) Then nitrogen is introduced, and 0.8g of potassium carbonate is added to react for 30min at room temperature;
(3) Removing the organic solvent after the reaction is finished, adding 100mL of water, stirring uniformly, adjusting the pH value to 3 by using 1M hydrochloric acid, and extracting by using 100mL of ethyl acetate to obtain ethyl acetate extract; repeating the extraction for 4 times, combining ethyl acetate extract liquid obtained after the extraction for 4 times, concentrating and drying to obtain solid, namely the D-fluorescein.
Comparative example 1 differs from example 1 in that the solvent used for the reaction in step (1) is different; comparative example 2 the solvent used was methanol; whereas in example 1, a mixed solvent composed of methanol and water was used.
Method for preparing comparative example 3D-fluorescein
(1) 1g of 2-cyano-6-hydroxybenzothiazole and 1.05g of D-cysteine hydrochloride hydrate are added into 36mL of mixed solvent consisting of methanol, dimethyl sulfoxide and water, and the mixed solvent is stirred uniformly; wherein the volume ratio of the methanol to the dimethyl sulfoxide to the water in the mixed solvent is 2:1:1;
(2) Then nitrogen is introduced, and 0.8g of potassium carbonate is added to react for 30min at room temperature;
(3) Removing the organic solvent after the reaction is finished, adding 100mL of water, stirring uniformly, adjusting the pH value to 3 by using 1M hydrochloric acid, and extracting by using 100mL of ethyl acetate to obtain ethyl acetate extract; repeating the extraction for 4 times, combining ethyl acetate extract liquid obtained after the extraction for 4 times, concentrating and drying to obtain solid, namely the D-fluorescein.
Comparative example 3 differs from example 2 in that the solvent used for the reaction in step (1) is different; comparative example 3 compared with example 1, dimethyl sulfoxide was added to the mixed solvent, and a mixed solvent composed of methanol, dimethyl sulfoxide and water was used; whereas example 2 was compared with example 1 in which acetonitrile was added to the mixed solvent, a mixed solvent composed of methanol, acetonitrile and water was used.
Method for preparing comparative example 4D-fluorescein
(1) 1g of 2-cyano-6-hydroxybenzothiazole and 1.05g of D-cysteine hydrochloride hydrate are added into 36mL of mixed solvent consisting of methanol, acetone and water, and stirred uniformly; wherein the volume ratio of the methanol to the acetone to the water in the mixed solvent is 2:1:1;
(2) Then nitrogen is introduced, and 0.8g of potassium carbonate is added to react for 30min at room temperature;
(3) Removing the organic solvent after the reaction is finished, adding 100mL of water, stirring uniformly, adjusting the pH value to 3 by using 1M hydrochloric acid, and extracting by using 100mL of ethyl acetate to obtain ethyl acetate extract; repeating the extraction for 4 times, combining ethyl acetate extract liquid obtained after the extraction for 4 times, concentrating and drying to obtain solid, namely the D-fluorescein.
Comparative example 4 differs from example 2 in that the solvent used for the reaction in step (1) is different; comparative example 4 in comparison with example 1, acetone was added to the mixed solvent, and a mixed solvent composed of methanol, acetone and water was used; whereas example 2 was compared with example 1 in which acetonitrile was added to the mixed solvent, a mixed solvent composed of methanol, acetonitrile and water was used.
The actual yield results of D-luciferin prepared by the preparation methods described in examples 1 to 2 and comparative examples 1 to 4 are shown in Table 1.
TABLE 1 actual yields of D-luciferin prepared by the methods
| Actual yield of D-luciferin | |
| Example 1 preparation of D-luciferin | 82.8％ |
| Example 2 preparation of D-luciferin | 93.1％ |
| Process for preparing comparative example 1D-fluorescein | 70.7％ |
| Comparative example 2D-fluorescein preparation method | 54.5％ |
| Method for preparing comparative example 3D-fluorescein | 74.3％ |
| Method for preparing comparative example 4D-fluorescein | 80.1％ |
As can be seen from the data in Table 1, the actual yield of D-luciferin obtained by the method for preparing D-luciferin of example 1 reaches 82.8%; significantly higher than the preparation method of comparative example 1D-fluorescein; this illustrates: in the process of preparing D-fluorescein by taking 2-cyano-6-hydroxybenzothiazole and D-cysteine hydrochloride hydrate as raw materials; the composition of the reaction solvent has an important influence on the actual yield of D-luciferin. The experimental result shows that the reaction is carried out in a mixed solvent consisting of methanol and water by taking the 2-cyano-6-hydroxybenzothiazole and the D-cysteine hydrochloride hydrate as raw materials, and compared with the reaction carried out in a water-insoluble organic solvent (dichloromethane), the actual yield of the D-fluorescein can be obviously improved.
As can be seen from the data of table 1, the actual yield of D-luciferin prepared by the preparation method of comparative example 2D-luciferin was only 54.5; is significantly lower than the preparation method of the D-fluorescein of comparative example 1. This illustrates: in the process of preparing D-fluorescein by taking 2-cyano-6-hydroxybenzothiazole and D-cysteine hydrochloride hydrate as raw materials; the composition of the reaction solvent has an important influence on the actual yield of D-luciferin. 2-cyano-6-hydroxybenzothiazole and D-cysteine hydrochloride hydrate are taken as raw materials to react in water-soluble organic solvent methanol only, and compared with the reaction in non-water-soluble organic solvent (dichloromethane), the actual yield of D-fluorescein cannot be improved; the actual yield of D-luciferin can be significantly improved only by carrying out the reaction in a mixed solvent composed of methanol and water.
As can be seen from the data in table 1, the actual yield of D-luciferin obtained by the method for preparing D-luciferin of example 2 reached 93.1%, which is significantly higher than that of D-luciferin of example 1; this illustrates: acetonitrile is added into the mixed solvent, and 2-cyano-6-hydroxybenzothiazole and D-cysteine hydrochloride hydrate are reacted in the mixed solvent consisting of methanol, acetonitrile and water, so that the actual yield of D-fluorescein can be further remarkably improved compared with the mixed solvent consisting of methanol and water.
As can be seen from the data in Table 1, the actual yields of D-luciferin obtained by the methods for producing D-luciferin of comparative examples 3 and 4 were not improved as compared with those of example 1; this illustrates:
only acetonitrile is further added into the mixed solvent, and only 2-cyano-6-hydroxy benzothiazole and D-cysteine hydrochloride hydrate are reacted in the mixed solvent consisting of methanol, acetonitrile and water, so that the actual yield of D-fluorescein can be further and obviously improved compared with the mixed solvent consisting of methanol and water; however, the addition of further organic solvents to the mixed solvent does not significantly further increase the actual yield of D-luciferin.
Claims (10)
1. The preparation method of the D-fluorescein takes 2-cyano-6-hydroxybenzothiazole and D-cysteine hydrochloride hydrate as raw materials for reaction preparation, and is characterized in that the reaction is prepared by the reaction in a mixed solvent consisting of a water-soluble organic solvent and water.
2. The method for producing D-luciferin according to claim 1, wherein the water-soluble organic solvent is methanol.
3. The method for producing D-luciferin according to claim 2, wherein the mixed solvent is composed of methanol and water; wherein the volume ratio of the methanol to the water is 2-3:1.
4. The method for producing D-luciferin according to claim 1, wherein the weight ratio of 2-cyano-6-hydroxybenzothiazole to D-cysteine hydrochloride hydrate is 1:1 to 1.6.
5. The method for preparing D-fluorescein according to claim 1, wherein the weight ratio of 2-cyano-6-hydroxybenzothiazole to D-cysteine hydrochloride hydrate is 1:1.05.
6. The method for producing D-luciferin according to claim 1, wherein the ratio of the mixed solvent to 2-cyano-6-hydroxybenzothiazole is 30 to 50 mL/1 g based on the amount of 2-cyano-6-hydroxybenzothiazole.
7. The method for producing D-luciferin according to claim 1, wherein the ratio of the mixed solvent to 2-cyano-6-hydroxybenzothiazole is 36 mL/1 g based on the amount of 2-cyano-6-hydroxybenzothiazole.
8. The method for producing D-luciferin according to claim 3, wherein acetonitrile is further added to the mixed solvent; the mixed solvent consists of methanol, acetonitrile and water.
9. The method for preparing D-fluorescein according to claim 8, wherein the volume ratio of methanol, acetonitrile and water is 2-3:1-2:1;
most preferably, the volume ratio of methanol, acetonitrile and water is 2:1:1.
10. A process for producing a potassium salt of D-luciferin, comprising the process for producing D-luciferin as claimed in any one of claims 1 to 9, further comprising the steps of:
dissolving D-fluorescein in a solvent, then adding KOH solution for reaction, and obtaining a product to obtain the D-fluorescein potassium salt.
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| CN202311631359.4A CN117603158A (en) | 2023-12-01 | 2023-12-01 | Preparation method of D-fluorescein and D-fluorescein potassium salt |
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