CN100344634C - Chemical synthetic process of D-fluorescein and device therefor - Google Patents
Chemical synthetic process of D-fluorescein and device therefor Download PDFInfo
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- CN100344634C CN100344634C CNB2004100517052A CN200410051705A CN100344634C CN 100344634 C CN100344634 C CN 100344634C CN B2004100517052 A CNB2004100517052 A CN B2004100517052A CN 200410051705 A CN200410051705 A CN 200410051705A CN 100344634 C CN100344634 C CN 100344634C
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Abstract
The present invention relates to a method and a synthesizing device for chemically synthesizing D-fluorescein, which belongs to the field of chemical synthesis. A D-fluorescein product is obtained by the method that high-purity 2-cyano-6-methoxybenzothiazole and pyridine hydrochlride are demethylated at high temperature, and then, reaction products in the demethylating reaction are extracted; next, chromatography and purification are carried out, and finally, a condensation reaction for the purified demethylated products and D-cysteine is carried out so as to obtain the D-fluorescein product. The present invention provides a method for synthesizing D-fluorescein under a medium scale condition, which has the advantage of simple operation, and a reaction device thereof. The defects of the existing D-fluorescein of complicated reaction, high requirement for reaction conditions and reaction apparatuses and difficult control are overcome. The yield of D-fluorescein is more than 60%, and purity is more than 50%; a luminous coefficient is higher than that of similar products in China, which reaches the level of SigmaL-9504 products, but prices are largely lower than that of D-fluorescein products in Sigma company.
Description
[affiliated technical field]
The invention belongs to the field of chemical synthesis, relate to the chemical synthesis process and the reaction unit of D-fluorescein.
[background technology]
The D-fluorescein is the substrate of the bioluminescence reaction of dependency ATP, and noctilcent principle is that luciferase is at Mg
2++, O
2Under the condition that exists with ATP, catalysis D-fluorescein forms the oxidized fluorescence element, sends an enzyme reaction process of photon in reaction process, and reaction formula is expressed as:
Because of the ATP in the reaction is all biological energy derives, under certain condition, the ATP amount of sending in light intensity and the reactive system is directly proportional, therefore biloluminescence method can be widely used in the detection of biomaterial, detection by quantitative and the sanitary condition assessment in the health detection and the rapid detection of microbe population as ultramicron in the medical jurisprudence, in addition since luciferase gene as a reporter gene, on molecular biology, be widely used, therefore, the application of luciferase-luciferin bioluminescence has very big market, the D-fluorescein is as a kind of substrate of bioluminescence reaction, and along with the increase that the noclilucence method is used, its demand is increasing.Because of the D-fluorescein is a kind of steric isomer, oxidation is untwisted and is seen photolysis easily in some solvents, and international and domestic product is the price height not only, and delivery date is long, and home products content is low, and luminous coefficient is not high.
(ρ-anisidine) synthetic by eight footworks, total yield was 5.8% with methyl oxyaniline in 1963 by people such as White at first in the chemosynthesis of D-fluorescein.Seto in 1963 also uses methyl oxyaniline, and (ρ-anisidine) synthesized the D-fluorescein by five-step approach, total yield are 6.5%.People such as White had synthesized the D-fluorescein from 2-amino-6-methoxyl group-benzothiazole by four step rule afterwards, yield reaches 13.9%, but this method in 1978 by people such as Seto prove a kind of very easily and the method for mass preparation D-fluorescein, its yield has kept the level of lab scale substantially, this method is a kind of carboxylic reagent of preparation earlier, in fact is total up to for five steps.1992, the people such as Yoshiaki Toya of Japan have synthesized the D-fluorescein from 2-amino-6-methoxyl group-benzothiazole by three-step approach, total yield reaches 36%, 1993, by the synthetic D-fluorescein of the four step rule of PEG hydrotropy, total yield is 49% to the people such as Nobutaka Suzuki of Japan from 2-amino-6-methoxyl group-benzothiazole.
In documents and materials, about the newer method of the chemosynthesis approach of D-fluorescein all is to be raw material with 2-amino-6-methoxyl group-benzothiazole, behind the cyano group demethylation, form by carrying out condensation with the D-halfcystine, about cyanogenation, in people's such as YoshiakiToya the synthetic method, cuprous and isoAmNO (nitrosification isopentyl ester) synthesizes a kind of nitrosyl mixture (CuCl with anhydrous chlorides of rase
2.NO), this material is acetonitrile (CH
3CN)-the Sandmyer response service of 2-aminobenzothiazole in polyoxyethylene glycol (PEG) 200 mixtures.The 2-aminobenzothiazole is not exclusively dissolving in acetonitrile solution, and the adding of PEG can make it dissolve fully.Acetonitrile (CH
3CN)-polyoxyethylene glycol (PEG) 200 obtained best result, when using CuBr
2Replaced C uCl
2The time, bromination reaction temperature (110 ℃) during than chlorination reaction (140 ℃) low, the highest yield of single step of chlorination-cyanogenation is 79%, and the highest yield of single step of bromination-cyanogenation is 70%.
For demethylating reaction, available pyridine hydrochloride method is carried out, initial method is the hydrochloric acid gas that feeds new system in pyridine solution, and adding 2-cyano group-6-methoxyl group benzo thiazole carries out, and newer method is that 2-cyano group-6-methoxyl group benzo thiazole and pyridine hydrochloride reaction 45min in the sealed tube of the logical nitrogen of high temperature (200 ℃) carry out relatively.
The reaction path complexity of above-mentioned preparation D-fluorescein requires severe reaction conditions, and conversion unit requires high, and is difficult to control, and the total yield of D-fluorescein is not high.
[summary of the invention]
At the existing problem of reaction of present preparation D-fluorescein, the purpose of this invention is to provide a kind of easy and simple to handle, can be high under medium scale condition yield ground synthesize the method and the reaction unit of D-fluorescein.
The present invention can be implemented under the comparatively easy condition efficient synthetic D-fluorescein, and luminous coefficient is higher than home products, reach the level of the L-9504 of Sigma company fluorescein product, and price is significantly less than the fluorescein product of Sigma company.
The present invention carries out according to the following steps:
1. the easy reaction unit of D-fluorescein chemosynthesis
This reaction unit comprises that one places the there-necked flask on the high temperature oil bath heating unit, there-necked flask one side openings is connected to and vacuumizes and the pipeline of logical nitrogen, another side openings sealing, intermediate openings connects a prolong, and the waste gas outlet of prolong is connected with a waste gas resorber.
2. the synthetic method of D-fluorescein
With 2-cyano group-6-methylbenzothiazole (2-Cyano-6-methoxybenzothiazoe) (C
9H
6N
2OS is after FW=190.2) (99%) and pyridine hydrochloride (Pyridine hydrochloride) (98%) at high temperature react demethylation, behind extraction and column chromatography purification, again with hydration D-halfcystine (D-Cystine.HCl.H
2O) in alkaline mixed solvent, be condensed into D-fluorescein (C
11H
8N
2O
3S
2, FW=280.3), acidifying can be synthesized available D-fluorescein by centrifugal vacuum-drying after extracting, and total yield reaches more than 40%.
Concrete steps are as follows:
2.1 demethylating reaction
The reaction starting material of D-fluorescein are 2-cyano group-6 methoxyl group benzo thiazole (99%, NEW JERSEY, USA1-800-ACROS-01) pyridine hydrochloride (98%, ACROS ORGANICS), reaction ratio is 2-cyano group-6 a methoxyl group benzo thiazole: pyridine hydrochloride=1: (1～3), temperature of reaction is 180～250 ℃, and environment is for vacuumizing logical nitrogen, and the reaction times is 0.5～3h.
2.2 the extraction of demethylation target product
Product behind the demethylating reaction is with 1: mixture is three times behind the ethyl acetate of (1～3) and the frozen water washing reaction, and each consumption is about raw-material 50～100 times of reaction.By carrying out concentrate drying with rotation vacuum-evaporation instrument behind the extraction organic layer.
2.3 the purifying of demethylation product
Target product purifying behind the demethylation is to carry out column chromatography with 80～400 purpose silochroms, but the adding pressure type chromatography purification post of the most handy band threeway of chromatography column.With 5: (1～3) chloroform/ethyl acetate is done eluting solvent, being that 25mm is high to diameter is the chromatography column of 250mm, and elution flow rate is 1～10ml/min, detects by HTLC (efficient thin-layer chromatography), collect the higher part of target product purity, be rotated the vacuum-evaporation concentrate drying after merging.
2.4 the condensation reaction of demethylation product behind the purifying and D-halfcystine
Demethylation product behind the purifying and D-halfcystine hydrate in alkaline mixed solvent under room temperature lucifuge reaction 10～30min, transfer pH to 1～6 with hydrochloric acid, by centrifugal 10～20min. collecting precipitation under 1000～15000r/min room temperature condition, and this is deposited under the vacuum condition dry, get final product to such an extent that purity is 40～60% D-fluorescein product.
2.5 the analysis of components of synthetic D-fluorescein product
Method with gas chromatography mass spectrometry is carried out analysis of components.
2.6 the luminescence analysis of synthetic D-fluorescein product
Use 50mmol/L, pH is the solution that 7.8 glycylglycine damping fluid is made into 0.5mg/mL, and after vacuumizing logical nitrogen,-20 ℃ of cryopreservation under the lucifuge condition, time spent thaws and afterwards add the luciferin solution of 60 μ L0.5mg/mL in luminescent system, places the noclilucence instrument to detect luminous value immediately.
[embodiment]
Embodiment 1: about the easy synthesizer of D-fluorescein
This reaction unit is made up of a temperature control unit and a high temperature oil bath heating system of can be in a big way comparatively accurately regulating (0～300 ℃), there-necked flask 2 reactive systems that vacuumize logical nitrogen gas regulation system and band prolong 4 and exhaust gas absorption device.The entire reaction device is except that nitrogen supply bottle, vacuum extractor and temperature control device, and all the other all place stink cupboard.
As shown in Figure 1, high temperature oil bath system wherein can be used on adjustable electric furnace 1 (500W) and goes up with Stainless Steel Kettle 6 Sheng Viscotrol C (about 2000mL), by temperature sensor 3 meticulous attemperation; Left side opening with there-necked flask 2 (500mL) vacuumizes logical nitrogen, and intermediate openings is used for application of sample and refrigeration cycle, and the right opening is used for sampling and detects (TLC detection); Refrigeration cycle gets final product with general water condensing tube 4, and waste gas absorbs available exsiccant calcium chloride 5 to carry out.
Embodiment 2: about the synthetic method of D-fluorescein
1. demethylating reaction
Take by weighing 940mg 2-cyano group-6-methoxyl group benzo thiazole (2-Cyano-6-methoxybenzothiazoe) (99%) and 1000～3060mg pyridine hydrochloride (Pyridine hydrochloride) (98%) is put into small test tube, vacuumize that put into behind the logical nitrogen can thermoregulated high temperature oil bath reaction flask, 190～220 ℃ the insulation 1～3 hour after, stop the termination reaction of heating, carry out the extraction of demethylation target product after cooling.
2. the extraction of demethylation target product
Add in the above-mentioned reaction tubes with 200mL～400mL frozen water, and adding 100mL～200mL ethyl acetate, shake even after, extract organic layer with separating funnel, water layer carries out extracted twice with 100mL～200mL ethyl acetate again, with 200mL～400mL frozen water washing organic layer once, behind the merging organic layer, by the dry 1480mg yellowish red color material that gets of rotation vacuum-evaporation instrument.
3. the purifying of demethylation product
At diameter is but that the 20mm height adds 80～120 purpose silochroms in organic pressurizing chromatographic column of 350mm, make the purifying chromatography column, above-mentioned dried yellowish red color demethylation product (810mg) is mixed the top that a small amount of silica gel places chromatography column, use chloroform: ethyl acetate is that 5: 1～3 mixed solvent is as eluting solvent, be to collect 50 pipes under the condition of 2.5ml/min at flow velocity, every pipe 10mL, vacuum concentration also uses thin layer chromatography (being called for short TLC) to detect, merge the 9th～14 manage faint yellow material 380mg, on the TLC plate, be single spot, merge other manage yellow substance 409mg, on the TLC plate two spots, both common 789mg, the rate of recovery reaches 97.4%.
4. the condensation reaction of demethylation product behind the purifying and D-halfcystine
Take by weighing (100～160) mg D-Cystine.HCl.H
2O (C
3H
7NO
2S.HCl, FW=175.6) demethylation product and behind the above-mentioned column chromatography purification of 100mg, after vacuumizing logical nitrogen, add 0.5mL～4mL alkaline ethanol solvent (also must vacuumize logical nitrogen) lucifuge reaction at room temperature 10～60min, transfer pH to 1～6 with hydrochloric acid, by centrifugal 10min. collecting precipitation under the 10000r/min room temperature condition, and this vacuum condition that is deposited in lucifuge is dry down, get final product 154.6mg D-fluorescein product, yield is 103.6%.
5. the analysis of components of synthetic D-fluorescein product
With synthetic D-fluorescein product (1
#With 2
#) and Sigma L9504D-fluorescein and Shanghai fluorescein sample carry out the gas chromatography mass spectrometry analysis of components, its result is as follows:
Table 1 synthetic D-fluorescein product gas chromatography mass spectrometry is analyzed
| Sample number into spectrum | ％(mg/mL) | Gas chromatography mass spectrometry method analytical results | ||
| R.T | M | ％of total | ||
| 1 # 2 # | 1.2 2.1 | 6.796 14.414 16.030 6.787 14.420 | 151 234 236+44 151 234 | 5.059 37.618 57.323 3.995 32.017 |
| Sigma L9504 Shanghai fluorescein | 0.95 0.32 | 16.074 6.805 14.412 16.039 6.867 11.122 | 236+44 151 234 236+44 151 208 | 63.988 5.579 40.300 54.121. 3.246 96.754 |
Annotate: R.T represents the retention time of material, and M represents the thing molecular weight, and 236+44 represents that molecular weight is 280 material, and the molecular weight of D-fluorescein is 280.2.
The result shows: M is that the material of 236+44 component is more than Sigma L9504 in the synthetic fluorescein, and the Shanghai fluorescein can not surveyed the material that M is the 236+44 component.
6. the luminescence analysis of synthetic D-fluorescein product
With Sigma L9504D-fluorescein, go up the marine products fluorescein and from synthetic D-fluorescein (1
#With 2
#), be made into the solution of 0.5mg/mL with 50mmol/LGB (glycylglycine damping fluid), and after vacuumizing logical nitrogen,-20 ℃ of cryopreservation under the lucifuge condition, add 60 μ l luciferin solution in luminescent system, and detect luminous value in the noclilucence instrument, it the results are shown in Table 2.
Table 2 synthesizes the luminous vitality test of D-uranine
| The fluorescein sample | Consumption (μ l) | CP6S | CPM | The fluorescein sample | Consumption (μ l) | CP6S | CPM |
| 1 # | 60 100 150 200 300 | 15755 21102 19989 22032 23571 | 157460 206714 194327 212902 227242 | *1 # | 60 80 150 | 12938 14095 21032 | 127585 139176 206788 |
| Shanghai fluorescein (0.5mg/ml) | 60 80 100 120 140 150 160 180 200 300 400 60 | 4353 4468 4943 5358 6042 4265/4846 * 5267/4929 4388 4833 * 4060 * 4671 * 12878 | 40054 40272 44465 48185 52313 36913/41851 * 46404/42678 38041 43586 35928 42131 130406 | 2 # | 60 80 100 120 140 160 180 200 300 | 12213 17678 19585 17496 18415 21817 20327 21820 22831 | 127063 173356 190838 171181 180824 213429 197409 213147 221202 |
| Sigma fluorescein L9504 | 60 80 150 | 12878 14149 24494 | 130406 141882 2746938 |
Annotate: data are in the table
*1
#For in-20 ℃ place 2 days 1
#, again and the simultaneously-measured luminous value of Sigma L9504 D-fluorescein of newly joining.
The result shows: the luminous vigor from synthetic D-uranine is luminous more many than energetic than last marine products uranine, compares with Sigma L9504 D-fluorescein, and is more better or be not less than Sigma L9504 D-fluorescein at least than vigor.
Embodiment 3: about the synthetic method of D-fluorescein
1. methyl reaction (2002,6/17)
Take by weighing 949mg 2-cyano group-6-methoxyl group benzo thiazole (2-Cyano-6-methoxybenzothiazoe) (99%) and 1000mg pyridine hydrochloride (Pyridine hydrochloride) (98%) is put into small test tube, vacuumize that put into behind the logical nitrogen can thermoregulated high temperature oil bath reaction flask, 200 ℃ the insulation 1 hour after, stop the termination reaction of heating, carry out the extraction of demethylation target product after cooling.
2. the extraction of methyl target product
Add in the above-mentioned reaction tubes with the 200mL frozen water, and adding 100mL ethyl acetate, shake even after, extract organic layer with separating funnel, water layer carries out extracted twice with the 100mL ethyl acetate again, with 200mL frozen water washing organic layer once, behind the merging organic layer, by the dry 776mg yellowish red color material that gets of rotation vacuum-evaporation instrument.
3. the purifying of demethylation product
At diameter is but that the 25mm height adds 200～300 purpose silochroms in organic pressurizing chromatographic column of 25cm, make the purifying chromatography column, above-mentioned dried yellowish red color demethylation product (739mg) is mixed the top that a small amount of silica gel places chromatography column, use chloroform: ethyl acetate is that 5: 1 mixed solvent is as eluting solvent, be to collect 30 pipes under the condition of 1.0ml/min at flow velocity, every pipe 10mL, vacuum concentration also uses thin layer chromatography (being called for short TLC) to detect, merge 13-14 manage faint yellow material 199mg, on the TLC plate, be single spot, get 497mg after the 15th～22 pipe material drying, both common 696mg, the rate of recovery reaches 94.2%.
4. the condensation reaction and the extraction of demethylation product behind the purifying and D-halfcystine
Take by weighing 100mg D-Cystine.HCl.H
2Demethylation product behind the above-mentioned column chromatography purification of O and 120mg, after vacuumizing logical nitrogen, add 4.0mL alkaline methanol solvent (also must vacuumize logical nitrogen) lucifuge reaction at room temperature 10min, transfer pH to 1.0 with hydrochloric acid, by centrifugal 10min. collecting precipitation under the 10000r/min room temperature condition, and this vacuum condition that is deposited in lucifuge is down dry, get final product 165.7mg D-fluorescein product, net yield is that 104.1% of theoretical value (is called for short 3
#).
5. the data analysis of synthetic D-fluorescein product
With Sigma L9504 D-fluorescein, last marine products fluorescein and synthetic D-fluorescein, be made into the solution of 0.5mg/mL with 50mmol/LGB (glycylglycine damping fluid), and after vacuumizing logical nitrogen,-20 ℃ of cryopreservation under the lucifuge condition, in luminescent system, add 60 μ l luciferin solution, and in the noclilucence instrument, detecting luminous value, it the results are shown in Table 3 in addition the gas chromatography mass spectrometry analysis of components to be carried out in Sigma L9504 D-fluorescein, last marine products fluorescein and the sampling of synthetic D-fluorescein.
Table 3 synthesizes the luminous vitality test of D-fluorescein
| Title | The mass spectrum result | Luminescence analysis result (CP6S) | |||
| Peak# | R.T(min) | M | ％of total | ||
| 3 # | 1 2 3 4 | 10.543 14.636 15.129 16.600 | 176 250 280 236(+44) | 2.694 20.338 37.702 39.267 | 14823 |
| The Shanghai fluorescein | 1 2 | 6.867 11.122 | 151 208 | 3.246 96.754 | 6798 |
| Sigma fluorescein L9504 | 1 2 3 | 6.805 14.412 16.039 | 151 234 236(+44) | 5.579 40.300 54.121. | 12878 |
Annotate: R.T represents the retention time of material, and M represents the thing molecular weight, and 280 and 236 (+44) expression molecular weight is 280 maybe may (because of at high temperature, 280 material can become 236 material, and emitting molecular weight simultaneously is 44 (CO for 280 material
2) material), both all can be the D-fluorescein.
The result shows: the luminous vigor from synthetic D-fluorescein is luminous more many than energetic than last marine products fluorescein, compare with SigmaL9504 D-fluorescein, luminous more better or be lower than the luminous vigor of Sigma L9504 D-fluorescein slightly than vigor, this is relevant with reaction and extraction conditions.
Claims (8)
1, the method for a kind of D-fluorescein chemosynthesis, at first it carries out the high temperature demethylating reaction by 2-cyano group-6-methoxyl group benzo thiazole and pyridine hydrochloride, extract the demethylating reaction product then, through column chromatography purification, demethylation product and D-halfcystine with purifying carries out can obtaining D-fluorescein product after the condensation reaction at last, the ratio that it is characterized in that demethylating reaction is 2-cyano group-6-methoxyl group benzo thiazole: pyridine hydrochloride=1: (1～3), reaction conditions is to vacuumize feeding nitrogen, temperature of reaction is 180～250 ℃, and the reaction times is 0.5～3h.
2, the method for the described D-fluorescein of claim 1 chemosynthesis, the extraction, the purge process that it is characterized in that the demethylating reaction product are: (1) adds the demethylating reaction product with ethyl acetate and frozen water, even back extraction organic layer shakes, water layer is used ethyl acetate extraction again, merge organic layer and wash organic layer with frozen water more at last, the organic layer material is by the dry yellowish red color material that gets of rotation vacuum-evaporation instrument; (2) make the purifying chromatography column with 80～400 order silochroms, with 5: the chloroform of (1～3): ethyl acetate is made eluting solvent, collects elutriant, detects with thin layer chromatography, collect the higher part of target product purity, be rotated the vacuum-evaporation concentrate drying after merging.
3, the described D-fluorescein of claim 2 chemical synthesis process is characterized in that: the ethyl acetate that leaching process adopted of demethylating reaction product and the ratio of frozen water are: 1: (1～3), each consumption are 50～100 times of reaction material; (2) but chromatography column with the band threeway the adding pressure type chromatography column, flow velocity is 1～5ml/min.
4, the method for the described D-fluorescein of claim 1 chemosynthesis, it is characterized in that the demethylation product behind the purifying and the condensation reaction of D-halfcystine are: the demethylating reaction product behind the purifying and D-halfcystine hydrate are vacuumized feed under the condition of nitrogen gas lucifuge reaction 10～30min under room temperature in the alkaline ethanol solvent, regulate pH to 1～6 with hydrochloric acid, high speed centrifugation 10～20min under 1000～15000r/min, collecting precipitation, lucifuge vacuum-drying.
5, the method for the described D-fluorescein of claim 2 chemosynthesis, it is characterized in that the demethylation product behind the purifying and the condensation reaction of D-halfcystine are: the demethylating reaction product behind the purifying and D-halfcystine hydrate are vacuumized feed under the condition of nitrogen gas lucifuge reaction 10～30min under room temperature in the alkaline ethanol solvent, regulate pH to 1～6 with hydrochloric acid, high speed centrifugation 10～20min under 1000～15000r/min, collecting precipitation, lucifuge vacuum-drying.
6, the method for the described D-fluorescein of claim 3 chemosynthesis, it is characterized in that the demethylation product behind the purifying and the condensation reaction of D-halfcystine are: the demethylating reaction product behind the purifying and D-halfcystine hydrate are vacuumized feed under the condition of nitrogen gas lucifuge reaction 10～30min under room temperature in the alkaline ethanol solvent, regulate pH to 1～6 with hydrochloric acid, high speed centrifugation 10～20min under 1000～15000r/min, collecting precipitation, lucifuge vacuum-drying.
7, a kind of reaction unit of finishing the chemosynthesis of the described D-fluorescein of claim 1, it is characterized in that comprising that one places the there-necked flask (500mL) on the high temperature oil bath heating unit, there-necked flask one side openings is connected to and vacuumizes and the pipeline of logical nitrogen, another side openings sealing, intermediate openings connects a prolong, and the waste gas outlet of prolong is connected with a waste gas resorber.
8, the reaction unit of the described D-fluorescein of claim 7 chemosynthesis, it is characterized in that the high temperature oil bath system, can be used on adjustable electric furnace (1) and (500W) go up, come meticulous attemperation by temperature sensor (3) with Stainless Steel Kettle (6) Sheng Viscotrol C (about 2000mL); Vacuumize logical nitrogen with there-necked flask (2) left side opening (500mL), intermediate openings is used for application of sample and refrigeration cycle, and the right opening is used for sampling and detects; Refrigeration cycle gets final product with general water condensing tube (4), and waste gas absorbs available exsiccant calcium chloride (5) to carry out.
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| CN102260132B (en) * | 2010-05-24 | 2014-03-05 | 湖南博瑞新特药有限公司 | Demethylating reagent, method for removing methyl and method for preparing luciferin by use of demethylating reagent |
| CN102532053B (en) * | 2011-12-30 | 2014-08-06 | 广东省微生物研究所 | Preparation and purification methods of high-purity D-fluorescein |
| CN102633742A (en) * | 2012-03-22 | 2012-08-15 | 盛世泰科生物医药技术(苏州)有限公司 | Synthesis method of sodium 2-(6-hydroxy benzo[d]thiazole-2-yl) thiazole-4-carboxylate |
| CN103012354A (en) * | 2012-12-24 | 2013-04-03 | 广州医药研究总院 | Preparation method of 5(6)-carboxyl carboxyl luciferin isomer |
| CN105384704B (en) * | 2015-12-11 | 2017-12-19 | 常州南京大学高新技术研究院 | A kind of demethylation technique of methoxybenzothiazole of 2 cyano group 6 and its method for preparing D fluoresceins |
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| Title |
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| 虫荧光素的合成及其鉴定 平学凡，等，第二军医大学学报，第11卷第3期 1990 * |
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