CN118221694A - Preparation method of high-purity 5, 6-carboxyfluorescein - Google Patents
Preparation method of high-purity 5, 6-carboxyfluorescein Download PDFInfo
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- CN118221694A CN118221694A CN202211634057.8A CN202211634057A CN118221694A CN 118221694 A CN118221694 A CN 118221694A CN 202211634057 A CN202211634057 A CN 202211634057A CN 118221694 A CN118221694 A CN 118221694A
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- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 title claims abstract description 65
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- NJYVEMPWNAYQQN-UHFFFAOYSA-N 5-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C21OC(=O)C1=CC(C(=O)O)=CC=C21 NJYVEMPWNAYQQN-UHFFFAOYSA-N 0.000 claims abstract description 36
- 238000000034 method Methods 0.000 claims abstract description 32
- 238000010828 elution Methods 0.000 claims abstract description 17
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 42
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 33
- 239000007787 solid Substances 0.000 claims description 28
- 239000003480 eluent Substances 0.000 claims description 22
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims description 18
- 239000007853 buffer solution Substances 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 14
- AFQIYTIJXGTIEY-UHFFFAOYSA-N hydrogen carbonate;triethylazanium Chemical compound OC(O)=O.CCN(CC)CC AFQIYTIJXGTIEY-UHFFFAOYSA-N 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 9
- 238000012856 packing Methods 0.000 claims description 9
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 claims description 9
- 239000000706 filtrate Substances 0.000 claims description 8
- 239000012074 organic phase Substances 0.000 claims description 8
- 239000002244 precipitate Substances 0.000 claims description 8
- 230000001105 regulatory effect Effects 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 4
- 238000011068 loading method Methods 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 3
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 3
- 239000000377 silicon dioxide Substances 0.000 claims description 3
- 238000001704 evaporation Methods 0.000 claims description 2
- 238000000746 purification Methods 0.000 abstract description 10
- 230000008901 benefit Effects 0.000 abstract description 4
- 239000000945 filler Substances 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 14
- 239000011521 glass Substances 0.000 description 9
- 238000001035 drying Methods 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- 239000012043 crude product Substances 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 description 4
- 230000005526 G1 to G0 transition Effects 0.000 description 3
- UAOMVDZJSHZZME-UHFFFAOYSA-N diisopropylamine Chemical compound CC(C)NC(C)C UAOMVDZJSHZZME-UHFFFAOYSA-N 0.000 description 3
- 238000011049 filling Methods 0.000 description 3
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical class O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000010791 quenching Methods 0.000 description 3
- 230000000171 quenching effect Effects 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- -1 Octadecylsilyl Chemical group 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000005281 excited state Effects 0.000 description 2
- 239000005457 ice water Substances 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 229940098779 methanesulfonic acid Drugs 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- SRPWOOOHEPICQU-UHFFFAOYSA-N trimellitic anhydride Chemical compound OC(=O)C1=CC=C2C(=O)OC(=O)C2=C1 SRPWOOOHEPICQU-UHFFFAOYSA-N 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 235000005811 Viola adunca Nutrition 0.000 description 1
- 240000009038 Viola odorata Species 0.000 description 1
- 235000013487 Viola odorata Nutrition 0.000 description 1
- 235000002254 Viola papilionacea Nutrition 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000011449 brick Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 229940043279 diisopropylamine Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 230000005283 ground state Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 239000003495 polar organic solvent Substances 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 125000006853 reporter group Chemical group 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/10—Spiro-condensed systems
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- 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
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- 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
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention provides a preparation method of high-purity 5, 6-carboxyfluorescein, in particular to a preparation method of high-purity 6-carboxyfluorescein by gradient elution with reversed-phase filler, which can completely separate high-purity 6-carboxyfluorescein and high-purity 5-carboxyfluorescein, greatly improves the purification efficiency of carboxyfluorescein, and has the advantages of multi-batch preparation and stable process.
Description
Technical Field
The invention belongs to the field of biochemistry, and particularly relates to a preparation method of high-purity 5, 6-carboxyfluorescein.
Background
The compounds on which fluorescent labels depend are called fluorescent substances. The fluorescent substance is a compound having a conjugated double bond system chemical structure, and is excited to an excited state when irradiated with ultraviolet light or blue-violet light, and emits fluorescence when the excited state is recovered to a ground state. Fluorescent labeling refers to the use of fluorescent substances covalently bound or physically adsorbed to a group of the molecule to be studied, and its fluorescent properties are used to provide information about the object under study. The fluorescent marker has the advantages of no radioactive contamination, simple operation and the like, so that the fluorescent marker is widely applied in a plurality of research fields.
TaqMan fluorescent probe is an oligonucleotide probe, the 5 'end of which carries a fluorescent group, such as FAM, TET, VIC, HEX, etc., and the 3' end of which carries a quenching group, such as TAMRA, BHQ, etc. During PCR amplification, a pair of primers is added, and a specific fluorescent probe is added, so that when the probe is complete, fluorescent signals emitted by the reporter group are absorbed by the quenching group; during PCR amplification, the 5'-3' exonuclease activity of Taq enzyme is used for carrying out enzyme digestion degradation on the probe to separate a report fluorescent group from a quenching fluorescent group, so that a fluorescence monitoring system can receive a fluorescence signal, namely, one fluorescence molecule is formed for each amplified DNA chain, and the accumulation of the fluorescence signal and the formation of a PCR product are completely synchronous.
The 5 (6) -carboxyfluorescein has good optical parameters, higher quantum yield and molar absorptivity, so that the 5 (6) -carboxyfluorescein is widely applied to the aspects of biomarkers and the like, and the 5 (6) -carboxyfluorescein mixture brings a plurality of limitations to the application of labeled probes, including probe purification and the like, so that the separation and purification of high-purity fluorescein isomers are important.
The main method for purifying the current fluorescein isomer is a crystallization method, a 5 (6) -carboxyl fluorescein mixture is dissolved in a solvent A, a solvent B is slowly added after the mixture is dissolved, the mixture is cooled to 0 ℃, diisopropylamine is slowly added to slowly wash out solids, the solids are left stand at low temperature, the solids are obtained after filtration, the solids are dissolved in ethyl acetate and subjected to acid regulation, and 6-carboxyl fluorescein is obtained after layering and concentration, the purity is 95% (refer to patent document CN 115197237A), the purity is still lower, and the practical application requirements are difficult to meet.
Disclosure of Invention
The invention relates to a preparation and purification method of fluorescein isomers, in particular to preparation and separation of 5 (6) -carboxyl fluorescein, which can be used for preparation and purification of 5 (6) -carboxyl-2 ', 4',5', 7' -hexachlorofluorescein.
In a first aspect of the present invention, there is provided a process for the preparation of 5 (6) -carboxyfluorescein (5, 6-carboxyfluorescein), said process comprising the steps of: the crude carboxyfluorescein mixture containing 6-carboxyfluorescein and 5-carboxyfluorescein was purified using reverse phase chromatography.
In another preferred embodiment, the reversed phase chromatography uses octadecylsilane chemically bonded silica packing.
In another preferred embodiment, the eluent of the reverse phase chromatography comprises: acetonitrile and/or triethylamine-carbonic acid buffer solution.
In another preferred embodiment, the upper column sample of the reverse phase chromatography is a crude carboxyfluorescein mixture dissolved in a potassium carbonate solution.
In another preferred embodiment, the concentration of the potassium carbonate solution is 0.5-2mol/L; preferably about 1mol/L.
In another preferred embodiment, the method comprises the steps of:
Loading the crude carboxyfluorescein mixture dissolved by potassium carbonate solution on a column, eluting by using an eluent, and collecting the 6-carboxyfluorescein and 5-carboxyfluorescein eluent in a fractional manner;
wherein the eluent is acetonitrile/triethylamine-carbonic acid buffer solution gradient eluent; the elution gradient is preferably set to 0 to 20% by volume of acetonitrile.
In another preferred embodiment, the method further comprises the steps of:
acid regulating the 6-carboxyl fluorescein eluent to precipitate solid, and collecting 6-carboxyl fluorescein solid precipitate; and/or
The 5-carboxyfluorescein eluate was acidified to precipitate a solid, and the 5-carboxyfluorescein solid precipitate was collected.
In another preferred embodiment, the method further comprises the steps of:
extracting with ethyl acetate to remove the filtrate of the solid precipitate of the 6-carboxyfluorescein, and evaporating the organic phase to obtain a solid of the 6-carboxyfluorescein; and/or
The filtrate of the solid precipitate of 5-carboxyfluorescein was removed by extraction with ethyl acetate and the organic phase was evaporated to dryness to obtain a solid of 5-carboxyfluorescein.
In another preferred embodiment, the method further comprises the step of pre-treating the reverse phase chromatography column prior to loading the column.
In another preferred embodiment, the step of pre-treating the reverse phase chromatography column comprises:
The column was then flushed 3-5 column volumes with triethylamine-carbonic acid buffer.
In another preferred embodiment, the triethylamine-carbonic acid buffer solution is about 0.05 to about 0.2M triethylamine-carbonic acid buffer solution; preferably, the triethylamine-carbonic acid buffer solution is about 0.1M triethylamine-carbonic acid buffer solution.
In another preferred embodiment, the 5-carboxyfluorescein is 5-carboxy-2 ', 4',5', 7' -hexachlorofluorescein; the 6-carboxyfluorescein is 6-carboxyl-2 ', 4',5', 7' -hexachlorofluorescein.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows the HPLC detection result of the crude carboxyfluorescein mixture prepared in example 1;
FIG. 2 shows the HPLC detection result of the prepared high purity 6-carboxyfluorescein;
FIG. 3 shows the HPLC detection result of the prepared high purity 5-carboxyfluorescein.
Detailed Description
Through extensive and intensive research, the inventor unexpectedly discovers that the high-purity 6-carboxyfluorescein and the high-purity 5-carboxyfluorescein can be completely separated by using the reversed-phase packing for gradient elution, the purification efficiency of the carboxyfluorescein is greatly improved, and the preparation process is stable in multiple batches. On the basis, the preparation method of the carboxyfluorescein with high yield and high purity is obtained.
Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.
In the invention, liquid chromatography is adopted to purify the carboxyfluorescein.
Liquid chromatography is classified into normal phase chromatography and reverse phase chromatography according to the relative polarities of mobile phase and stationary phase. The case where the mobile phase polarity is greater than the stationary phase polarity is called Reverse Phase Chromatography (RPC). Reversed Phase Chromatography (RPC) uses a nonpolar reversed phase medium as a stationary phase, an aqueous solution of a polar organic solvent as a mobile phase, and elution chromatography for separating and purifying a solute according to the difference in polarity (hydrophobicity) of the solute.
In the process of the present invention, carboxyfluorescein is preferably purified by reverse phase chromatography. More preferably, the carboxyfluorescein is purified using a C18 reverse phase chromatography column (C18 column).
The C18 column is a commonly used reversed-phase chromatographic column, also called ODS column, and the packing material is octadecylsilane chemically bonded silica packing (Octadecylsilyl, ODS for short). Because C18 (ODS) is a long-chain alkyl bonding phase, has higher carbon content and better hydrophobicity, has stronger adaptability to various biological macromolecules, and is widely applied to biochemical analysis work.
The general synthesis method of carboxyfluorescein comprises the following steps:
taking trimellitic anhydride and resorcinol, and methanesulfonic acid as a solvent, and reacting for 6 hours at 80 ℃.
Post-treatment: adding into ice water, extracting with ethyl acetate, concentrating and drying the extract.
In a preferred embodiment, the purification method for preparing high-purity carboxyfluorescein provided by the invention is as follows:
Purifying with C18 column, eluting with 0.1M triethylamine-carbonic acid buffer solution (mobile phase A) and acetonitrile, dissolving crude carboxyfluorescein with 1M K 2CO3 solution, loading, gradient eluting, collecting 6-carboxyfluorescein and 5-carboxyfluorescein eluate (about 3% of acetonitrile peak, 6-carboxyfluorescein, and 10% peak, 5-carboxyfluorescein). The pH of the eluent of 6-carboxyfluorescein and 5-carboxyfluorescein is respectively regulated to about 1 by hydrochloric acid, precipitated solids are collected, and filtrate is concentrated and evaporated to dryness after extraction by ethyl acetate. The obtained solid substance is the high-purity carboxyfluorescein.
The invention has the main advantages that:
(1) Purification of carboxyfluorescein using reverse phase chromatography was first proposed;
(2) The method can greatly improve the purification efficiency of the carboxyfluorescein;
(3) The existing 6-carboxyfluorescein purification method is at the cost of discarding 5-carboxyfluorescein, and the method can be used for preparing high-purity 6-carboxyfluorescein and 5-carboxyfluorescein simultaneously, so that the comprehensive benefit is higher.
The present invention will be described in further detail with reference to the following examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods for which detailed conditions are not noted in the following examples are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.
Example 1
50G of trimellitic anhydride is weighed at room temperature, added into a 2L three-necked flask, added with a magnetic stirrer, then added with 103g of resorcinol and 500mL of methanesulfonic acid in sequence, and reacted for 6 hours at the temperature of 80 ℃.
The reaction phenomenon is that the materials are gradually dissolved and changed into a brick red solution from light yellow.
After the reaction is finished, cooling the reaction liquid to room temperature, adding the reaction liquid into 2L of ice water, adding 500mL multiplied by 3 ethyl acetate for extraction, combining organic phases, drying by anhydrous sodium sulfate, and concentrating to obtain a carboxyfluorescein mixture crude product.
6-Carboxyfluorescein as detected by HPLC: 5-carboxyfluorescein was 50:49. The HPLC detection results are shown in FIG. 1.
The crude product was purified by reverse C18 packing column, a 36mm x 460mm medium pressure glass column was taken, ethanol was added to the silica gel matrix reverse C18 packing, stirred, poured into the glass column, and after filling, the glass column was flushed with 4 column volumes using 0.1M TEAB aqueous solution (triethylamine-carbonic acid buffer, mobile phase a).
After the C18 column was washed clean, the crude product was dissolved in 200mL of 1mol/L potassium carbonate solution, loaded with an eluent of 0.1M TEAB aqueous solution and acetonitrile, and eluted in a gradient of 0 to 20% by volume of acetonitrile.
The elution gradient is about 3%, and the elution peak is 6-carboxyfluorescein; the elution gradient reaches about 10%, the elution peak is 5-carboxyfluorescein, and the 6-carboxyfluorescein and the 5-carboxyfluorescein eluent are collected in parts.
The pH value of the eluent of the 6-carboxyfluorescein and the 5-carboxyfluorescein is respectively regulated by dilute hydrochloric acid until solid is separated out, and the solid is filtered by a Buchner funnel to obtain filter residues.
Extracting the filtrate with 400ml ethyl acetate for three times to obtain organic phase, spin drying, mixing with the residue, and oven drying in vacuum oven to obtain 6-carboxyfluorescein product 44.3g with yield 45%, purity 99.5%, and HPLC chart shown in figure 2; 41.35g of 5-carboxyfluorescein with 42 percent of yield and 99.8 percent of purity, and the HPLC chart is shown in figure 3.
Example 2
Crude carboxyfluorescein mixture (6-carboxyfluorescein: 5-carboxyfluorescein 50:49) was purified by reverse C18 packed column.
A 36mm size medium pressure glass column was taken, ethanol was added to the silica gel matrix reversed phase C18 packing, stirred, poured into the glass column, and after filling, the glass column was flushed with 4 column volumes using 0.1M aqueous TEAB (triethylamine-carbonic acid buffer, mobile phase a).
After the C18 column was washed clean, the crude product was dissolved in 200mL of 1mol/L potassium carbonate solution, loaded with an eluent of 0.1M TEAB aqueous solution and acetonitrile, and eluted in a gradient of 0 to 20% by volume of acetonitrile.
The elution gradient is about 3%, and the elution peak is 6-carboxyfluorescein; the elution gradient reaches about 10%, the elution peak is 5-carboxyfluorescein, and the 6-carboxyfluorescein and the 5-carboxyfluorescein eluent are collected in parts.
The pH value of the eluent of the 6-carboxyfluorescein and the 5-carboxyfluorescein is respectively regulated by dilute hydrochloric acid until solid is separated out, and the solid is filtered by a Buchner funnel to obtain filter residues.
Extracting the filtrate with 400ml ethyl acetate for three times to obtain an organic phase, spin-drying, mixing with the filter residue, and drying in a vacuum drying oven to obtain 43.8g of 6-carboxyfluorescein product with purity of 99.6%; 40.6g of 5-carboxyfluorescein with the purity of 99.5 percent.
Example 3
Crude carboxyfluorescein mixture (6-carboxyfluorescein: 5-carboxyfluorescein 50:49) was purified by reverse C18 packed column.
A 36mm size medium pressure glass column was taken, ethanol was added to the silica gel matrix reversed phase C18 packing, stirred, poured into the glass column, and after filling, the glass column was flushed with 4 column volumes using 0.1M aqueous TEAB (triethylamine-carbonic acid buffer, mobile phase a).
After the C18 column was washed clean, the crude product was dissolved in 200mL of 1mol/L potassium carbonate solution, loaded with an eluent of 0.1M TEAB aqueous solution and acetonitrile, and eluted in a gradient of 0 to 20% by volume of acetonitrile.
The elution gradient is about 3%, and the elution peak is 6-carboxyfluorescein; the elution gradient reaches about 10%, the elution peak is 5-carboxyfluorescein, and the 6-carboxyfluorescein and the 5-carboxyfluorescein eluent are collected in parts.
The pH value of the eluent of the 6-carboxyfluorescein and the 5-carboxyfluorescein is respectively regulated by dilute hydrochloric acid until solid is separated out, and the solid is filtered by a Buchner funnel to obtain filter residues.
Extracting the filtrate with 400ml ethyl acetate for three times to obtain an organic phase, spin-drying, mixing with the filter residue, and drying with a vacuum drying oven to obtain 47.2g of 6-carboxyfluorescein product with purity of 99.5%; 44.6g of 5-carboxyfluorescein with the purity of 99.5 percent.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Claims (10)
1. A process for the preparation of 5 (6) -carboxyfluorescein, said process comprising the steps of: the crude carboxyfluorescein mixture containing 6-carboxyfluorescein and 5-carboxyfluorescein was purified using reverse phase chromatography.
2. The method of claim 1, wherein the reversed phase chromatography uses octadecylsilane chemically bonded silica packing.
3. The method of claim 1, wherein the eluent of the reverse phase chromatography comprises: acetonitrile and triethylamine-carbonic acid buffer.
4. The method of claim 1, wherein the column sample of the reverse phase chromatography is a crude carboxyfluorescein mixture dissolved in a potassium carbonate solution.
5. The method of claim 4, wherein the potassium carbonate solution has a concentration of 0.5 to 2mol/L.
6. The method according to claim 4, wherein the method comprises the steps of:
Dissolving the crude carboxyfluorescein mixture with potassium carbonate solution, loading on a column, eluting with eluent, and collecting 6-carboxyfluorescein and 5-carboxyfluorescein eluents in parts;
Wherein the eluent is acetonitrile/triethylamine-carbonic acid buffer solution gradient eluent.
7. The method of claim 6, wherein the elution gradient is set to 0 to 20% by volume of acetonitrile.
8. The method of claim 6, wherein the method further comprises the step of:
acid regulating the 6-carboxyl fluorescein eluent to precipitate solid, and collecting 6-carboxyl fluorescein solid precipitate; and/or
The 5-carboxyfluorescein eluate was acidified to precipitate a solid, and the 5-carboxyfluorescein solid precipitate was collected.
9. The method of claim 6, wherein the method further comprises the step of:
extracting with ethyl acetate to remove the filtrate of the solid precipitate of the 6-carboxyfluorescein, and evaporating the organic phase to obtain a solid of the 6-carboxyfluorescein; and/or
The filtrate of the solid precipitate of 5-carboxyfluorescein was removed by extraction with ethyl acetate and the organic phase was evaporated to dryness to obtain a solid of 5-carboxyfluorescein.
10. The method of claim 6, wherein the triethylamine-carbonic acid buffer solution is about 0.05M to about 0.2M triethylamine-carbonic acid buffer solution.
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