CN116354980A - 2' -phenyl-5 (6) -carboxyfluorescein and preparation method thereof - Google Patents

2' -phenyl-5 (6) -carboxyfluorescein and preparation method thereof Download PDF

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CN116354980A
CN116354980A CN202111618040.9A CN202111618040A CN116354980A CN 116354980 A CN116354980 A CN 116354980A CN 202111618040 A CN202111618040 A CN 202111618040A CN 116354980 A CN116354980 A CN 116354980A
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pivalate
carboxyfluorescein
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fluorescein
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苗茹
张明
陆阳
陆林海
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Suzhou Novikon Biotechnology Co ltd
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Abstract

The invention relates to 2' -phenyl-5 (6) -carboxyl fluorescein and a preparation method thereof, wherein the fluorescein is directly used on a DNA automatic synthesizer after being derivatized to generate phosphoramidite monomer for modifying the 5' end of a DNA probe, and can be loaded on CPG or PS after being derivatized to directly mark the 3' end of the DNA probe. And the synthesis method of the 2' -phenyl-5 (6) -carboxyfluorescein is simple and is easy for large-scale production.

Description

2' -phenyl-5 (6) -carboxyfluorescein and preparation method thereof
Technical Field
The invention belongs to the technical field of fluorescent dye synthesis, and particularly relates to 2' -phenyl-5 (6) -carboxyl fluorescein and a preparation method thereof.
Background
The fluorescent probe uses fluorescent substances as indicators, and the fluorescent substances generate fluorescence under excitation of a certain wavelength, and the detected substances are quantitatively or qualitatively analyzed by detecting the generated fluorescence. Compared with other detection methods, the fluorescent probe has the advantages of high sensitivity, high selectivity, quick response, good repeatability, simple operation, low cost and the like in the aspect of biomedical detection, so that the fluorescent probe is widely applied to the aspects of molecular diagnosis, gene detection, antibody immunoassay and the like. The fluorescent probe consists of three parts, namely a recognition group, a fluorescent group and a connector, wherein the recognition group determines the selectivity and the specificity of the probe, the fluorescent group determines the sensitivity of the probe, and the connector part plays a role of a connecting hinge in the probe. The fluorescent dye exists in the fluorescent probe in the identity of a fluorescent group, and the photophysical property of the fluorescent dye is an important expression of the performance of the probe.
Currently, fluorescent probes for labeling or derivatization mainly include rhodamine, fluorescein, phthalaldehyde and the like, wherein the fluorescein compound occupies an important position in the field of biological research. The structural formula of the fluorescein dye is shown in fig. 1, and the substituent r1=r2, r3=r4 or r1=r2=r3=r4 is called symmetrical fluorescein; in the structural formula shown in fig. 1, the substituents r1+.r2, r3+.r4, or r1+.r2+.r3+.r4 are called asymmetric fluorescein. The fluorescein dye with the structural formula shown in figure 1 can be directly used on a DNA automatic synthesizer after being derived into phosphoramidite monomer for modifying the 5 'end of a DNA probe, and can be simultaneously loaded on CPG or PS after being derived for directly marking the 3' end of the DNA probe.
Wherein, the asymmetric fluorescein dye is generally synthesized by condensing 1 equivalent of resorcinol derivative and 1 equivalent of phthalic anhydride into a phenone derivative 2, and the condensation method generally uses the Friedel-crafts reaction AlCl 3 And then the benzophenone derivative and another molecule of resorcinol derivative are condensed into the asymmetric fluorescein dye under the acidic condition at more than 80 degrees. However, with AlCl 3 The reaction solvent used for the Friedel-crafts reaction of the catalyst is generally selected from dichloromethane, dichloroethane, nitromethane, nitrobenzene and the like, and some resorcinol derivatives and phthalic anhydride derivatives have poor normal-temperature solubility in the solvent, and can have certain solubility only by heating, so that the reaction yield is low, the reaction is incomplete and byproducts are many. Therefore, the method for searching for the green synthetic asymmetric fluorescein dye has very important significance.
Disclosure of Invention
In order to solve the problems, the application provides 2' -phenyl-5 (6) -carboxyl fluorescein and a preparation method thereof, and the preparation method has high yield and high purity.
To achieve the above object, the present application provides a 2' -phenyl-5-carboxyfluorescein having the following structural formula:
Figure BDA0003437113410000021
in order to achieve the above object, the present application further provides a 2' -phenyl-6-carboxyfluorescein having the following structural formula:
Figure BDA0003437113410000022
in order to achieve the above object, the present application also provides a method for preparing 2 '-phenyl-5-carboxyfluorescein or 2' -phenyl-6-carboxyfluorescein, comprising the steps of:
s1, dissolving trimellitic acid glycoside and resorcinol in methane sulfonic acid, heating for reaction, and quenching with ice water to obtain 5 (6) -carboxyl fluorescein;
s2, dissolving 5 (6) -carboxyl fluorescein into pyridine, fully reacting with pivaloyl chloride, adding water for quenching, decompressing and distilling out a solvent, retaining a residue, adding dichloromethane into the residue, fully mixing, washing with water and hydrochloric acid in sequence, collecting an organic phase, drying, and evaporating out the solvent to obtain 5 (6) -carboxyl fluorescein pivalate;
s3, dissolving 5 (6) -carboxyl fluorescein pivalate into ethanol, fully reacting with diisopropylamine, crystallizing at low temperature to obtain 5-carboxyl fluorescein pivalate diisopropylamine salt or 6-carboxyl fluorescein pivalate diisopropylamine salt, acidifying the 5-carboxyl fluorescein pivalate diisopropylamine salt to obtain 5-carboxyl fluorescein pivalate, and acidifying the 6-carboxyl fluorescein pivalate diisopropylamine salt to obtain 6-carboxyl fluorescein pivalate;
s4, adding alkali into the 5-carboxyl fluorescein pivalate, fully dissolving, heating to react completely, pouring into ice water, regulating acidity with acid, and collecting solid to obtain 4- (2, 4-dihydroxybenzophenone) isophthalic acid; adding alkali into 6-carboxyl fluorescein pivalate, fully dissolving, heating to react completely, pouring into ice water, regulating acidity with acid, and collecting solid to obtain 2- (2, 4-dihydroxybenzophenone) terephthalic acid;
s5, dissolving 4- (2, 4-dihydroxybenzophenone) isophthalic acid and 4-phenylresorcinol in methane sulfonic acid, heating for reaction, pouring into ice water, and separating out solid to obtain 2' -phenyl-5-carboxyfluorescein;
2- (2, 4-dihydroxyphenyl ketone) terephthalic acid and 4-phenyl resorcinol are dissolved in methane sulfonic acid, heated and reacted, poured into ice water, and solid is separated out, thus obtaining 2' -phenyl-6-carboxyl fluorescein. Further, in step S1, the temperature of the heating reaction is 110-150 ℃.
Further, in step S2, pivaloyl chloride is sufficiently reacted in such a manner that pivaloyl chloride is dropwise added to a mixed solution of 5 (6) -carboxyfluorescein and pyridine until the reaction is completed.
Further, in step S2, the temperature at which the pivaloyl chloride is sufficiently reacted is 0℃to 15 ℃.
Further, in the step S3, the addition amount of the diisopropylamine is 1 to 4 times equivalent of 5 (6) -carboxyfluorescein pivalate.
Further, in step S4, the base is LiOH, naOH, KOH, na 2 CO 3 、K 2 CO 3 、 K 3 PO 4 The temperature of the heating treatment is 100-150 ℃.
Further, in the step S4, the mass percentage of the alkali is 10% -60%.
Further, in step S4, the acid is any one of hydrochloric acid, sulfuric acid, and acetic acid.
The application has the beneficial effects that the 2' -phenyl-5 (6) -carboxyl fluorescein is directly used on a DNA automatic synthesizer after being derivatized to generate phosphoramidite monomer for modifying the 5' end of a DNA probe, and can be loaded on CPG or PS after being derivatized to directly mark the 3' end of the DNA probe. And the synthesis method of the 2' -phenyl-5 (6) -carboxyfluorescein is simple and is easy for large-scale production.
Drawings
FIG. 1 is a structural formula of a conventional fluorescein dye;
FIG. 2 is a schematic illustration of the chemical reaction process of 2' -phenyl-5-carboxyfluorescein in the present application;
FIG. 3 is a schematic illustration of the chemical reaction process of 2' -phenyl-6-carboxyfluorescein in the present application;
Detailed Description
For the purposes, technical solutions and advantages of the present application, the technical solutions of the present application will be clearly and completely described below with reference to specific embodiments and drawings of the present application. It should be apparent that the described embodiments are only some, but not all, of the embodiments of the present application and are not intended to limit the scope of the present invention. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.
The application provides 2' -phenyl-5-carboxyfluorescein, which has the following structural formula:
Figure BDA0003437113410000041
the application also provides 2' -phenyl-6-carboxyfluorescein, which has the following structural formula:
Figure BDA0003437113410000042
the application also provides a preparation method of the 2 '-phenyl-5-carboxyfluorescein or the 2' -phenyl-6-carboxyfluorescein, and the preparation method refers to the flow charts of fig. 2 and 3, and comprises the following steps:
s1, preparation of 5 (6) -carboxyfluorescein (compound 5):
weighing a certain amount of trimellitic acid glycoside (compound 3) and resorcinol (compound 4) in a dry flask, adding a certain amount of methane sulfonic acid, heating to react completely, pouring the reaction solution into ice water for quenching, filtering, collecting solid, naturally drying the solid to obtain a 5 (6) -carboxyfluorescein crude product, and using the crude product for the next reaction.
S2, preparation of 5 (6) -carboxyfluorescein pivalate (Compound 6):
weighing a certain amount of 5 (6) -carboxyl fluorescein crude product (compound 5) into a dry flask, adding a certain amount of pyridine for dissolution, dropwise adding pivaloyl chloride at a low temperature, after the reaction is completed, adding water for quenching reaction, distilling off the solvent under reduced pressure, adding methylene dichloride into residues, sequentially washing with water and dilute acid, collecting an organic phase, drying, and distilling off the solvent to obtain 5 (6) -carboxyl fluorescein pivalate (compound 6), wherein the product is directly used for the next crystallization to separate isomers.
S3, preparing 5-carboxyfluorescein pivalate (compound 7) or 6-carboxyfluorescein pivalate (compound 7):
weighing a certain amount of 5 (6) -carboxyl fluorescein pivalate (compound 6) into a dry flask, adding ethanol for dissolution, then adding a certain amount of diisopropylamine, and crystallizing the mixed solution at a low temperature to obtain 5-carboxyl fluorescein pivalate diisopropylamine salt or 6-carboxyl fluorescein pivalate diisopropylamine salt. Then acidifying the 5-carboxyfluorescein pivalate diisopropylamine salt to obtain 5-carboxyfluorescein pivalate (compound 7), and acidifying the 6-carboxyfluorescein pivalate diisopropylamine salt to obtain 6-carboxyfluorescein pivalate (compound 7).
S4 preparation of 4- (2, 4-dihydroxybenzophenone) -isophthalic acid (Compound 8) or 2- (2, 4-dihydroxybenzophenone) -terephthalic acid (Compound 8)
Weighing a certain amount of 5-carboxyfluorescein pivalate (compound 7) in a dry flask, adding alkali for dissolution, then heating for reaction, pouring into ice water after the reaction is finished, adjusting the acid with acid, and filtering and collecting solid to obtain 4- (2, 4-dihydroxybenzophenone group) isophthalic acid (compound 8).
Weighing a certain amount of 6-carboxyfluorescein pivalate (compound 7) in a dry flask, adding alkali for dissolution, then heating for reaction, pouring into ice water after the reaction is finished, adjusting the acid with acid, and filtering and collecting solid to obtain 2- (2, 4-dihydroxybenzophenone group) terephthalic acid (compound 8).
S5 preparation of 2 '-phenyl-5-carboxyfluorescein (Compound 9) or 2' -phenyl-6-carboxyfluorescein (Compound 9)
Weighing a certain amount of 4- (2, 4-dihydroxybenzophenone) isophthalic acid (compound 8) and 4-phenylresorcinol in a dry flask, adding methane sulfonic acid, heating for reaction, pouring into ice water after the reaction is completed, separating out solids, filtering, collecting the solids, and naturally airing to obtain the asymmetric fluorescein dye 2' -phenyl-5-carboxyfluorescein (compound 9).
Weighing a certain amount of 2- (2, 4-dihydroxybenzophenone) terephthalic acid (compound 8) and 4-phenylresorcinol in a dry flask, adding methane sulfonic acid, heating for reaction, pouring into ice water after the reaction is completed, separating out solids, filtering, collecting the solids, and naturally airing to obtain the asymmetric fluorescein dye 2' -phenyl-6-carboxyfluorescein (compound 9).
In some embodiments, in step S1, the temperature of the heating reaction is 110 ℃ to 150 ℃. In the step S2, the temperature of the reaction with pivaloyl chloride is 0-15 ℃. In the step S3, the addition amount of the diisopropylamine is 1-4 times equivalent of 5 (6) -carboxyl fluorescein pivalate. In step S4, the alkali is LiOH, naOH, KOH, na 2 CO 3 、K 2 CO 3 、K 3 PO 4 The temperature of the heating treatment is 100-150 ℃, the mass percentage of the alkali is 10-60%, and the acid is any one of hydrochloric acid, sulfuric acid and acetic acid.
It should be noted that: in this application 2' -phenyl-5 (6) -carboxyfluorescein represents 2' -phenyl-5-carboxyfluorescein or 2' -phenyl-6-carboxyfluorescein.
Example 1
(1) 5 g of trimellitic acid glycoside (compound 3) and 5.7 g of resorcinol (compound 4) are weighed into a dry flask, 100 ml of methane sulfonic acid is added to react to completion at 135 ℃, the reaction solution is poured into ice water to quench, filtration is carried out, the solid is collected, and the solid is naturally dried to obtain a 5 (6-) carboxyfluorescein crude product which is used for the next reaction.
(2) 9 g of 5 (6-) carboxyfluorescein (compound 5) is weighed into a dry flask, 90 ml of pyridine is added for dissolution, 11 g of pivaloyl chloride is dropwise added at 5 ℃, after the reaction is completed, water is added for quenching reaction, the solvent is distilled off under reduced pressure, 300 ml of dichloromethane is added for dissolution of residues, 300 ml of water and 300 ml of 2MHCl are sequentially used for washing, an organic phase is collected, the solvent is distilled off after drying, and 5 (6-) carboxyfluorescein pivalate is obtained, and the product is directly used for the next crystallization and separation of isomers.
(3) 12 g of 5 (6-) carboxyfluorescein pivalate (compound 6) is weighed into a dry flask, 70 ml of ethanol is added for dissolution, then 30 ml of diisopropylamine is added, the mixed solution is placed in a refrigerator at-20 ℃ for overnight crystallization, and the solid is collected by filtration, so that 6-carboxyfluorescein pivalate diisopropylamine salt is obtained. Then, after acidifying 6-carboxyfluorescein pivalate diisopropylamine salt with 2M hydrochloric acid, nuclear magnetic resonance detection was performed on the acidified substance, and it was found that the acidified substance was 6-carboxyfluorescein pivalate (compound 7). The nuclear magnetic detection data are as follows: 1 H NMR(DMSO-d6)δ8.30(dd,1H),8.22(d,1H),7.85(s,1H),7.30(d,2H), 6.96(d,2H),6.94(dd,2H),1.33(s,18H)。
(4) 5 g of 6-carboxyfluorescein pivalate (compound 7) was weighed into a dry flask, 70 ml of 40% NaOH was added for dissolution, then the mixture was heated to 130 ℃ for reaction, after the reaction was completed, the mixture was poured into ice water, the mixture was made acidic by hydrochloric acid, and the solid was collected by filtration, and the obtained solid was 2- (2, 4-dihydroxybenzophenone) terephthalic acid (compound 8) as detected by nuclear magnetic data. The nuclear magnetic detection data are as follows: 1 H NMR(DMSO-d6)δ12.02(s,1H),10.78(s,1H),8.17(dd,2H),7.87(s,1H),7.00(d, 1H),6.35(s,1H),6.31(dd,1H)。
(5) 1 g of 2- (2, 4-dihydroxybenzophenone) terephthalic acid and 615 mg of 4-phenylresorcinol are weighed into a dry flask, 20 ml of methanesulfonic acid is added, the reaction is heated to 120 ℃, after the reaction is completed, the mixture is poured into ice water, solids are separated out, the solids are collected by filtration and naturally dried, the nuclear magnetic detection is carried out, the substance detected by the nuclear magnetic resonance is 2 '-phenyl-6-carboxyfluorescein (compound 9), and the maximum wavelength of the 2' -phenyl-6-carboxyfluorescein is lambda max=505 nm. The nuclear magnetic detection data are as follows: 1 H NMR(DMSO-d6)δ 10.52(s,1H),10.22(s,1H),8.24(dd,1H),8.13(d,1H),7.74(s,1H),7.25-7.35(m, 5H),6.96(s,1H),6.93(s,1H),6.66(dd,2H)。
example 2
(1) 5 g of trimellitic acid glycoside (compound 3) and 5.7 g of resorcinol (compound 4) are weighed into a dry flask, 100 ml of methane sulfonic acid is added to react to completion at 135 ℃, the reaction solution is poured into ice water to quench, filtration is carried out, the solid is collected, and the solid is naturally dried to obtain a 5 (6-) carboxyfluorescein crude product which is used for the next reaction.
(2) 9 g of 5 (6-) carboxyfluorescein (compound 5) is weighed into a dry flask, 90 ml of pyridine is added for dissolution, 11 g of pivaloyl chloride is dropwise added at 5 ℃, after the reaction is completed, water is added for quenching reaction, the solvent is distilled off under reduced pressure, 300 ml of dichloromethane is added for dissolution of residues, 300 ml of water and 300 ml of 2MHCl are sequentially used for washing, an organic phase is collected, the solvent is distilled off after drying, and 5 (6-) carboxyfluorescein pivalate is obtained, and the product is directly used for the next crystallization and separation of isomers.
(3) 12 g of 5 (6-) carboxyfluorescein pivalate (compound 6) was weighed into a dry flask, 70 ml of ethanol was added for dissolution, then 30 ml of diisopropylamine was added, the mixture was placed in a-20 ℃ refrigerator for overnight crystallization, and the remaining material after collecting the solid by filtration was 5-carboxyfluorescein pivalate diisopropylamine salt. Then, the 5-carboxyfluorescein pivalate diisopropylamine salt is acidified by using 2M hydrochloric acid to obtain the 5-carboxyfluorescein pivalate.
(4) 5 g of 5-carboxyfluorescein pivalate (compound 7) was weighed into a dry flask, 70 ml of 40% NaOH was added for dissolution, then the mixture was heated to 130 ℃ for reaction, after the reaction was completed, the mixture was poured into ice water, hydrochloric acid was used for acidity adjustment, and the solid was collected by filtration, thus obtaining 4- (2, 4-dihydroxybenzophenone) isophthalic acid as a solid.
(5) 1 g of 4- (2, 4-dihydroxybenzophenone) -isophthalic acid and 615 mg of 4-phenylresorcinol were weighed into a dry flask, 20 ml of methanesulfonic acid was added, the mixture was heated to 120℃for reaction, and after completion of the reaction, the mixture was poured into ice water, and solids were separated out, collected by filtration, and naturally dried to obtain 2' -phenyl-5-carboxyfluorescein (compound 9).
In summary, the application provides a 2' -phenyl-5 (6) -carboxyfluorescein and a preparation method thereof, wherein the fluorescein is directly used on a DNA automatic synthesizer after being derived into phosphoramidite monomer for modifying the 5' end of a DNA probe, and can be loaded on CPG or PS after being derived to directly label the 3' end of the DNA probe.
In the preparation method of the 2 '-phenyl-5 (6) -carboxyfluorescein, a fluorescein dye (compound 1) with a symmetrical structure is synthesized, a 5-or 6-carboxyl isomer is separated through crystallization by a simple method, then the 6-carboxyl isomer (or the 5-carboxyl isomer) is subjected to ring opening under alkaline conditions to generate a benzophenone derivative (compound 2) with a single isomer, and finally the benzophenone derivative is condensed with another resorcinol derivative to obtain the p-2' -phenyl-5 (6) -carboxyfluorescein with the single isomer.
Although the present disclosure describes embodiments, not every embodiment is described in terms of a single embodiment, and such description is for clarity only, and one skilled in the art will recognize that the embodiments may be combined in any suitable manner to form other embodiments that will be apparent to those skilled in the art.
The above list of detailed descriptions is only specific to practical embodiments of the present invention, and they are not intended to limit the scope of the present invention, and all equivalent embodiments or modifications that do not depart from the spirit of the present invention should be included in the scope of the present invention.

Claims (10)

1. 2' -phenyl-5-carboxyfluorescein, characterized in that it has the following structural formula:
Figure FDA0003437113400000011
2. 2' -phenyl-6-carboxyfluorescein, characterized in that it has the following structural formula:
Figure FDA0003437113400000012
3. a method for preparing 2 '-phenyl-5-carboxyfluorescein or 2' -phenyl-6-carboxyfluorescein, which is characterized by comprising the following steps:
s1, dissolving trimellitic acid glycoside and resorcinol in methane sulfonic acid, heating for reaction, and quenching with ice water to obtain 5 (6) -carboxyl fluorescein;
s2, dissolving 5 (6) -carboxyl fluorescein into pyridine, fully reacting with pivaloyl chloride, adding water for quenching, decompressing and distilling out a solvent, retaining a residue, adding dichloromethane into the residue, fully mixing, washing with water and hydrochloric acid in sequence, collecting an organic phase, drying, and evaporating out the solvent to obtain 5 (6) -carboxyl fluorescein pivalate;
s3, dissolving 5 (6) -carboxyl fluorescein pivalate into ethanol, fully reacting with diisopropylamine, crystallizing at low temperature to obtain 5-carboxyl fluorescein pivalate diisopropylamine salt or 6-carboxyl fluorescein pivalate diisopropylamine salt, acidifying the 5-carboxyl fluorescein pivalate diisopropylamine salt to obtain 5-carboxyl fluorescein pivalate, and acidifying the 6-carboxyl fluorescein pivalate diisopropylamine salt to obtain 6-carboxyl fluorescein pivalate;
s4, adding alkali into the 5-carboxyl fluorescein pivalate, fully dissolving, heating to react completely, pouring into ice water, regulating acidity with acid, and collecting solid to obtain 4- (2, 4-dihydroxybenzophenone) isophthalic acid; adding alkali into 6-carboxyl fluorescein pivalate, fully dissolving, heating to react completely, pouring into ice water, regulating acidity with acid, and collecting solid to obtain 2- (2, 4-dihydroxybenzophenone) terephthalic acid;
s5, dissolving 4- (2, 4-dihydroxybenzophenone) isophthalic acid and 4-phenylresorcinol in methane sulfonic acid, heating for reaction, pouring into ice water, and separating out solid to obtain 2' -phenyl-5-carboxyfluorescein;
2- (2, 4-dihydroxyphenyl ketone) terephthalic acid and 4-phenyl resorcinol are dissolved in methane sulfonic acid, heated and reacted, poured into ice water, and solid is separated out, thus obtaining 2' -phenyl-6-carboxyl fluorescein.
4. The method according to claim 3, wherein in the step S1, the temperature of the heating reaction is 110℃to 150 ℃.
5. The process according to claim 3, wherein in step S2, pivaloyl chloride is sufficiently reacted by dropwise addition to a mixed solution of 5 (6) -carboxyfluorescein and pyridine until the reaction is completed.
6. The process according to claim 3, wherein the reaction temperature with pivaloyl chloride in step S2 is 0℃to 15 ℃.
7. The process according to claim 3, wherein the diisopropylamine is added in an amount of 1 to 4 equivalents of 5 (6) -carboxyfluorescein pivalate in step S3.
8. The method of claim 3, wherein in step S4, the base is LiOH, naOH, KOH, na 2 CO 3 、K 2 CO 3 、K 3 PO 4 The temperature of the heating treatment is 100-150 ℃.
9. The method according to claim 3, wherein in the step S4, the alkali is 10-60% by mass.
10. The method according to claim 3, wherein in step S4, the acid is any one of hydrochloric acid, sulfuric acid and acetic acid.
CN202111618040.9A 2021-12-27 2021-12-27 2' -phenyl-5 (6) -carboxyfluorescein and preparation method thereof Pending CN116354980A (en)

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