CN111943940A - Preparation method of rhodamine activated ester - Google Patents
Preparation method of rhodamine activated ester Download PDFInfo
- Publication number
- CN111943940A CN111943940A CN202010832231.4A CN202010832231A CN111943940A CN 111943940 A CN111943940 A CN 111943940A CN 202010832231 A CN202010832231 A CN 202010832231A CN 111943940 A CN111943940 A CN 111943940A
- Authority
- CN
- China
- Prior art keywords
- rhodamine
- activated ester
- preparing
- ester
- set forth
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
- C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
Abstract
The invention provides a preparation method of rhodamine activated ester, which comprises the following steps: firstly, reacting N-hydroxysuccinimide with trifluoroacetic anhydride to prepare trifluoroacetic acid-N-hydroxysuccinimide ester; and secondly, dissolving the rhodamine dye and trifluoroacetic acid-N-hydroxysuccinimide ester in an organic solvent, fully reacting, removing the organic solvent, and purifying to prepare the rhodamine activated ester with high purity. By applying the preparation method, the byproducts are few, the prepared rhodamine activated ester does not contain DCU impurities, the product purity is high, the yield is high, and the production cost is reduced.
Description
Technical Field
The invention belongs to the technical field of biological analysis and detection, and particularly relates to a preparation method of rhodamine activated ester.
Background
Most of biomolecules have no fluorescence or weak fluorescence and poor detection sensitivity, so people label or derive an object to be detected by using a strong-fluorescence labeling reagent or a fluorescence generation reagent to generate a covalent or non-covalent combined substance with high fluorescence intensity, so that the detection limit is greatly reduced, namely the fluorescence probe technology. Fluorescence analysis methods have found wide application in analytical chemistry, particularly in bioassays, due to their high sensitivity, good selectivity and low sample volumes. With the continuous development of biotechnology, fluorescent probe technology has shown great potential in protein, nucleic acid, polypeptide, cell detection and immunoassay.
At present, fluorescent probes for labeling or derivation mainly comprise rhodamine compounds, fluorescein compounds, phthalic aldehyde compounds and the like, wherein the rhodamine compounds occupy important positions in the biological research field. Rhodamine is a dye with excellent optical properties, and compared with other common fluorescent dyes, the rhodamine fluorescent dye has the advantages of good photostability, long-wavelength absorption, large absorption coefficient, high photostability in an open-loop form, insensitivity to pH, wider wavelength range, higher fluorescence quantum yield, long fluorescence lifetime and the like. Therefore, the fluorescent dye is widely applied to the aspects of pharmacology, physiology, molecular biology, cell biology, molecular genetics, environmental chemistry, single molecule detection, information science, fluorescent labeling, laser dye and the like, and is the most commonly used fluorescent dye in the fields of analytical chemistry and biomedical science in the biotechnology field.
However, in the preparation process of the rhodamine derivative, a method of condensation of the carbodiimide (DCC for short) is usually adopted, such as 6-carboxytetramethylrhodamine activated ester and the like, the carbodiimide serving as a condensation agent is converted into dicyclohexylurea (DCU for short) in the reaction process, and the DCU is characterized by being slightly soluble in an organic solvent, most of the DCU can be removed by a filtering method, but a small amount of DCU is not easy to be removed completely, so that the labeling efficiency is low when the DCU is labeled with biomolecules, and the yield and the quality of downstream products are influenced to a certain extent.
Disclosure of Invention
The invention aims to solve the technical problems that in the current method for preparing rhodamine activated ester by adopting DCC condensing agent, a small amount of DCU (dichlorodifluoromethane) can be remained in the product, the DCU impurity is difficult to be thoroughly removed, the marking efficiency of downstream products is influenced, and the method for preparing the rhodamine activated ester for product marking, which has higher purity and does not contain DCU impurity, is provided.
In order to achieve the aim, the application provides a preparation method of rhodamine activated ester, which comprises the following steps: s1, placing N-hydroxysuccinimide and trifluoroacetic anhydride at room temperature, fully reacting, and removing the solvent to obtain trifluoroacetic acid-N-hydroxysuccinimide ester; s2, firstly, fully dissolving the rhodamine dye in an organic solvent; secondly, continuously adding the trifluoroacetic acid-N-hydroxysuccinimide ester in the step S1 into the organic solvent, fully reacting, and removing the organic solvent to obtain crude rhodamine activated ester; s3, purifying the crude rhodamine activated ester in the step S2.
As a further improvement of the present application, in step S2, the organic solvent is any one of DMF, dichloromethane, and acetonitrile.
As a further improvement of the application, the rhodamine dye is any one of rhodamine B, rhodamine 6G, rhodamine 123 and rhodamine derivatives.
As a further improvement of the application, the rhodamine derivative is any one of 5-carboxytetramethyl rhodamine and 6-carboxytetramethyl rhodamine.
As a further improvement of the present application, in step S2, the purification method is an extraction method.
As a further improvement of the present application, in step S1, the method for removing the solvent is a rotary evaporation method; in step S2, the method for removing the organic solvent is a rotary evaporation method.
As a further improvement of the application, the process of purifying the crude rhodamine activated ester in the step S2 by using the extraction method is as follows: firstly, dissolving the crude rhodamine activated ester in the step S2 by using chloroform to obtain chloroform mixed solution; and secondly, transferring the chloroform mixed solution into a separating funnel, washing with water and saturated sodium chloride in sequence, layering, collecting an organic phase, and evaporating to dryness to obtain the high-purity rhodamine activated ester.
As a further improvement of the application, the rhodamine activated ester has a purity of greater than 95%.
As a further improvement of the present application, in step S1, the reaction time is 1h to 2 h.
As a further improvement of the present application, in step S1, the method for removing the solvent is a reduced pressure rotary evaporation method.
The method has the beneficial effects that a brand-new rhodamine activated ester preparation method is provided, DCC condensing agent is not needed in the method, but an intermediate trifluoroacetic acid-N-hydroxysuccinimide ester (NHS-OTf) is synthesized firstly, and the intermediate is directly reacted with rhodamine dye to generate the rhodamine activated ester, so that byproducts are reduced, the product purity and yield are improved, the production cost is reduced, and the marking efficiency of downstream products is finally improved.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be described in detail and completely with reference to the specific embodiments of the present application. It should be apparent that the described embodiments are only some of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
In order to realize the preparation of the rhodamine activated ester which has higher purity and does not contain DCU impurities and is used for product marking, the invention provides a preparation method of the rhodamine activated ester, which comprises the following steps:
s1, fully reacting N-hydroxysuccinimide (NHS) with trifluoroacetic anhydride at room temperature, and removing the solvent to obtain trifluoroacetic acid-N-hydroxysuccinimide ester (NHS-OTf), wherein the trifluoroacetic acid-N-hydroxysuccinimide ester is a white solid, and a reaction container can be a dry flask; synthesis of trifluoroacetic acid-N-hydroxysuccinimide ester:
s2, firstly, fully dissolving the rhodamine dye in an organic solvent, and further, enabling a reaction container to be a dry flask; secondly, continuously adding the trifluoroacetic acid-N-hydroxysuccinimide ester in the step S1 into the organic solvent, fully reacting, and removing the organic solvent to obtain crude rhodamine activated ester;
s3, purifying the rhodamine activated ester in the step S2.
In the present application, the process of purifying the crude rhodamine activated ester in step S2 by using the extraction method is as follows: firstly, dissolving the crude rhodamine activated ester in the step S2 by using chloroform to obtain chloroform mixed solution; and secondly, transferring the chloroform mixed solution into a separating funnel, washing with water and saturated sodium chloride in sequence, layering, collecting an organic phase, and evaporating to dryness to obtain the rhodamine activated ester. The purity of the rhodamine activated ester is more than 95%.
In the present application, in step S1, the solvent removal method is a rotary evaporation method; further preferably, the reduced pressure rotary evaporation method is adopted, and the vacuum pump is arranged on the rotary evaporation equipment to exhaust air so as to enable the reaction to be in a negative pressure state; in step S1, the reaction time is 1-2 h. In step S2, the organic solvent is any one of DMF, dichloromethane, and acetonitrile, and more preferably DMF; the rhodamine dye is any one of rhodamine B, rhodamine 6G, rhodamine 123 and rhodamine derivatives; as a further improvement of the application, the rhodamine derivative is any one of 5-carboxytetramethyl rhodamine and 6-carboxytetramethyl rhodamine; the method for removing the organic solvent is a rotary evaporation method.
The working principle of the rotary evaporation is that the distillation flask rotates at a constant speed under the most appropriate speed through the control of a motor to increase the evaporation area, and meanwhile, the evaporation flask is in a negative pressure state through the air suction of a vacuum pump, so that the boiling point of the solution is reduced, and the evaporation speed is increased. Vacuum evaporators serve as a means of evaporation because lowering the pressure above the liquid lowers the boiling point of the component liquid therein, thereby accelerating the rate of evaporation to some extent. The evaporation bottle is arranged in a water bath kettle for heating at a constant temperature and simultaneously rotates, and the solution in the bottle is heated, diffused and evaporated under the negative pressure condition in the rotating bottle. The evaporation system can be sealed and decompressed to 400-600 mm Hg, the solvent in the distillation flask is heated by a heating bath, the heating temperature can be close to the boiling point of the solvent, the rotation can be carried out at the same time, the speed is 50-160 r/min, the solvent forms a thin film to increase the evaporation area, and in addition, the hot steam can be quickly liquefied under the action of a high-efficiency condensing pipe to accelerate the distillation rate.
The main components of rotary steaming are as follows: 1) a rotary motor for driving the evaporation bottle by rotation; 2) the evaporation pipeline is a vacuum sealing pipeline which is used for supporting the rotary shaft of the evaporation bottle and extracting steam from the material; 3) the vacuum system is used for reducing the pressure in the evaporator system and lowering the boiling point of the material; 4) heating the bath kettle, wherein water or oil is usually used for heating the materials; 5) a condenser, which is generally made into a double-snake surrounding type to accelerate the condensation efficiency, and condensing the sample by adding condensing agents such as dry ice and acetone; 6) a condensed water collecting bottle at the bottom of the condenser, which is used for capturing the distilled solvent after recondensation and collecting a sample; 7) the lifting device, a mechanical or motorized mechanism, can quickly lift the evaporation flask from the heating bath.
In the application, 6-carboxytetramethylrhodamine (TAMRA for short) is a rhodamine dye widely applied to research of bioluminescent probes, has high quantum yield, high light stability and small pH dependence, and 6-carboxytetramethylrhodamine activated ester (TAMRA-NHS) derived from the dye reacts with primary amine and secondary amine on protein, polypeptide, nucleic acid and other biomolecules to form various bioluminescent probe molecules, so that the dye can be used for fluorescence microscopy, flow cytometry, PCR (polymerase chain reaction) technology and immunofluorescence-based analysis. Therefore, the 6-carboxyl tetramethyl rhodamine is selected as a raw material to prepare the rhodamine activated ester, and the TAMRA-NHS synthetic reaction formula is as follows:
the application also compares the purity of carboxyl tetramethyl rhodamine activated ester prepared by respectively applying the carbodiimide and trifluoroacetic acid-N-hydroxysuccinimide ester. The specific embodiment is as follows:
examples
The method comprises the following steps: putting 11 g of N-hydroxysuccinimide (100mmol) into a 100ml dry flask A, adding 21g of trifluoroacetic anhydride (100mmol), stirring at room temperature for reaction until white solids in the flask disappear, wherein the reaction time is 1-2 h, and removing the solvent in the flask by using a rotary evaporation method after the reaction is finished to obtain 21g of white solids, namely trifluoroacetic acid N-hydroxysuccinimide ester (NHS-OTf).
Step two: placing 1g of 6-carboxytetramethylrhodamine (TAMRA) into a dry flask B, adding 20ml of N, N-dimethylformamide into the flask B, fully dissolving the 6-carboxytetramethylrhodamine, then adding 0.6g of trifluoroacetic acid N-hydroxysuccinimide ester (NHS-OTf) into the flask B, stirring at room temperature for reaction, monitoring the reaction process by TCL, after the reaction is finished, removing the solvent by reduced pressure rotary evaporation, dissolving the residue by a certain amount of chloroform, and transferringTransferring into an extraction separating funnel, washing with water and saturated sodium chloride in sequence, layering, collecting an organic phase, and drying to obtain 1.1g of purple black solid, wherein the yield reaches 85%, and through nuclear magnetic detection and analysis, the product does not contain DCU, the content of impurities in the product is extremely low, and the purity of the product is more than 95%. The nuclear magnetic detection data are as follows:1H NMR(400MHz,DMSO-d6):8.47(m,2H)，7,91(m,1H)，7.04(m,4H)，6.97(s,2H),3.31(s,12H),2.59(s,4H)。
comparative example
Placing 1g of 6-carboxytetramethylrhodamine (TAMRA) and 0.7g of carbodiimide (DCC) in a dry flask B, adding 20ml of N, N-dimethylformamide into the flask B, fully dissolving the 6-carboxytetramethylrhodamine, then adding 0.3g N-hydroxysuccinimide ester (NHS) into the flask B, stirring and reacting at room temperature, monitoring the reaction process by using TCL, removing the solvent by reduced pressure rotary evaporation after the reaction is finished, dissolving the residue by using a certain amount of organic solvent, extracting, drying to obtain 1g of purple black solid, wherein the yield is about 75%, and the product slightly contains DCU, a small amount of DCU is not suitable to be removed and has the purity of about 85% by nuclear magnetic detection and analysis.
In summary, the application provides a preparation method of rhodamine activated ester, which does not adopt a DCC condensing agent, but synthesizes an intermediate trifluoroacetic acid-N-hydroxysuccinimide ester (NHS-OTf) first, and the intermediate reacts with rhodamine dye directly to generate the rhodamine activated ester, thereby reducing byproducts, improving the product purity and yield, reducing the production cost and finally improving the marking efficiency of downstream products.
The present application has been described in connection with only the presently preferred embodiments with the understanding that the present disclosure is not to be considered as limiting, and the present application is not limited to the examples described above, but rather, it is to be understood that changes, modifications, additions or substitutions that are within the spirit and scope of the application by one of ordinary skill in the art are included.
Claims (10)
1. A preparation method of rhodamine activated ester is characterized by comprising the following steps:
s1, placing N-hydroxysuccinimide and trifluoroacetic anhydride at room temperature, fully reacting, and removing the solvent to obtain trifluoroacetic acid-N-hydroxysuccinimide ester;
s2, firstly, fully dissolving the rhodamine dye in an organic solvent; secondly, continuously adding the trifluoroacetic acid-N-hydroxysuccinimide ester in the step S1 into the organic solvent, fully reacting, and removing the organic solvent to obtain crude rhodamine activated ester;
s3, purifying the crude rhodamine activated ester in the step S2.
2. The method for preparing rhodamine activated ester as set forth in claim 1, wherein in step S2, the organic solvent is any one of DMF, dichloromethane and acetonitrile.
3. The method for preparing rhodamine activated ester as set forth in claim 1, wherein the rhodamine-based dye is any one of rhodamine B, rhodamine 6G, rhodamine 123 and rhodamine derivatives.
4. The method for preparing rhodamine activated ester as defined in claim 3 wherein said rhodamine derivative is any one of 5-carboxytetramethylrhodamine and 6-carboxytetramethylrhodamine.
5. The method for preparing rhodamine activated ester as set forth in claim 1, wherein in step S2, the purification method is an extraction method.
6. The method for preparing rhodamine activated ester as set forth in claim 1 wherein in step S1, the method for removing the solvent is a rotary evaporation method; in step S2, the method for removing the organic solvent is a rotary evaporation method.
7. The method for preparing the rhodamine activated ester as set forth in claim 5, wherein the process for purifying the crude rhodamine activated ester in step S2 by applying the extraction method comprises the following steps:
firstly, dissolving the crude rhodamine activated ester in the step S2 by using chloroform to obtain chloroform mixed solution; and secondly, transferring the chloroform mixed solution into a separating funnel, washing with water and saturated sodium chloride in sequence, layering, collecting an organic phase, and evaporating to dryness to obtain the high-purity rhodamine activated ester.
8. The method of preparing activated esters of rhodamine of claim 7 wherein said activated esters of rhodamine have a purity of greater than 95%.
9. The method for preparing rhodamine activated ester as set forth in claim 1, wherein in step S1, the reaction time is 1h to 2 h.
10. The method for preparing rhodamine activated ester as set forth in claim 6 wherein in step S1, said solvent removal method is reduced pressure rotary evaporation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010832231.4A CN111943940A (en) | 2020-08-18 | 2020-08-18 | Preparation method of rhodamine activated ester |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010832231.4A CN111943940A (en) | 2020-08-18 | 2020-08-18 | Preparation method of rhodamine activated ester |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111943940A true CN111943940A (en) | 2020-11-17 |
Family
ID=73343071
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010832231.4A Pending CN111943940A (en) | 2020-08-18 | 2020-08-18 | Preparation method of rhodamine activated ester |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111943940A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040171034A1 (en) * | 2002-05-03 | 2004-09-02 | Brian Agnew | Compositions and methods for detection and isolation of phosphorylated molecules |
| US20120077778A1 (en) * | 2010-09-29 | 2012-03-29 | Andrea Bourdelais | Ladder-Frame Polyether Conjugates |
| CN103224483A (en) * | 2013-05-06 | 2013-07-31 | 西北工业大学 | Fluorescent compound used for labeling oligosaccharide, and preparation method thereof |
| US10239853B1 (en) * | 2018-06-12 | 2019-03-26 | Aat Bioquest, Inc. | Polycyclic phosphoramidtes and their applications |
| US20200166460A1 (en) * | 2018-11-28 | 2020-05-28 | Promega Corporation | Reactive peptide labeling |
-
2020
- 2020-08-18 CN CN202010832231.4A patent/CN111943940A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040171034A1 (en) * | 2002-05-03 | 2004-09-02 | Brian Agnew | Compositions and methods for detection and isolation of phosphorylated molecules |
| US20120077778A1 (en) * | 2010-09-29 | 2012-03-29 | Andrea Bourdelais | Ladder-Frame Polyether Conjugates |
| CN103224483A (en) * | 2013-05-06 | 2013-07-31 | 西北工业大学 | Fluorescent compound used for labeling oligosaccharide, and preparation method thereof |
| US10239853B1 (en) * | 2018-06-12 | 2019-03-26 | Aat Bioquest, Inc. | Polycyclic phosphoramidtes and their applications |
| US20200166460A1 (en) * | 2018-11-28 | 2020-05-28 | Promega Corporation | Reactive peptide labeling |
Non-Patent Citations (1)
| Title |
|---|
| HARLAN FK 等: "Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities", 《PLOS ONE》, vol. 11, no. 5, 26 May 2016 (2016-05-26), pages 1 - 19 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109867611B (en) | Water-soluble two-photon hydrogen sulfide fluorescent probe for detecting red wine and hydrogen sulfide in vivo and preparation method and application thereof | |
| CN113214672B (en) | Amide substituted azaindole-pentamethine cyanine dye, its synthesis method and application | |
| CN102061103A (en) | Type I boron fluoride complex dipyrromethene dye, and preparation method and application thereof | |
| CN113683631B (en) | Organic boric acid glucose probe and preparation method and application thereof | |
| CN113845519B (en) | Microenvironment sensitive type fluorescent probe and preparation method and application thereof | |
| CN108473505A (en) | Polyfluorene simultaneously [4,5-cde] oxepin conjugate and its purposes in analyte detection method | |
| CN109575042B (en) | Chiral fluorescent probe with spiropyran characteristic and preparation method and application thereof | |
| CN108997772B (en) | Mitochondria positioning near-infrared fluorescent dye THX-Np and preparation method and application thereof | |
| CN108774226B (en) | Fluorescent probe for detecting silver ions and preparation method and application thereof | |
| CN111943940A (en) | Preparation method of rhodamine activated ester | |
| CN110357896B (en) | Compound, preparation and application thereof in detecting divalent copper ions and strong acid pH | |
| CN101845003A (en) | N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid compound and preparation method thereof | |
| CN101666748A (en) | New method for detecting TNT in water phase by fluorescence | |
| CN113735757B (en) | Micro-environment sensitive fluorescent organic small molecular compound and synthetic method and application thereof | |
| CN110981715B (en) | Protein chemical cross-linking agent and preparation method and application thereof | |
| CN112250700B (en) | BODIPY (boron dipyrromethene) protein misfolding probe Halo-BODIPY as well as preparation method and application thereof | |
| CN111533724B (en) | His-tag fluorescent probe with near-infrared property and preparation and application thereof | |
| CN113880760A (en) | Preparation method of lysosome targeted two-photon hydrogen sulfide fluorescent probe | |
| CN107163017B (en) | A kind of preparation process of tetramethylrhodamine | |
| CN106432236B (en) | Using naphthalimide as binary channels copper ion probe of core and its preparation method and application | |
| CN110669350A (en) | Piperidyl BODIPY red-light fluorescent dye and preparation method and application thereof | |
| CN112110913A (en) | Preparation and application of novel fluorescent probe and test paper for hydrazine hydrate detection | |
| CN114671893B (en) | Iron ion probe based on open-closed loop and synthesis method thereof | |
| CN111333576B (en) | High-stability wash-free Halo-tag probe and synthetic method and biological application thereof | |
| CN111333621B (en) | 488nm excited washing-free Halo-tag probe and synthesis and biological application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |