CN117105813A - Compounds with enhanced immunogenicity for use as chimeric antigens, fluorescent markers thereof, preparation method and uses thereof - Google Patents
Compounds with enhanced immunogenicity for use as chimeric antigens, fluorescent markers thereof, preparation method and uses thereof Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
- C07C237/28—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a non-condensed six-membered aromatic ring of the carbon skeleton
- C07C237/42—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a non-condensed six-membered aromatic ring of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
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- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/022—Boron compounds without C-boron linkages
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
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Abstract
The invention discloses a compound 5- ((2-carbamoyl-4-chlorophenyl) amino) -3, 3-dimethyl-5-oxopentanoic acid with enhanced immunogenicity, which is used as chimeric antigen, a structure shown as a formula 1, a fluorescent marker thereof, a preparation method and application thereof. The invention relates to the field of pharmaceutical chemistry, and the preparation method comprises the step of reacting a compound with a structure shown as a formula 2 with a compound with a structure shown as a formula 3 in a solvent of 1, 4-dioxane. The small molecule is easy to combine with CD91 and has the characteristic of fast entering DC cell.
Description
Technical Field
The invention relates to the field of pharmaceutical chemistry, in particular to a small molecular compound, a fluorescent marker thereof, a preparation method and application thereof.
Background
Cellular immunotherapy is of increasing interest in the treatment of infectious diseases or cancers because of its better therapeutic effect. The main principle of the method is as follows: after the antigen of the bacteria/virus or tumor is mesophilic in antigen presenting cells (such as DC cells, namely dendritic cells, which are the most important antigen presenting cells), the antigen of the bacteria/virus or tumor is further presented to T cells and activates the T cells, and the T cells are activated and amplified, so that the aim of removing the bacteria/virus or killing the tumor cells is finally achieved. CD91 is an important molecule that aids in the intracellular antigenicity of DCs.
Patent CN109535228B discloses a fusion protein as an immunogen enhancer for enhancing antigen-specific T cell responses. The fusion cell comprises: (a) An Antigen Presenting Cell (APC) binding domain or a CD91 receptor binding domain; (b) a protein transduction domain; and (C) an antigen of a pathogen, wherein the APC binding domain or CD91 receptor binding domain is located at the N-terminus of the fusion protein and the antigen of the pathogen is located at the C-terminus of the protein transduction domain. The protein transduction domain is selected from the group consisting of: (i) A fusion polypeptide comprising a T cell sensitization signal transduction peptide, a linker, and a translocation peptide; (it) T cell sensitization signal transduction peptide; and (iii) a translocation peptide of 34-112 amino acid residues in length. The fusion protein in this patent is relatively bulky and has a certain degradation time in the cell.
US2007/0003555A1 discloses a complex and a method for connecting proteins by using p95 fragments or recombinant forms of HSP proteins, which can be connected with CD91 polypeptide fragments, CD91 and carry different antigens and other substances into DC cells through endophilic action, and can be used for treating immune diseases, hyperplasia diseases and infectious diseases.
In summary, in the prior art, the CD91 is combined with protein macromolecules, which have large molecular weight, large volume, inconvenient preservation and relatively high cost; therefore, designing a small molecule compound that can bind to CD91 to replace the protein macromolecule to bring antigen into DC cells is a current need to be addressed.
Disclosure of Invention
The object of the present invention is to provide a compound 5- ((2-carbamoyl-4-chlorophenyl) amino) -3, 3-dimethyl-5-oxopentanoic acid with enhanced immunogenicity of chimeric antigen, and the aforementioned compound is confirmed to be able to bind efficiently and activate CD91 molecules on DC cells, enter into DC cells rapidly, and up-regulate the expression of MHC-I molecules presenting antigen by fluorescence labeling.
In order to achieve the above object, the present invention provides the following technical solutions:
a compound 5- ((2-carbamoyl-4-chlorophenyl) amino) -3, 3-dimethyl-5-oxopentanoic acid, having a structure as shown in formula 1.
A process for preparing a compound as claimed in claim 1, which comprises: the compound with the structure shown in the formula 2 and the compound with the structure shown in the formula 3 are reacted in a solvent of 1, 4-dioxane to obtain the compound.
Preferably, the molar ratio of the compound having the structure shown in formula 2 to the compound having the structure shown in formula 3 is 1: (1.5-2.0).
Preferably, the compound of formula 2 is reacted with the compound of formula 3 at 100-120 ℃ for 18-20 hours.
In another aspect, the use of a 5- ((2-carbamoyl-4-chlorophenyl) amino) -3, 3-dimethyl-5-oxopentanoic acid compound in cellular immunotherapy, by fluorescence labeling methods, demonstrates that said compound is capable of reaching the goal of entering DC cells by activating a CD91 molecule.
A compound 5- ((2-carbamoyl-4-chlorophenyl) amino) -3, 3-dimethyl-5-oxopentanoic acid BODIPY fluorescent marker with a structure shown in formula 6.
A method for preparing a 5- ((2-carbamoyl-4-chlorophenyl) amino) -3, 3-dimethyl-5-oxopentanoic acid BODIPY fluorescent marker, comprising the steps of:
(1) obtaining a compound (intermediate I) having the structure of formula 4 from the reaction of a compound having the structure of formula 1 with 4-Dimethylaminopyridine (DMAP);
(2) dissolving intermediate I in dichloromethane and reacting with trifluoroacetic acid (TFA) to obtain intermediate II having the structure of formula 5;
(3) from intermediate II and a BODIPY-CO dye 2 H) The reaction yields a fluorescent label having the structure of formula 6.
Preferably, the specific step in the step (1) includes: adding the compound shown in the formula 1 and DMAP into a dry flask, adding N-BOC-1, 4-butanediamine, cooling in an ice bath, adding EDCI and TEA, keeping the temperature and stirring for 30min, and then moving to room temperature for reaction.
Preferably, the specific step in the step (2) includes: the intermediate I was added to a dry flask, dissolved in dichloromethane, TFA was added and the reaction was stirred at room temperature.
Preferably, the specific step in the step (3) includes: intermediate II, BODIPY-CO 2 H and BtOH are added into a dry flask, dissolved by THF and cooled by ice bath; EDCI and TEA were added, stirred for 30min at room temperature, and then allowed to react at room temperature.
By adopting the technical scheme, compared with the prior art, the small molecule in the invention has the characteristics of small molecular weight, easy combination with CD91 and rapid entering into DC cells by way of example.
Drawings
FIG. 1 is a nuclear magnetic resonance spectrum of compound formula 1.
FIG. 2 is a nuclear magnetic resonance spectrum of compound formula 4.
FIG. 3 is a nuclear magnetic resonance spectrum of compound formula 6.
FIG. 4 shows the difference between the 3 sequences in qPCR analysis on CD91 RNA interference.
FIG. 5 is a flow assay to confirm pAVE3578 sequence interference with CD91 RNA.
FIG. 6 is a photograph of DC cytofluorescence after treatment with the compounds provided in the examples of the present invention.
FIG. 7 is a graph showing the change in fluorescence intensity of flow cytometry analysis.
FIG. 8 is a graph showing the MHC expression level of qPCR assay according to an embodiment of the present invention.
Detailed Description
A small molecule capable of carrying antigen for DC uptake by CD91, as disclosed herein, is described in further detail below with reference to the accompanying drawings and examples. It should be noted that the technical features or combinations of technical features described in the following embodiments should not be regarded as being isolated, and they may be combined with each other to achieve a better technical effect. In the drawings of the embodiments described below, like reference numerals appearing in the various drawings represent like features or components and are applicable to the various embodiments. Thus, once an item is defined in one drawing, no further discussion thereof is required in subsequent drawings.
Example 1
Preparation of 5- ((2-carbamoyl-4-chlorophenyl) amino) -3, 3-dimethyl-5-oxopentanoic acid
2-amino-5-chlorobenzamide 1, 3-dimethylglutaric anhydride was added to a dry 50mL round bottom flask and dissolved in 1, 4-dioxane and reacted by heating at 100℃for 18h.
The reaction was monitored by thin layer chromatography until complete consumption of 2-amino-5-chlorobenzamide. Developing agent: DCM: meoh=20:1.
The solvent was removed by rotary evaporation and the product 5- ((2-carbamoyl-4-chlorophenyl) amino) -3, 3-dimethyl-5-oxopentanoic acid was obtained as a white solid in 90% yield by column chromatography over silica gel (DCM: meoh=20:1).
1 H-NMR(600MHz，DMSO-d 6 )δ12.11(bs,1H),11.50(s,1H),8.46(d,J＝9.0Hz,1H),8.37(bs,1H),7.87-7.82(m,2H),7.55(bd,J＝9.0Hz, 1H), 2.40 (s, 2H), 2.31 (s, 2H), 1.09 (s, 6H). Referring to FIG. 1, a nuclear magnetic resonance spectrum of the compound of formula 1 is shown.
Example 2
Preparation of tert-butyl (4- (5- (2-carbamoyl-4-chlorophenyl) amino) -3, 3-dimethyl-5-oxopentylamino) carbamate (intermediate I)
The compound 5- ((2-carbamoyl-4-chlorophenyl) amino) -3, 3-dimethyl-5-oxopentanoic acid, 4-Dimethylaminopyridine (DMAP) was added to a dry 10mL round bottom flask, dissolved in chloroform, N-BOC-1, 4-butanediamine was added, ice-cooled to 0-5 ℃, 1-ethyl-3 (3-dimethylpropylamine) carbodiimide) (EDCI) and Triethylamine (TEA) were added, stirred for 30min with heat preservation, and then moved to room temperature for reaction for 12h.
The reaction was monitored by thin layer chromatography until the substrate was completely consumed. Developing agent: DCM, meoh=30:1, the product was developed with basic potassium permanganate solution and ninhydrin.
The solvent was removed by rotary evaporation and isolated using silica gel column chromatography (DCM: meoh=30:1) to give intermediate I as a white solid in 96% yield.
Referring to FIG. 2, a nuclear magnetic resonance spectrum of the compound of formula 4 is shown. The nuclear magnetic spectrum is: 1H NMR (600 MHz, CDCl 3) δ12.21 (bs, 1H), 8.54 (bd, J=9.0 Hz, 1H), 7.58 (bs, 1H), 7.47 (bd, J=9.0 Hz, 1H), 7.39 (bs, 1H), 6.56 (bs, 1H), 5.90 (bs, 1H), 4.70 (bs, 1H), 3.29-3.24 (m, 2H), 3.15-3.09 (m, 2H), 2.40 (bs, 2H), 2.29 (bs, 2H), 1.56-1.49 (m, 4H), 1.43 (s, 9H), 1.13 (s, 6H).
Example 3
Preparation of intermediate II
Intermediate II
Intermediate I was added to a dry 5-mL round bottom flask, dissolved in dichloromethane, trifluoroacetic acid (TFA) was added and stirred at room temperature.
The reaction was monitored by thin layer chromatography until complete consumption of compound 4. Developing agent: DCM, meoh=30:1, the product was developed with basic potassium permanganate solution and ninhydrin.
The solvent was removed by rotary evaporation of the reaction solution to give crude intermediate II which was used directly in the next step without isolation.
Also included is the preparation of a BODIPY dye (BODIPY) label of 5- ((2-carbamoyl-4-chlorophenyl) amino) -3, 3-dimethyl-5-oxopentanoic acid.
Example 4
Intermediate II, BODIPY-CO 2 H and BtOH were added to a dry 5-mL round bottom flask, dissolved in THF, and cooled to 0-5℃in an ice bath.
EDCI and TEA were added, stirred for 30min at room temperature, and then allowed to react for 12h.
The reaction was monitored by thin layer chromatography until complete consumption of compound 6. Developing agent: PE ea=1:3, the product has stronger fluorescence (rf=0.1).
The reaction solution was subjected to rotary evaporation to remove the solvent, and the mixture was separated by silica gel column chromatography (PE: ea=1:3) to obtain a fluorescent label. The yield was 62%.
Referring to FIG. 3, a nuclear magnetic resonance spectrum of the compound of formula 6 is shown. The nuclear magnetic spectrum is: 1H NMR (600 MHz, CDCl 3) delta 10.98 (s, 1H), 8.43 (d, J=9.0 Hz, 1H), 7.45 (d, J=2.4 Hz, 1H), 7.38-7.30 (m, 1H), 7.13 (bs, 1H), 7.01 (s, 1H), 6.80 (d, J=4.2 Hz, 1H), 6.52 (bs, 1H), 6.19 (d, J=4.2 Hz, 1H), 6.09-6.01 (m, 2H), 5.05 (bs, 1H), 3.24-3.12 (m, 4H), 2.48 (s, 3H), 2.34 (s, 3H), 2.21 (s, 3H), 2.18 (s, 3H), 1.41-1.32 (m, 4H), 1.18 (s, 6H).
Example 5
Cell culture
DC cells were normally cultured in 10% FBS+RPMI-1640 medium and when grown to 80% or more, passaged. The cell culture supernatant was discarded, and the cells were washed with 2mL of PBS and discarded. 700uL of 0.25% trypsin was added, and the mixture was placed in a CO2 incubator for about 1.5min, and the morphology of the cells was observed under a microscope to round, and the flask was gently tapped to detach the cells. Cells were completely detached by adding 2mL of 10% FBS+RPMI-1640 medium. The cell mixture was pipetted into a sterilized 15mL centrifuge tube and centrifuged at 1000rpm for 3min at room temperature. The supernatant was discarded, resuspended in 1mL of complete medium, and 10uL of the cell suspension was added to 1uL of trypan blue, mixed well, and counted in a cell counting plate.
Example 6
On the basis of example 5, the cell viability is ensured to be more than 90%, 6 pore plates are paved, and each pore is 6 multiplied by 10 5 Individual cells. Cell wall-attached supernatant was changed to complete medium, adenovirus in which the following 3 CD91 interference sequences pAVE-3576, pAVE-3577 and pAVE-3578 were respectively packed and Control plasmid pAVE-Control adenovirus supernatant in which the Control plasmid pAVE-Control adenovirus was packed were added, the samples were collected after 24 hours of culture, total RNA was extracted, cDNA was reverse transcribed, and qPCR analysis was performed using the CD91 primer sequences shown in the following table and GAPDH inner primer sequences, to verify the interference effect of the 3 CD91 interference sequences on CD 91.
Table 13 CD91 interference sequences are listed below
The primer sequences of the genes in Table 2 are as follows:
| gene | Forward(5’-3’) | Reverse(5’-3’) |
| CD91 | CTGGCCTATCACCGTGGCTG | GACGGTCTCACGCTCGAAGG |
| MHC-I | CTACAACCAGAGCGAGGCCG | AATCCTTGCCGTCGTAGGCG |
| GAPDH | GCGGGGCTCTCCAGAACATC | TCCACCACTGACACGTTGGC |
Referring to FIG. 4, the differences in CD91 RNA interference from qPCR analysis of 3 sequences are shown. The figure shows the RNA transcription level of each group of CD91, and experimental results show that the 3 rd sequence pAVE3578 has the best interference effect, and can be used in subsequent experiments.
Example 7
Flow analysis confirmed the interfering effect of pAVE3578 plasmid on CD91 RNA.
On the basis of example 6, after 24 hours of culture, the pAVE3578 group with the best interference effect was sampled and the CD91 expression level was detected in a flow manner.
The experimental steps are as follows:
1. cells were harvested, centrifuged at 1000rpm for 5 minutes at 4℃and the supernatant discarded.
2. Cells were resuspended in 50mL staining Buffer and counted and assayed for cell viability using Trypan Blue (Trypan Blue).
3. Centrifuging the cell fluid and discarding the supernatant; resuspension of cells with a starting Buffer to adjust the cell concentration to 2X 10 7 And each mL.
4. 50. Mu.L of diluted primary antibody (diluted to the appropriate concentration with a starting Buffer) was added to each flow tube; 50 μ of a Buffer for stabilizing was added to the blank tube.
5. To each tube, 50. Mu.L of the cell suspension was added and gently mixed.
6. Incubate for 20 min at 4℃in the dark.
7. After the incubation was completed, centrifugation was carried out at 1000rpm for 5 minutes at 4℃and the supernatant was discarded.
8. The wash was repeated 3 times with 100 μ L Staining Buffer.
9. The cells were resuspended with 100. Mu. L Staining Buffer and then detected by an upflow instrument.
Data analysis was performed by graphpad prism 8, significance analysis was performed with Bonferroni's multiple comparisons test, alpha=5.000%, and then statistical analysis was performed. Referring to FIG. 5, a flow assay is shown to confirm pAVE3578 sequence versus CD91 RNA interference. After treatment of DC cells with interfering adenoviruses, pAVE3578 showed significantly lower expression compared to the non-interfering group CD91 compared to the control group.
Example 8
Total RNA extraction experiments
The method comprises the following steps:
the culture solution in the 6-well plate was aspirated, 1ml Trizol Reagent was added to each well, and the cells were completely lysed by blowing with a gun head.
The lysate was transferred to a 1.5ml EP tube and left at room temperature for 10 minutes.
200. Mu.L of chloroform was added thereto, mixed with vigorous shaking, and left at room temperature for 10 minutes.
Centrifuge at 12000rpm at 4℃for 10min, aspirate the supernatant into a new centrifuge tube, add an equal volume of isopropanol and pellet at room temperature for 10min.
Centrifuge at 12000rpm at 4℃for 15 min, discard supernatant.
The precipitate was washed once with 500 μl of 75% ethanol. Centrifugation was carried out at 12000rpm at 4℃for 5 minutes, the precipitate was recovered, and the supernatant was discarded. And (5) at normal temperature, inversely airing for 10 minutes.
With 20. Mu. LDEPC-H 2 O was dissolved and precipitated, OD260 and OD280 were measured, and the RNA concentration was calculated.
Agarose electrophoresis checked the integrity of the RNA.
Example 9
Real-time PCR analysis
The primer design is shown in Table 2,
| gene | Forward(5’-3’) | Reverse(5’-3’) |
| CD91 | CTGGCCTATCACCGTGGCTG | GACGGTCTCACGCTCGAAGG |
| MHC-I | CTACAACCAGAGCGAGGCCG | AATCCTTGCCGTCGTAGGCG |
| GAPDH | GCGGGGCTCTCCAGAACATC | TCCACCACTGACACGTTGGC |
Reverse transcription reaction system
The reverse transcription procedure was: 30min at 42 ℃; and at 85 ℃ for 10min.
Real-time fluorescent quantitative reaction system
The quantitative PCR reaction procedure was: denaturation at 95℃for 5 min; 95 ℃ for 12 seconds; cycling at 60 ℃,40 seconds and 40 cycles; 95 ℃ for 10 seconds; 60 ℃ for 10 seconds; 40℃for 30 seconds.
The above was performed by a real-time fluorescent quantitative PCR instrument (Applied Biosystems,7900, u.s.).
Example 10
The fluorescent-labeled small molecule compounds treat DC cells.
Ensuring the cell viability to be more than 90 percent, paving 6 pore plates, and ensuring each pore to be 6 multiplied by 10 5 Individual cells. Removing supernatant after cell adhesion, changing into complete culture medium, respectively adding Control plasmid pAVE-Control adenovirus supernatant and 3 rd sequence pAVE3578 adenovirus supernatant, and mixing; after incubation for 24h, cells (60. Mu.M) were treated with the fluorescent labeling compound, photographed at 3, 6, 12, 24, 48 hours, and cell samples were collected and the fluorescence intensity of the cells was measured using a flow cytometer.
Referring to FIG. 6, a fluorescent photograph of DC cells after treatment with the compounds provided in the examples of the present invention is shown. As shown, photographs of the same field of view under white light and fluorescence at each time point are shown. The difference between 24 and 48 hours after DC cells treated with small molecule compounds was most visible to the naked eye.
Example 11
Flow detection of cell fluorescence
On the basis of example 8, DC cell samples were collected at various time points and the fluorescence intensity of cells was measured by flow cytometry.
The experimental procedure was as follows:
cells were harvested, centrifuged at 1000rpm for 5 minutes at 4℃and the supernatant discarded.
Cells were resuspended in 100. Mu.L PBS and counted and assayed for cell viability using Trypan Blue (Trypan Blue).
Centrifuging the cell fluid and discarding the supernatant; the cells were resuspended in PBS and the cell concentration was adjusted to 2X 10 7 And each mL. The cells were detected by flow up meter. Significance analysis was performed at α=0.05.
The change in fluorescence intensity is analyzed by flow cytometry as shown in FIG. 7. After RNA interference of CD91 (pAVE-3587 group) by treating DC cells with small molecule compound for 24 and 48h, the fluorescence intensity was significantly higher than that of the CD91 RNA non-interference group (pAVE-Control group) (p < 0.05), and the fluorescence intensity of the experimental group and the Control group at the rest of time points were not significantly different (p > 0.05).
Example 12
Detection of MHC expression level
MHC (major histocompatibility complex) is a tool for presenting antigens to DC cells; upregulation of expression of MHC in DC cells is an important marker for enhanced antigen presenting capacity.
DC cells were treated with fluorescent-labeled small molecule compounds, and DC cell samples were collected at 6,16,24 hours on the basis of example 8, total RNA extraction was performed as described in example 8, real-time PCR analysis was performed as described in example 9, and MHC-I primer sequences are shown in Table 2.
Referring to FIG. 8, a qPCR detection MHC expression level graph is provided according to an embodiment of the present invention. MHC expression levels on DC cells were examined at 6,16,24 hours and found to be up-regulated at 16 hours after the application of small molecules.
Although the exemplary aspects of the present disclosure have been described for illustrative purposes, those skilled in the art will appreciate that the foregoing description is merely illustrative of preferred embodiments of the invention and is not intended to limit the scope of the invention in any way, including additional implementations in which functions may be performed out of the order of presentation or discussion. Any alterations and modifications of the present invention, which are made by those of ordinary skill in the art based on the above disclosure, are intended to be within the scope of the appended claims.
Claims (10)
1. A compound of 5- ((2-carbamoyl-4-chlorophenyl) amino) -3, 3-dimethyl-5-oxopentanoic acid compound having the structure shown in formula 1, which is useful as a chimeric antigen with enhanced additional immunogenicity.
2. A process for the preparation of a compound as claimed in claim 1,
the method comprises the following steps: the compound having the structure shown in formula 2 and the compound having the structure shown in formula 3 are reacted in a solvent of 1, 4-dioxane.
3. The preparation method according to claim 2, characterized in that: the molar ratio of the compound having the structure shown in formula 2 to the compound having the structure shown in formula 3 is 1: (1.5-2.0).
4. The preparation method according to claim 2, characterized in that: the compound with the structure shown in the formula 2 reacts with the compound with the structure shown in the formula 3 for 18-20 hours at the temperature of 100-120 ℃.
5. Use of a 5- ((2-carbamoyl-4-chlorophenyl) amino) -3, 3-dimethyl-5-oxopentanoic acid compound according to claim 1 in cellular immunotherapy, wherein: by fluorescence labelling, it was demonstrated that the compounds were able to reach the purpose of entering DC cells by activating the CD91 molecule.
6. A compound, namely a BODIPY fluorescent marker of 5- ((2-carbamoyl-4-chlorophenyl) amino) -3, 3-dimethyl-5-oxopentanoic acid, has a structure shown in a formula 6.
7. A preparation method of a 5- ((2-carbamoyl-4-chlorophenyl) amino) -3, 3-dimethyl-5-oxopentanoic acid BODIPY fluorescent marker,
the method is characterized by comprising the following steps of:
(1) obtaining an intermediate I with a structure of formula 4 by reacting a compound with a structure of formula 1 with 4-Dimethylaminopyridine (DMAP);
(2) dissolving intermediate I in a solvent and reacting with trifluoroacetic acid (TFA) to obtain intermediate II having the structure of formula 5;
(3) from intermediate II and a BODIPY-CO dye 2 H) The reaction yields a fluorescent label having the structure of formula 6.
8. The method of manufacturing according to claim 6, wherein: the specific steps in the step (1) comprise: adding the compound shown in the formula 1 and DMAP into a dry flask, adding N-BOC-1, 4-butanediamine, cooling in an ice bath, adding EDCI and TEA, keeping the temperature and stirring for 30min, and then moving to room temperature for reaction.
9. The method of manufacturing according to claim 6, wherein: the specific steps in the step (2) comprise: the intermediate I was added to a dry flask, dissolved in dichloromethane, TFA was added and the reaction was stirred at room temperature.
10. The method of manufacturing according to claim 6, wherein: the specific steps in the step (3) comprise: intermediate II, BODIPY-CO 2 H and BtOH are added into a dry flask, dissolved by THF and cooled by ice bath; EDCI and TEA were added, stirred for 30min at room temperature, and then allowed to react at room temperature.
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