A method for achieving protein or virus labeling

Abstract

The present invention provides a method for realizing protein or virus labeling, using quantum dots or fluorescent proteins to label proteins or viruses, coupling hydrazine fragments with amino groups on proteins or viruses, coupling aldehyde fragments with amino groups on quantum dots or fluorescent proteins, and then coupling aldehyde fragments with hydrazine fragments connected with different components to realize the labeling of proteins or viruses; hydrazine fragments are prepared with 6-chloronicotinic acid as raw materials. The synthetic routes of hydrazine fragments and aldehyde fragments of the present invention, no high cytotoxic byproducts are produced, high safety factor, high reaction yield, simple separation and purification; the method has wide applicability, mild reaction conditions, good selectivity, high coupling efficiency, less fluorescence quenching of fluorescent proteins or quantum dots, and can realize the visualization of proteins; realize the directional coupling of fluorescent protein-protein, fluorescent protein-virus, quantum dot-protein or quantum dot-virus, and no by-product production of the complex coupled between protein and protein, virus and virus, quantum dot and quantum dot.

Description

Technical Field

[0001] The invention belongs to the field of organic synthesis, and in particular relates to a method for realizing protein or virus labeling. Background Art

[0002] Proteins are the most important functional molecules in life activities. The specific interactions between proteins constitute an important part of the intracellular biochemical reaction network. The study of the interaction mechanism between proteins can deepen our understanding of the laws of life activities. Quantum dots (QDs) are semiconductor nanocrystals with high brightness and photostability. They are ideal detection and tracking labels that can be used for visual tracking of proteins. Similarly, the cross-linking technology of proteins (or viruses) and fluorescent proteins is widely used in the fields of cell biology, immunology, molecular biology, etc. For example, by binding fluorescent protein-labeled antibodies to specific proteins, the localization and expression level of proteins can be detected; by binding fluorescent protein-labeled antibodies to cells, cell tracking and imaging can be achieved.

[0003] Protein or virus labeling technology uses specific chemical reagents to fix and bind proteins (viruses) or proteins (viruses) to other substances. During the cross-linking process, the formation of covalent bonds may be accompanied by the elimination of some groups or the addition of some groups. Currently, commonly used chemical cross-linkers are divided into four categories. The first category, cross-linkers that react with primary amine groups (such as N-hydroxysuccinimide), are widely used to form stable covalent bonds with the N-terminus and ε-amino groups on protein lysine residues, and have high reaction efficiency. The second category, thiol-reactive cross-linkers, can modify the thiol groups on the target protein by alkylation (haloacetyl) or disulfide exchange (maleimide, pyridine dithiol). The third category of cross-linkers can cross-link the C-terminal carboxyl group of the protein with the side chain carboxyl groups of aspartic acid and glutamic acid (such as formaldehyde, glutaraldehyde). Finally, the fourth category is mainly photoreactive cross-linkers (diazides, aromatic azides) that form cross-links through ultraviolet light activation. Its advantage is that the reaction strength can be adjusted by controlling the time and intensity of the light source. The use of the above-mentioned chemical cross-linking agents will result in many by-products, such as by-products of complexes between antibodies and complexes between fluorescent proteins, etc., and the coupling efficiency and selectivity are low, which can easily cause problems such as fluorescence quenching of luminescent materials.

[0004] In summary, developing a method for protein or virus labeling with low cytotoxicity, high selectivity, simple synthesis, low cost, and low fluorescence quenching has become an urgent problem to be solved. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for achieving protein or virus labeling. The method provided by the present invention has the advantages of low cost and energy consumption, safe production, no highly cytotoxic byproducts, and achieves directional labeling of proteins or viruses, without the byproducts of complexes between antibodies and other complexes such as between fluorescent proteins. To achieve this purpose of the invention, the present invention adopts the following technical solutions:

[0006] The object of the present invention is to provide a method for achieving protein or virus labeling, wherein the protein or virus is labeled by using quantum dots or fluorescent proteins, a hydrazine fragment is coupled with an amino group on the protein or virus, an aldehyde fragment is coupled with an amino group on the quantum dots or fluorescent proteins, and then an aldehyde fragment connected with different components is coupled with a hydrazine fragment, thereby achieving protein or virus labeling; the hydrazine fragment is prepared using 6-chloronicotinic acid as a raw material;

[0007]

[0008] Wherein, R1 and R2 are each independently selected from H, alkyl, nitro, trifluoromethyl, trifluoroethyl, -SO4Na, -CH2SO4Na, -C2H4SO4Na, wherein the number of carbon atoms in the alkyl is not greater than 6.

[0009] The present invention marks proteins or viruses, thereby realizing visualization of proteins or viruses, and can realize coupling of fluorescent protein-protein, fluorescent protein-virus, quantum dot-protein, and quantum dot-virus. The protein can be selected from one of human serum albumin (HSA), calf serum albumin (BSA), chicken ovalbumin (OVA), etc.; the virus can be selected from one of influenza A virus (H9N2, H1N1), tobacco mosaic virus (TMV), etc.; the fluorescent protein can be selected from one of green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), etc.; quantum dots (QDs) are amino-modified semiconductor quantum dots, which can be selected from one of cadmium selenide quantum dots (CdSe QDs-NH2), cadmium sulfide quantum dots (CdS QDs-NH2), zinc selenide quantum dots (ZnSe QDs-NH2), etc.; the types of the above proteins, viruses, fluorescent proteins and quantum dots are not limited to the types listed above, and other types not listed are also applicable.

[0010] Specifically, the preparation method of the hydrazine fragment comprises the following steps:

[0011] (1) subjecting 6-chloronicotinic acid to a substitution reaction with hydrazine hydrate to obtain intermediate A;

[0012] (2) adding acetone and p-toluenesulfonic acid to intermediate A for condensation to obtain intermediate B;

[0013] (3) reacting the intermediate B, the R1-substituted N-hydroxysuccinimide and a condensing agent to generate a hydrazine fragment;

[0014]

[0015] Preferably, in step (1), the molar ratio of 6-chloronicotinic acid to hydrazine hydrate is 1:(2-2.5);

[0016] Preferably, in step (1), the reaction temperature is reflux temperature, and the reaction time is 4-6 h; more preferably, the reaction time can be 4 h, 4.2 h, 4.4 h, 4.6 h, 4.8 h, 5 h, 5.2 h, 5.4 h, 5.6 h, 5.8 h or 6 h, but is not limited to the values ​​listed above, and other values ​​not listed within the above numerical range are also applicable.

[0017] Preferably, in step (2), the molar ratio of the intermediate A to p-toluenesulfonic acid is 1:(0.05-0.1).

[0018] Preferably, in step (2), acetone is used as the raw material and the solvent, and the mass volume ratio of the intermediate A to the acetone is 1 g:(8-12) mL.

[0019] Preferably, in step (2), the reaction temperature is reflux temperature, and the reaction time is 8-12 h; more preferably, the reaction time can be 8 h, 8.5 h, 9 h, 9.5 h, 10 h, 10.5 h, 11 h, 11.5 h or 12 h, etc., but are not limited to the values ​​listed above, and other values ​​not listed within the above numerical range are also applicable.

[0020] Preferably, in step (3), the molar ratio of the intermediate B, R1-substituted N-hydroxysuccinimide and the condensing agent is 1:(1.1-1.2):(1.2-1.5).

[0021] Preferably, the condensing agent is one or more selected from DCC, EDCI, HBTU, and CDI;

[0022] Preferably, in step (3), the reaction temperature is 25-30°C, and the reaction time is 3-6h; more preferably, the reaction temperature can be 25°C, 26°C, 27°C, 28°C, 29°C or 30°C, and the reaction time can be 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6h, but are not limited to the values ​​listed above, and other values ​​not listed within the above numerical range are also applicable.

[0023] Specifically, the preparation method of the aldehyde fragment comprises the following steps: 4-formylbenzoic acid, R2-substituted N-hydroxysuccinimide and a condensing agent react to generate an aldehyde fragment;

[0024]

[0025] Preferably, the molar ratio of the 4-formylbenzoic acid, the R2-substituted N-hydroxysuccinimide and the condensing agent is 1:(1.1-1.2):(1.2-1.5);

[0026] Preferably, the condensing agent is one or more selected from DCC, EDCI, HBTU, and CDI;

[0027] Preferably, the reaction temperature is 25-30°C, and the reaction time is 3-6h; more preferably, the reaction temperature can be 25°C, 26°C, 27°C, 28°C, 29°C or 30°C, and the reaction time can be 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6h, but are not limited to the values ​​listed above, and other values ​​not listed within the above numerical range are also applicable.

[0028] Compared with the prior art, the present invention provides a method for achieving protein or virus labeling, which has the following characteristics:

[0029] Beneficial effects:

[0030] (1) The present invention prepares hydrazine fragments and aldehyde fragments, which are used to couple with amino groups on proteins (viruses) and fluorescent proteins (quantum dots), respectively, to achieve labeling of proteins or viruses;

[0031] (2) The synthetic routes of the hydrazine fragment and the aldehyde fragment of the present invention do not produce highly cytotoxic byproducts, have a high safety factor, a high reaction yield, and are simple to separate and purify; the method for labeling such proteins or viruses has wide applicability, mild reaction conditions, good selectivity, high coupling efficiency, little fluorescence quenching of fluorescent proteins or quantum dots, and can achieve protein visualization; the directional coupling of fluorescent protein-protein, fluorescent protein-virus, quantum dot-protein, or quantum dot-virus is achieved, and no byproduct of complexes coupled between proteins, viruses, or quantum dots is produced. DETAILED DESCRIPTION

[0032] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0033] Example 1

[0034] This embodiment provides a method for achieving protein or virus labeling, comprising the following steps:

[0035] The preparation method of the hydrazine fragment is as follows:

[0036] (1) 6-Chloronicotinic acid (10 g, 63.47 mmol, 1.0 eq), hydrazine hydrate (8.03 g, 126.94 mmol, 2.0 eq, wt: 80%) and water were mixed and reacted at reflux temperature for 4 h. DCM was added for extraction three times. The organic phases were combined, dried, filtered and concentrated to obtain a crude product. Excessive methanol solution of hydrogen chloride was added to form a salt, and 11 g of a white solid, namely intermediate A, was obtained by filtration. The yield was 92%.

[0037] (2) Add intermediate A (12 g, 63.29 mmol, 1.0 eq) and p-toluenesulfonic acid (1.09 g, 6.33 mmol, 0.05 eq) to 100 mL of acetone, heat under reflux for 8 hours, and after the reaction is complete, concentrate and remove the solvent, add DCM to redissolve, add saturated sodium bicarbonate solution to wash, separate the liquids, extract the aqueous phase with DCM three times, combine the organic phases and dry them to obtain 10.88 g of white solid, namely intermediate B, with a yield of 89%;

[0038] (3) Sodium N-hydroxysuccinimide-sulfonate (9.89 g, 45.57 mmol, 1.1 eq) and DCC (10.26 g, 49.72 mmol, 1.2 eq) were added to the intermediate B (8 g, 41.43 mmol, 1.0 eq), and the mixture was reacted at room temperature for 3 h. The mixture was filtered, washed with saturated sodium bicarbonate solution, separated, and the aqueous phase was extracted with DCM three times. The organic phases were combined, dried, concentrated, and desolventized. MeOH was added to slurry to obtain 14.78 g of a white solid, i.e., the hydrazine fragment. Yield: 91%.

[0039] The preparation method of the aldehyde fragment is as follows: 4-formylbenzoic acid (10 g, 66.65 mmol, 1.0 eq) and sodium N-hydroxysuccinimide-sulfonate (15.92 g, 73.32 mmol, 1.1 eq) and DCC (16.50 g, 79.98 mmol, 1.2 eq) were reacted at room temperature for 3 h, filtered, washed with saturated sodium bicarbonate solution, separated, and the aqueous phase was extracted with DCM three times. The organic phases were combined, dried, concentrated and desolvated, and MeOH was added to slurry to generate 20.25 g of aldehyde fragment. Yield: 87%.

[0040] The method for achieving protein or virus labeling comprises the following steps:

[0041] Add 5 μL of 50 mM hydrazine fragment solution to 500 μg protein, mix well, incubate at 25 ° C for 2 h, then transfer the reaction solution to an ultrafiltration centrifuge tube, add 500 μL of PBS (pH = 7.4) buffer, mix well and centrifuge, repeat this operation 5 times, add PBS (pH = 7.4) buffer to 30 μL, and obtain hydrazine fragment-protein; similarly, add 5 μL of 50 mM aldehyde fragment solution to 500 μg fluorescent protein, mix well, incubate at 25 ° C for 2 h, then transfer the reaction solution to an ultrafiltration centrifuge tube, add 500 μL of PBS (pH = 7.4) buffer, mix well and centrifuge, repeat this operation 5 times, add PBS (pH = 7.4) buffer to 30 μL, and obtain aldehyde fragment-fluorescent protein. Finally, mix the hydrazine fragment with different components and the aldehyde fragment, incubate at room temperature for 2 h to achieve protein-fluorescent protein coupling, and obtain Thus, protein labeling is achieved.

[0042] Example 2

[0043] This example provides a method for achieving protein or virus labeling, which is basically the same as Example 1, except that the steps for preparing the hydrazine fragment are as follows:

[0044] (1) 6-Chloronicotinic acid (10 g, 63.47 mmol, 1.0 eq), hydrazine hydrate (10.04 g, 158.68 mmol, 2.5 eq, wt: 80%) and water were mixed and reacted at reflux temperature for 4 h. DCM was added for extraction three times. The organic phases were combined, dried, filtered and concentrated to obtain a crude product. Excessive methanol solution of hydrogen chloride was added to form a salt, and 11.24 g of a white solid, namely intermediate A, was obtained by filtration. The yield was 94%;

[0045] (2) Intermediate A (12 g, 63.29 mmol, 1.0 eq) and p-toluenesulfonic acid (2.18 g, 12.66 mmol, 0.1 eq) were added to 150 mL of acetone and heated under reflux for 10 hours. After the reaction was completed, the mixture was concentrated and desolventized, and DCM was added for redissolution. Saturated sodium bicarbonate solution was added for washing, and the mixture was separated. The aqueous phase was extracted with DCM for 3 times. The organic phases were combined and dried to obtain 11.25 g of a white solid, namely, intermediate B. The yield was 92%.

[0046] (3) Sodium N-hydroxysuccinimide-sulfonate (10.79 g, 49.71 mmol, 1.2 eq) and DCC (11.12 g, 53.86 mmol, 1.3 eq) were added to the intermediate B (8 g, 41.43 mmol, 1.0 eq), and the mixture was reacted at room temperature for 5 h. The mixture was filtered, washed with saturated sodium bicarbonate solution, separated, and the aqueous phase was extracted with DCM three times. The organic phases were combined, dried, concentrated, and desolventized. MeOH was added to slurry to obtain 14.94 g of a white solid, i.e., the hydrazine fragment. Yield: 92%.

[0047] Example 3

[0048] This example provides a method for achieving protein or virus labeling, which is basically the same as Example 1, except that the steps for preparing the hydrazine fragment are as follows:

[0049] (1) 6-Chloronicotinic acid (10 g, 63.47 mmol, 1.0 eq), hydrazine hydrate (8.83 g, 139.63 mmol, 2.2 eq, wt: 80%) and water were mixed and reacted at reflux temperature for 4 h. DCM was added for extraction three times. The organic phases were combined, dried, filtered and concentrated to obtain a crude product. Excessive methanol solution of hydrogen chloride was added to form a salt, and 11.12 g of a white solid, namely intermediate A, was obtained by filtration. The yield was 93%;

[0050] (2) Add intermediate A (12 g, 63.29 mmol, 1.0 eq) and p-toluenesulfonic acid (1.79 g, 9.50 mmol, 0.075 eq) to 100 mL of acetone, heat under reflux for 12 hours, and after the reaction is complete, concentrate and remove the solvent, add DCM to redissolve, add saturated sodium bicarbonate solution to wash, separate the liquids, extract the aqueous phase with DCM three times, combine the organic phases and dry them to obtain 11.00 g of white solid, namely intermediate B, with a yield of 90%;

[0051] (3) Sodium N-hydroxysuccinimide-sulfonate (10.34 g, 47.64 mmol, 1.15 eq) and DCC (12.83 g, 62.15 mmol, 1.5 eq) were added to the intermediate B (8 g, 41.43 mmol, 1.0 eq), and the mixture was reacted at room temperature for 5 h. The mixture was filtered, washed with saturated sodium bicarbonate solution, separated, and the aqueous phase was extracted with DCM three times. The organic phases were combined, dried, concentrated, and desolventized. MeOH was added to slurry to obtain 15.26 g of a white solid, i.e., the hydrazine fragment. Yield: 94%.

[0052] Example 4

[0053] This example provides a method for achieving protein or virus labeling, which is basically the same as Example 1, except that the steps for preparing the aldehyde fragment are as follows:

[0054] 4-Formylbenzoic acid (10 g, 66.65 mmol, 1.0 eq) was reacted with sodium N-hydroxysuccinimide-sulfonate (15.92 g, 73.32 mmol, 1.1 eq) and EDCI (17.75 g, 93.32 mmol, 1.4 eq) at room temperature for 3 h, filtered, washed with saturated sodium bicarbonate solution, separated, and the aqueous phase was extracted with DCM three times. The organic phases were combined, dried, concentrated and desolvated, and MeOH was added to slurry to generate 20.72 g of aldehyde fragments. Yield: 89%.

[0055] Example 5

[0056] This example provides a method for achieving protein or virus labeling, which is basically the same as Example 1, except that the steps for preparing the hydrazine fragment are as follows:

[0057] (3) Add N-hydroxysuccinimide-isopropyl (7.49 g, 47.64 mmol, 1.15 eq) and DCC (12.83 g, 62.15 mmol, 1.5 eq) to the intermediate B (8 g, 41.43 mmol, 1.0 eq), react at room temperature for 4 h, filter, add saturated sodium bicarbonate solution for washing, separate the liquids, extract the aqueous phase with DCM three times, combine the organic phases, dry, concentrate and desolventize, add MeOH for pulping, and obtain 12.94 g of white solid, i.e., hydrazine fragment Yield: 94%.

[0058] The method for achieving protein or virus labeling comprises the following steps:

[0059] Add 5 μL of 50 mM hydrazine fragment solution to 500 μg of virus, mix well, incubate at 25 ° C for 2 h, then transfer the reaction solution to an ultrafiltration centrifuge tube, add 500 μL of PBS (pH = 7.4) buffer, mix well and centrifuge, repeat this operation 5 times, add PBS (pH = 7.4) buffer to 30 μL, and obtain hydrazine fragment-virus; similarly, add 5 μL of 50 mM aldehyde fragment solution to 500 μg of quantum dots, mix well, incubate at 25 ° C for 2 h, then transfer the reaction solution to an ultrafiltration centrifuge tube, add 500 μL of PBS (pH = 7.4) buffer, mix well and centrifuge, repeat this operation 5 times, add PBS (pH = 7.4) buffer to 30 μL, and obtain aldehyde fragment-quantum dots. Finally, mix the hydrazine fragment with different components and the aldehyde fragment, incubate at room temperature for 2 h to achieve virus-quantum dot coupling, and obtain This achieves virus labeling.

[0060] Example 6

[0061] This example provides a method for achieving protein or virus labeling, which is basically the same as Example 1, except that the steps for preparing the hydrazine fragment are as follows:

[0062] (3) Add N-hydroxysuccinimide-pentyl (8.44 g, 45.57 mmol, 1.1 eq) and DCC (12.83 g, 62.15 mmol, 1.5 eq) to the intermediate B (8 g, 41.43 mmol, 1.0 eq), react at room temperature for 6 h, filter, add saturated sodium bicarbonate solution for washing, separate the liquids, extract the aqueous phase with DCM three times, combine the organic phases, dry, concentrate and desolventize, add MeOH for pulping, and obtain 13.74 g of white solid, i.e., hydrazine fragment Yield: 92%.

[0063] The method for achieving protein or virus labeling comprises the following steps:

[0064] Add 5 μL of 50 mM hydrazine fragment solution to 500 μg protein, mix well, incubate at 25 ° C for 2 h, transfer the reaction solution to an ultrafiltration centrifuge tube, add 500 μL of PBS (pH = 7.4) buffer, mix well and centrifuge, repeat this operation 5 times, add PBS (pH = 7.4) buffer to 30 μL, and obtain hydrazine fragment-protein; similarly, add 5 μL of 50 mM aldehyde fragment solution to 500 μg quantum dots, mix well, incubate at 25 ° C for 2 h, transfer the reaction solution to an ultrafiltration centrifuge tube, add 500 μL of PBS (pH = 7.4) buffer, mix well and centrifuge, repeat this operation 5 times, add PBS (pH = 7.4) buffer to 30 μL, and obtain aldehyde fragment-quantum dots. Finally, mix the hydrazine fragment with different components and the aldehyde fragment, incubate at room temperature for 2 h to achieve protein-quantum dot coupling, and obtain Thus, protein labeling is achieved.

[0065] Example 7

[0066] This example provides a method for achieving protein or virus labeling, which is basically the same as Example 1, except that the steps for preparing the hydrazine fragment are as follows:

[0067] (3) N-hydroxysuccinimide-trifluoromethyl (8.34 g, 45.57 mmol, 1.1 eq) and DCC (12.83 g, 62.15 mmol, 1.5 eq) were added to the above intermediate B (8 g, 41.43 mmol, 1.0 eq), reacted at room temperature for 5 h, filtered, washed with saturated sodium bicarbonate solution, separated, and the aqueous phase was extracted with DCM three times. The organic phases were combined, dried, concentrated and desolvated, and MeOH was added to slurry to obtain 13.80 g of white solid, i.e., hydrazine fragment Yield: 93%.

[0068] The method for achieving protein or virus labeling comprises the following steps:

[0069] Add 5 μL of 50 mM hydrazine fragment solution to 500 μg of virus, mix well, incubate at 25 ° C for 2 h, transfer the reaction solution to an ultrafiltration centrifuge tube, add 500 μL of PBS (pH = 7.4) buffer, mix well and centrifuge, repeat this operation 5 times, add PBS (pH = 7.4) buffer to 30 μL, and obtain hydrazine fragment-virus; similarly, add 5 μL of 50 mM aldehyde fragment solution to 500 μg of fluorescent protein, mix well, incubate at 25 ° C for 2 h, transfer the reaction solution to an ultrafiltration centrifuge tube, add 500 μL of PBS (pH = 7.4) buffer, mix well and centrifuge, repeat this operation 5 times, add PBS (pH = 7.4) buffer to 30 μL, and obtain aldehyde fragment-fluorescent protein. Finally, mix the hydrazine fragment with different components and the aldehyde fragment, incubate at room temperature for 2 h to achieve virus-fluorescent protein coupling, and obtain This achieves virus labeling.

[0070] Comparative Example 1

[0071] This comparative example provides a method for achieving protein or virus labeling, which is basically the same as Example 1, except that the preparation steps of the hydrazine fragment are as follows:

[0072] (1) 6-Chloronicotinic acid (10 g, 63.47 mmol, 1.0 eq), hydrazine hydrate (6.0 g, 95.21 mmol, 1.5 eq, wt: 80%) and water were mixed and reacted at reflux temperature for 4 h. DCM was added for extraction three times. The organic phases were combined, dried, filtered and concentrated to obtain a crude product. Excessive amount of methanolic hydrogen chloride solution was added to form a salt. The crude product was filtered to obtain 7.53 g of a white solid, namely, intermediate A, with a yield of 63%.

[0073] Comparative Example 2

[0074] This comparative example provides a method for achieving protein or virus labeling, which is basically the same as Example 1, except that the preparation steps of the hydrazine fragment are as follows:

[0075] (1) 6-Chloronicotinic acid (10 g, 63.47 mmol, 1.0 eq), hydrazine hydrate (12.0 g, 190.42 mmol, 3 e.q., wt: 80%) and water were mixed and reacted at reflux temperature for 4 h, and DCM was added for extraction three times. The organic phases were combined, dried, filtered and concentrated to obtain a crude product, and then an excess of hydrogen chloride methanol solution was added to form a salt, and 10.8 g of a white solid was obtained by filtration, namely, intermediate A, with a yield of 90%. Experiments have found that the addition of hydrazine hydrate is not beneficial to the improvement of the yield.

[0076] Comparative Example 3

[0077] This comparative example provides a method for achieving protein or virus labeling, which is basically the same as Example 1, except that the preparation steps of the hydrazine fragment are as follows:

[0078] (2) Add intermediate A (12 g, 63.29 mmol, 1.0 eq) and p-toluenesulfonic acid (0.55 g, 3.17 mmol, 0.025 eq) to 100 mL of acetone and heat under reflux for 12 hours. After the reaction is complete, concentrate and remove the solvent. Add DCM to redissolve the solvent, add saturated sodium bicarbonate solution to wash, separate the liquids, extract the aqueous phase with DCM three times, combine the organic phases and dry them to obtain 7.33 g of white solid, namely intermediate B, with a yield of 60%.

[0079] Comparative Example 4

[0080] This comparative example provides a method for achieving protein or virus labeling, which is basically the same as Example 1, except that the preparation steps of the hydrazine fragment are as follows:

[0081] (2) Intermediate A (12 g, 63.29 mmol, 1.0 eq) and p-toluenesulfonic acid (0.55 g, 3.17 mmol, 0.2 eq) were added to 100 mL of acetone, heated under reflux for 12 hours, and the reaction was completed. The mixture was concentrated and desolventized, and DCM was added for redissolution. The mixture was washed with saturated sodium bicarbonate solution, separated, and the aqueous phase was extracted with DCM for 3 times. The organic phases were combined and dried to obtain 10.76 g of white solid, namely, intermediate B, with a yield of 88%. It was found that the addition of p-toluenesulfonic acid did not improve the yield.

[0082] Comparative Example 5

[0083] This comparative example provides a method for achieving protein or virus labeling, which is basically the same as Example 1, except that the preparation steps of the aldehyde fragment are as follows: 4-formylbenzoic acid (10 g, 66.65 mmol, 1.0 eq) and sodium N-hydroxysuccinimide-sulfonate (15.92 g, 73.32 mmol, 1.1 eq) and DCC (13.75 g, 66.65 mmol, 1 e.q.) are reacted at room temperature for 3 h, filtered, washed with saturated sodium bicarbonate solution, separated, and the aqueous phase is extracted with DCM 3 times, the organic phases are combined, dried, concentrated and desolvated, and MeOH is added for pulping to generate 15.59 g of aldehyde fragments. Yield: 67%.

[0084] The applicant declares that the present invention illustrates a method of realizing protein or virus labeling through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0085] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0086] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for labeling proteins or viruses, using quantum dots or fluorescent proteins to label proteins or viruses, characterized in that: The hydrazine fragment is coupled with the amino group on the protein or virus, the aldehyde fragment is coupled with the amino group on the quantum dot or the fluorescent protein, and then the aldehyde fragment connected with different components is coupled with the hydrazine fragment to achieve the labeling of the protein or virus; the hydrazine fragment is prepared with 6-chloronicotinic acid as a raw material; Wherein, R1 and R2 are each independently selected from H, alkyl, nitro, trifluoromethyl, trifluoroethyl, -SO4Na, -CH2SO4Na, -C2H4SO4Na, wherein the number of carbon atoms in the alkyl is not greater than 6.

2. The method according to claim 1, characterized in that: The preparation method of the hydrazine fragment comprises the following steps: (1) subjecting 6-chloronicotinic acid to a substitution reaction with hydrazine hydrate to obtain intermediate A; (2) adding acetone and p-toluenesulfonic acid to intermediate A for condensation to obtain intermediate B; (3) reacting the intermediate B, the R1-substituted N-hydroxysuccinimide and a condensing agent to generate a hydrazine fragment; 3. The method according to claim 2, characterized in that: In step (1), the molar ratio of 6-chloronicotinic acid to hydrazine hydrate is 1:(2-2.5); Preferably, in step (1), the reaction temperature is reflux temperature and the reaction time is 4-6 hours.

4. The method according to claim 2, characterized in that: In step (2), the molar ratio of the intermediate A to p-toluenesulfonic acid is 1:(0.05-0.1).

5. The method according to claim 2, characterized in that: In step (2), acetone is used as the raw material and the solvent, and the mass volume ratio of the intermediate A to the acetone is 1 g:(8-12) mL.

6. The method according to claim 2, characterized in that: In step (2), the reaction temperature is reflux temperature and the reaction time is 8-12h.

7. The method according to claim 2, characterized in that: In step (3), the molar ratio of the intermediate B, R1-substituted N-hydroxysuccinimide and the condensing agent is 1:(1.1-1.2):(1.2-1.5).

8. The method according to claim 2, characterized in that: The condensing agent is one or more selected from DCC, EDCI, HBTU, and CDI; Preferably, in step (3), the reaction temperature is 25-30° C. and the reaction time is 3-6 h.

9. The method according to claim 1, characterized in that: The preparation method of the aldehyde fragment comprises the following steps: 4-formylbenzoic acid, R2-substituted N-hydroxysuccinimide and a condensing agent react to generate the aldehyde fragment; 10. The method according to claim 9, characterized in that: The molar ratio of the 4-formylbenzoic acid, the R2-substituted N-hydroxysuccinimide and the condensing agent is 1:(1.1-1.2):(1.2-1.5); Preferably, the condensing agent is one or more selected from DCC, EDCI, HBTU, and CDI; Preferably, the reaction temperature is 25-30°C and the reaction time is 3-6h.

Images