Carboxyl modified terpyridyl ruthenium complex as well as preparation method and application thereof

Abstract

The invention discloses a carboxyl-modified terpyridyl ruthenium complex as well as a preparation method and application thereof, and belongs to the field of polymer synthetic chemistry, and the carboxyl-modified terpyridyl ruthenium complex is shown as (I). The invention also discloses a preparation method of the carboxyl-modified terpyridyl ruthenium complex. The preparation method is low in preparation cost, simple in purification and relatively high in efficiency. The terpyridyl ruthenium complex provided by the invention can be used for specific modification of protein due to the introduction of carboxyl, and can also be used as an electrochemical luminescence reagent. According to the method, DMT-MM is used as a condensing agent, efficient labeling of the terpyridyl ruthenium complex and the protein is achieved, and the method is high in labeling efficiency, easy and convenient to operate and low in cost and has wide application prospects in the field of labeled immunoassay. # imgabs0 #

Description

Technical Field

[0001] The invention relates to the field of polymer synthesis chemistry, and in particular to a carboxyl-modified terpyridine ruthenium complex and a preparation method and application thereof. Background Art

[0002] Electrochemiluminescence immunoassay (ECLIA) is a labeling immunoassay method that combines highly sensitive electrochemiluminescence with highly specific immune reactions. ECLIA has the characteristics of high sensitivity, wide linear range, long validity period of markers, no radioactivity, and easy automation. It is widely used in clinical diagnosis, food, environmental protection, military and other fields to detect proteins, hormones, fatty acids, vitamins, drugs, viruses and other components.

[0003] As a commonly used electrochemiluminescent agent, terpyridine ruthenium has the characteristics of high luminescence intensity, high quantum yield, and good stability. It is one of the electrochemiluminescent systems with a wide range of applications. Currently, the commonly used terpyridine ruthenium on the market can be directly coupled to proteins after being modified with N-hydroxysuccinimide ester. However, N-hydroxysuccinimide ester-modified terpyridine ruthenium is prone to hydrolysis during the synthesis and preparation process, resulting in a low yield. Summary of the invention

[0004] The present invention aims to provide a carboxyl-modified terpyridine ruthenium complex, which can be used for specific modification of proteins by introducing carboxyl groups and can also be used as an electrochemiluminescent reagent. The terpyridine ruthenium complex of the present invention has the characteristics of low preparation cost, simple purification and high efficiency.

[0005] One of the purposes of the present invention is to provide a carboxyl-modified terpyridine ruthenium complex, the structural formula of which is shown in (I):

[0006]

[0007] The second object of the present invention is to provide a method for preparing the above-mentioned carboxyl-modified terpyridine ruthenium complex, comprising the following steps:

[0008] Step 1, preparing a ruthenium intermediate: (2,2'-bipyridine)-4,4'-disulfonate and ruthenium trichloride (RuCl3) are mixed in dimethylformamide (DMF) and lithium chloride (LiCl), and the mixture is heated, stirred and refluxed under nitrogen protection, and after cooling, the mixture is added to tertiary methyl ether at room temperature, and centrifuged to dry to obtain a dark purple solid, which is a ruthenium intermediate;

[0009] Step 2: Mix the intermediate of the ruthenium product in step 1 with 4-methyl-(2,2'-bipyridine)-4-hexanoic acid in ethanol (EtOH), heat the mixture, stir and reflux under nitrogen protection, cool it, add excess acid at room temperature, and add excess acetone under stirring, collect the orange-red flocculent precipitate, add a small amount of water to dissolve it, spin dry it, and purify the product by column chromatography to obtain a carboxyl-modified terpyridine ruthenium complex.

[0010] Wherein, the molar ratio of (2,2'-bipyridine)-4,4'-disulfonate to RuCl3 in step 1 is 2:1.

[0011] Wherein, each millimole of RuCl3 in the step 1 corresponds to 5-6 mL of DMF.

[0012] Wherein, the molar amount of LiCl in step 1 is 6-7 mmol.

[0013] Wherein, the heating temperature in step 1 is 140-145°C.

[0014] Wherein, the reflux time in step 1 is 20 to 24 hours.

[0015] Wherein, the molar ratio of the ruthenium intermediate in step 2 to 4-methyl-(2,2'-bipyridine)-4-hexanoic acid is 1:1.2 mmol.

[0016] Wherein, each millimole of the ruthenium intermediate in step 2 corresponds to 1.6-1.8 mL of EtOH.

[0017] Wherein, the heating temperature in step 2 is 80-85°C.

[0018] Wherein, the reflux time in the step 2 is 20 to 24 hours.

[0019] Wherein, the acid added in the step 2 is preferably one of ammonium hexafluorophosphate (NH4PF6) or trifluoroacetic acid.

[0020] The third object of the present invention is to provide a method for labeling proteins with a carboxyl-modified terpyridine ruthenium complex, comprising the following steps:

[0021] (1) dissolving the carboxyl-modified terpyridine ruthenium complex in morpholineethanesulfonic acid (MES) buffer, adding 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholinium chloride hydrate (DMT-MM) and the protein to be labeled, mixing well and incubating with shaking;

[0022] (2) adding the reaction solution into an ultrafiltration centrifuge tube and centrifuging for purification;

[0023] (3) collecting the terpyridine ruthenium and protein markers in the filter membrane of the ultrafiltration centrifuge tube, i.e., ruthenium-labeled protein.

[0024] Wherein, the MES buffer is 100mM, pH 6.0.

[0025] The concentration of DMT-MM is 200 mg / mL, and the DMT-MM is added at a molar ratio of terpyridine ruthenium complex to DMT-MM of 25:1.

[0026] The protein to be labeled can be antigen, antibody, streptavidin or other detection proteins used in the field of biotechnology, and the initial concentration of the protein to be labeled is 2-20 mg / mL.

[0027] The protein to be labeled and the carboxyl-modified terpyridine ruthenium complex are added at a molar ratio of 1:150, and the final concentration of the protein to be labeled is 1-2 mg / mL.

[0028] Among them, the shaking incubation conditions are 25°C, 700-800rpm, 2-3h.

[0029] Wherein, the molecular retention rate of the ultrafiltration centrifuge tube filter membrane is 30kDa.

[0030] The centrifugal speed is 1000g and the time is 8 to 10 minutes.

[0031] The invention also relates to the application of the carboxyl-modified terpyridine ruthenium complex in electrochemiluminescence immunoassay.

[0032] In the application, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholinium chloride hydrate (DMT-MM) is used as a condensation agent, and a carboxyl-modified terpyridine ruthenium complex is efficiently labeled with a protein to obtain a ruthenium-labeled protein.

[0033] The application in electrochemiluminescence immunoassay comprises the following steps:

[0034] S1, the capture protein was added to the graphene electrode plate and incubated overnight at 4 °C;

[0035] S2, remove uncoated capture proteins through a washing step, add blocking solution, and block at room temperature;

[0036] S3, remove excess blocking solution through washing steps, add the protein to be tested, and incubate with shaking at room temperature;

[0037] S4, remove unbound test protein through washing step, add ruthenium labeled protein, shake at room temperature

[0038] Incubation;

[0039] S5, free ruthenium-labeled protein is removed by a washing step, tri-n-propylamine is added as a photogenerated co-reactant, and the signal is detected in an electrochemiluminescence analyzer.

[0040] The coating diluent was 10 mM PBS buffer, pH 7.2.

[0041] The coating concentration of the captured protein is 1 to 20 μg / mL.

[0042] The coating incubation time is 16 to 20 hours.

[0043] The washing solution was PBST buffer (10 mM PBS, 0.05% Tween-20, pH 7.2).

[0044] The blocking solution was 10 mM PBS, 3% BSA, pH 7.2.

[0045] The closure time is 1 to 2 hours.

[0046] Among them, the shaking incubation conditions are 500-600 rpm, 1-2h.

[0047] The mass percentage of tri-n-propylamine added is 5% to 6%.

[0048] The present invention has the following beneficial effects:

[0049] The carboxyl-modified terpyridine ruthenium complex of the present invention is formed by the coordination of a polypyridine ligand and a ruthenium (II) center. The complex exhibits a strong absorption band in an aqueous solution, has orange-red luminescence in the visible light region, has a large Stokes shift, and has a good luminescence quantum yield. The terpyridine ruthenium complex of the present invention can be used for specific modification of proteins due to the introduction of a carboxyl group. The complex has unique photophysical properties and the advantage of being easy to be modified by groups, providing new possibilities for applications in the fields of chemical sensing, biological detection, and materials science.

[0050] The present invention utilizes DMT-MM as a condensing agent to achieve efficient labeling of terpyridine ruthenium complex and protein and applies it to electrochemiluminescence immunoassay. This method has the advantages of high labeling efficiency, simple operation, low cost, etc., and has broad application prospects in the field of labeled immunoassay. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the H NMR spectrum of the carboxyl-modified terpyridine ruthenium complex.

[0052] Figure 2 This is the carbon NMR spectrum of the carboxyl-modified terpyridine ruthenium complex.

[0053] Figure 3 The dose-response curve of the marker protein is shown in Figure 2 . DETAILED DESCRIPTION

[0054] In order to facilitate understanding by those skilled in the art, the present invention is further described below with reference to the embodiments, but the contents mentioned in the embodiments are not intended to limit the present invention.

[0055] Example 1 Preparation of carboxyl-modified terpyridine ruthenium complex

[0056] It is synthesized through two-step reaction, and the reaction equation is as follows:

[0057]

[0058] Step 1: Mix (2,2'-bipyridine)-4,4'-disulfonate (2.00mmol) and RuCl3 (1mmol) in DMF (6ml, 99.5%) and LiCl (6.0mmol), stir vigorously with a magnetic stirrer, gradually heat to 143°C, and reflux under nitrogen for 24h. Then, cool the reaction solution naturally, add the mixture to methyl tert-ether at room temperature, and centrifuge to dry to obtain a dark purple solid, which is the intermediate of ruthenium Ru(L1)2Cl2, wherein L1 is (2,2'-bipyridine)-4,4'-disulfonate ligand, and the intermediate structure of ruthenium is shown in (II).

[0059]

[0060] Step 2: Mix the intermediate of ruthenium (1.00mmol) of the product in step 1 with 4-methyl-(2,2'-bipyridine)-4-hexanoic acid (1.2mmol) in EtOH (1.6ml, 99.5%), stir vigorously with a magnetic stirrer, heat the mixture to 80°C, and reflux for 24h under nitrogen. Then, cool the reaction solution naturally to room temperature, add 3mmol of NH4PF6 and excess acetone under stirring, collect the orange-red flocculent precipitate, add a small amount of water to dissolve and spin dry, and finally use column chromatography to remove the product under the condition of ACN:H2O of 10:1, which is the carboxyl-modified terpyridine ruthenium complex. The structural formula is as follows:

[0061]

[0062] Attached Figure 1 and Figure 2 The hydrogen nuclear magnetic resonance spectrum and carbon nuclear magnetic resonance spectrum of the carboxyl-modified terpyridine ruthenium complex are shown respectively.

[0063] Proton spectrum data: 1H NMR(400MHz,D2O)δ8.59(s,4H),8.35(d,J=6.4Hz,2H),7.93–7.78(m,4H),7.67(dd,J=11.8,5.8Hz,2H),7.41(q,J=5.8Hz,4H),7.28– 7.18(m,2H),4.37(s,8H),3.59(s,1H),2.81(t,J=7.4Hz,2H),2.52(s,3H),2.42–2.29(m,2H),1.77–1.55(m,4H),1.44–1.28(m,2H).

[0064] Carbon spectrum data: 13 C NMR(101MHz,D2O)δ177.35,157.01,156.94,156.84,151.48,151.14,150.78,150.65,150.34,142.87,142. 63,128.66,127.38,125.59,123.91,55.78,52.64,52.00,34.53,34.21,33.43,28.77,27.43,23.89,20.42.

[0065] From the above data, it can be seen that the structure of the prepared compound is correct.

[0066] Example 2 Labeling of Carboxylated Terpyridine Ruthenium Complex with Mouse Anti-Human κ Light Chain Antibody

[0067] Dilute DMT-MM to 200 mg / mL working solution with 100 mM MES buffer (pH 6.0). Weigh a sufficient amount of terpyridine ruthenium complex according to the amount of terpyridine ruthenium complex used, and dilute it to 10 mg / mL (10 mM) working solution with 100 mM MES buffer (pH 6.0). Add a sufficient amount of 200 mg / mL DMT-MM working solution (prepared immediately before use) according to the molar ratio of terpyridine ruthenium complex to DMT-MM of 25:1, and vortex to mix. Add a sufficient amount of mouse anti-human κ light chain antibody to be labeled according to the molar ratio of mouse anti-human κ light chain antibody to terpyridine ruthenium complex of 1:150, and dilute the added mouse anti-human κ light chain antibody to be labeled to 2 mg / mL with 100 mM MES buffer (pH6.0), vortex to mix, and incubate at 25°C in the dark with shaking at 800 rpm for 2 hours. After the reaction is completed, transfer the above mixture to a 30kDa ultrafiltration centrifuge tube, centrifuge at 1000g for 10min at 4°C, and discard the filtrate at the bottom of the tube (Note: keep the solution in the middle filter tube and do not discard it). Add 2mL10mM PBS buffer (pH7.2) to the middle filter tube, centrifuge at 1000g for 10min at 4°C, and discard the filtrate at the bottom of the tube. Repeat this step 3 times. Carefully collect all the solutions in the middle filter tube (the labeled terpyridine ruthenium complex and protein conjugate solution is usually light yellow) into the centrifuge tube. Add 200μL10 mM PBS buffer (pH7.2) to the middle filter tube to reabsorb the residual conjugate (the carboxylated terpyridine ruthenium complex and the marker of the mouse anti-human κ light chain antibody, i.e., the ruthenium-labeled antibody) and let it stand for 2min. Pipette all the solutions in the middle filter tube and add them to the centrifuge tube. The conjugate should be stored at 2-8℃ away from light, and at -20℃ for long-term storage.

[0068] Example 3

[0069] 1. Application of carboxylated terpyridine ruthenium complex and mouse anti-human κ light chain antibody marker in electrochemiluminescence immunoassay

[0070] (1) Dilute mouse anti-human IgG (Fc fragment) antibody to 2 μg / mL with 10 mM PBS buffer (pH 7.2), and add 100 μL to each well of the graphene electrode plate and incubate the coating at 4°C for 16 h.

[0071] (2) After coating, the coating solution in each well was discarded, and 300 μL PBST (10 mM PBS, 0.05% Tween-20, pH 7.2) buffer was added to each well for washing three times.

[0072] (3) Add 200 μL of blocking solution (10 mM PBS, 3% BSA, pH 7.2) to each well and block at room temperature for 2 h. After blocking, discard the blocking solution in each well and add 300 μL of PBST (10 mM PBS, 0.05% Tween-20, pH 7.2) buffer to each well for three washes.

[0073] (4) Dilute the anti-PD-1 human antibody to 0, 0.1, 1, 10, and 100 ng / mL with 10 mM PBS, 1% Casein, pH 7.2 buffer, and add 100 μL of each to the reaction wells, incubate at room temperature for 1 h, 600 rpm. After the reaction is completed, discard the reaction solution in each well, and add 300 μL of PBST (10 mM PBS, 0.05% Tween-20, pH 7.2) buffer to each well for washing 3 times.

[0074] (5) Dilute the carboxylated terpyridine ruthenium complex and the marker of mouse anti-human κ light chain antibody (i.e., ruthenium-labeled antibody) 10,000-fold with 10 mM PBS, 1% Casein, pH 7.2 buffer, and add 100 μL of the diluted ruthenium-labeled antibody to each well and incubate at room temperature for 1 h at 600 rpm.

[0075] (6) After the reaction is completed, the reaction solution in each well is discarded, and each well is washed three times with 300 μL PBST (10 mM PBS, 0.05% Tween-20, pH 7.2) buffer. Finally, 100 μL of 5% tri-n-propylamine solution is added to each well of the plate, and the electrochemiluminescence value of each well is measured with an electrochemiluminescence analyzer.

[0076] 2. Application of N-hydroxysuccinimide esterified terpyridine ruthenium complex and mouse anti-human κ light chain antibody marker in electrochemiluminescence immunoassay

[0077] The mouse anti-human κ light chain antibody to be labeled and the N-hydroxysuccinimide-esterified terpyridine ruthenium complex were mixed at a molar ratio of 1:25, and incubated at 25°C in the dark with shaking at 800 rpm for 2 hours. After the reaction is completed, the above mixture is transferred to a 30kDa ultrafiltration centrifuge tube, centrifuged at 1000g for 10 minutes at 4°C, and the filtrate at the bottom of the tube is discarded. Add 2mL10mM PBS buffer (pH 7.2) to the middle filter tube, centrifuge at 1000g for 10 minutes at 4°C, and discard the filtrate at the bottom of the tube. Repeat this step 3 times. Carefully collect all the solutions in the middle filter tube into the centrifuge tube. Add 200μL10 mM PBS buffer (pH7.2) to the middle filter tube to reabsorb the residual conjugate (N-hydroxysuccinimide-esterified terpyridine ruthenium complex and the label of the mouse anti-human κ light chain antibody) and let it stand for 2 minutes. Pipette all the solution in the middle filter tube and add it to the centrifuge tube. The conjugate should be stored at 2-8℃ away from light. If it is stored for a long time, it needs to be kept at -20℃.

[0078] The steps of labeling the N-hydroxysuccinimide-esterified terpyridine ruthenium complex and mouse anti-human κ light chain antibody in electrochemiluminescent immunoassay are the same as those of labeling the carboxylated terpyridine ruthenium complex and mouse anti-human κ light chain antibody in electrochemiluminescent immunoassay, except that in step (5), the N-hydroxysuccinimide-esterified terpyridine ruthenium complex and mouse anti-human κ light chain antibody are used instead of the carboxylated terpyridine ruthenium complex and mouse anti-human κ light chain antibody.

[0079] The electrochemiluminescence values ​​of proteins labeled with N-hydroxysuccinimide-esterified terpyridine ruthenium and carboxylated terpyridine ruthenium of the present application were compared, and the dose-response curves were shown in FIG. Figure 3 As shown, carboxylated terpyridine ruthenium has a better electrochemiluminescent signal than N-hydroxysuccinimide esterified terpyridine ruthenium at high concentration. The carboxylated terpyridine ruthenium of the present invention can label proteins under the action of DMT-MM, and has high labeling efficiency and high luminescence efficiency, and can be used as an electrochemiluminescent reagent.

Claims

1. A carboxyl-modified terpyridine ruthenium complex, characterized in that: Its structural formula is shown in (I):

2. A method for preparing the carboxyl-modified terpyridine ruthenium complex as claimed in claim 1, characterized in that: The following steps are involved: Step 1, preparing a ruthenium intermediate: (2,2'-bipyridine)-4,4'-disulfonate and ruthenium trichloride (RuCl3) are mixed in dimethylformamide (DMF) and lithium chloride (LiCl), and the mixture is heated, stirred and refluxed under nitrogen protection, and after cooling, the mixture is added to tertiary methyl ether at room temperature, and centrifuged to dry to obtain a dark purple solid, which is a ruthenium intermediate; Step 2: Mix the intermediate of the ruthenium product in step 1 with 4-methyl-(2,2'-bipyridine)-4-hexanoic acid in ethanol (EtOH), heat the mixture, stir and reflux under nitrogen protection, cool it, add excess acid at room temperature, and add excess acetone under stirring, collect the orange-red flocculent precipitate, add a small amount of water to dissolve it, spin dry it, and purify the product by column chromatography to obtain a carboxyl-modified terpyridine ruthenium complex.

3. The method for preparing the carboxyl-modified terpyridine ruthenium complex according to claim 2, characterized in that: The molar ratio of (2,2'-bipyridine)-4,4'-disulfonate to RuCl3 in step 1 is 2:1; Each millimole of RuCl3 in step 1 corresponds to 5-6 mL of DMF; The molar amount of LiCl in the step 1 is 6-7 mmol.

4. The method for preparing the carboxyl-modified terpyridine ruthenium complex according to claim 2, characterized in that: The heating temperature in the step 1 is 140-145° C. and the reflux time is 20-24 hours.

5. The method for preparing the carboxyl-modified terpyridine ruthenium complex according to claim 2, characterized in that: The molar ratio of the ruthenium intermediate in step 2 to 4-methyl-(2,2'-bipyridine)-4-hexanoic acid is 1:1.2 mmol; Each millimole of the ruthenium intermediate in step 2 corresponds to 1.6-1.8 mL of EtOH; The heating temperature in the step 2 is 80-85°C; The reflux time in step 2 is 20 to 24 hours; The acid added in step 2 is one of ammonium hexafluorophosphate or trifluoroacetic acid.

6. A method for labeling proteins using the carboxyl-modified terpyridine ruthenium complex as claimed in claim 1, characterized in that: The following steps are involved: (1) dissolving the carboxyl-modified terpyridine ruthenium complex in MES buffer, adding 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholinium chloride hydrate DMT-MM and the protein to be labeled, mixing thoroughly and incubating with shaking; (2) adding the reaction solution into an ultrafiltration centrifuge tube and centrifuging for purification; (3) collecting the terpyridine ruthenium and protein markers in the filter membrane of the ultrafiltration centrifuge tube, i.e., ruthenium-labeled protein.

7. The method for labeling a protein according to claim 6, characterized in that: The MES buffer is 100 mM, pH 6.0; The concentration of DMT-MM is 200 mg / mL, and the DMT-MM is added at a molar ratio of terpyridine ruthenium complex to DMT-MM of 25:1; The protein to be labeled is an antigen, antibody, streptavidin or other detection protein used in the field of biotechnology, and the initial concentration of the protein to be labeled is 2-20 mg / mL; The protein to be labeled and the carboxyl-modified terpyridine ruthenium complex are added at a molar ratio of 1:150, and the final concentration of the protein to be labeled is 1-2 mg / mL; The shaking incubation conditions were 25°C, 700-800 rpm, 2-3 h; The molecular retention rate of the ultrafiltration centrifuge tube filter membrane is 30kDa; The centrifugal speed is 1000g and the time is 8 to 10 minutes.

8. Use of the carboxyl-modified terpyridine ruthenium complex as claimed in claim 1 in electrochemiluminescence immunoassay.

9. The use according to claim 8, characterized in that: Using 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholinium chloride hydrate DMT-MM as a condensation agent, carboxyl-modified terpyridine ruthenium complex and protein were efficiently labeled to obtain ruthenium-labeled protein.

10. The use according to claim 9, characterized in that: The application in electrochemiluminescence immunoassay, The following steps are involved: S1, the capture protein was added to the graphene electrode plate and incubated overnight at 4 °C; S2, remove uncoated capture proteins through a washing step, add blocking solution, and block at room temperature; S3, remove excess blocking solution through a washing step, add the protein to be tested, and incubate with shaking at room temperature; S4, remove unbound test protein through washing steps, add ruthenium-labeled protein, and incubate with shaking at room temperature; S5, free ruthenium-labeled protein is removed by a washing step, tri-n-propylamine is added as a photogenerated co-reactant, and signal detection is performed in an electrochemiluminescence analyzer; The coating diluent is 10 mM PBS buffer, pH 7.2; the coating concentration of the captured protein is 1 to 20 μg / mL; the coating incubation time is 16 to 20 h; The washing solution is PBST buffer, which is 10 mM PBS, 0.05% Tween-20, pH 7.2; The blocking solution is 10 mM PBS, 3% BSA, pH 7.2; the blocking time is 1 to 2 hours; Among them, the shaking incubation conditions are 500-600 rpm, 1-2 h; The mass percentage of tri-n-propylamine added is 5% to 6%.

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