CN115286618A - Lanthanide complex, preparation method thereof, fluorescent labeling reagent and fluorescent labeling method - Google Patents
Lanthanide complex, preparation method thereof, fluorescent labeling reagent and fluorescent labeling method Download PDFInfo
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Abstract
The invention belongs to the technical field of coordination compounds, and particularly relates to a lanthanide complex and a preparation method, a fluorescence labeling reagent and a fluorescence labeling method thereof, wherein the structure is as follows:
wherein A is any one of polyethyleneimine and polypropylene imine; f is 2,6-bis (N-pyrazole) pyridinecarboxylic acids, comprising the following structure:
wherein R4 is ester group, amido, aryl with active substitution, heteroAny one of a cyclic group, an alkynyl group, or a heterocyclic group having an active substituent; ln is a lanthanide metal, including Td 3+ 、Eu 3+ (ii) a According to the lanthanide complex, the preparation method of the lanthanide complex, the fluorescence labeling reagent and the fluorescence labeling method, the problem of small molar extinction coefficient of the 2,6-di (N-pyrazole) pyridine amino carboxylic acid lanthanide complex is solved by connecting a plurality of lanthanide complexes together to form a polymer, and meanwhile, the lanthanide complex prepared by the method has the advantages of good stability and high brightness.
Description
Technical Field
The invention belongs to the technical field of coordination compounds, and particularly relates to a lanthanide complex and a preparation method, a fluorescent labeling reagent and a fluorescent labeling method thereof.
Background
In numerous immunoassays, homogeneous phase time-resolved immunoassays do not require repeated washing to remove free labels, greatly simplify the operation steps, accelerate the detection speed, and make the detection process easier to realize automated operation, and at the same time, it also has the advantages of high detection sensitivity, strong reliability, low false positive/negative rate of experimental results, and the like, and thus, it has been extensively studied as a leading-edge technique in immunoassays for a long time.
The lanthanide fluorescent complex has the characteristics of small molecular weight, convenient modification, strong bleaching resistance and the like, and is widely used in homogeneous immunoassay, and mainly comprises crown ether lanthanide complexes, beta-diketone lanthanide complexes, polydentate carboxylic acid lanthanide complexes, cryptate lanthanide complexes, calixarene lanthanide complexes and the like. Although various lanthanide complexes have been reported at home and abroad, the lanthanide fluorescent complexes do not meet the requirements of practical application, and the ideal lanthanide fluorescent complexes have high molar extinction coefficient, high fluorescence quantum yield, long fluorescence life, good solubility in aqueous solution and high stability in aqueous solution.
Although the French Cisbio company developed Eu for drug screening 3+ ，Tb 3+ The cryptate complex and the product are commercialized, but the compound is very expensive (about 2 ten thousand/50 nM), meanwhile, most of the synthetic raw materials of the cryptate complex are not commercialized, the synthetic route is long, the operation process is complicated, the product yield is extremely low, and the compound has low fluorescence quantum yield (the compound has the advantages of low fluorescence quantum yield: (the salt is a new product of the amino acid, and the salt is a new product of the amino acid, the salt and the salt is a new product of the amino acid, and the salt is a new product of the amino acid<0.4 Poor solubility in aqueous solution, complex labeling process, poor reproducibility, and the like.
2,6-twoCompared with the cryptate terbium complex, the (N-pyrazole) pyridine amino carboxylic acid compound has the advantages of short synthetic route, high yield, good water solubility, long fluorescence lifetime and high fluorescence quantum yield>0.6 ) but the molar extinction coefficient of the lanthanide series complex and the cryptate lanthanide series complex is small and is all 20000M -1 cm -1 About, the fluorescence brightness is not high in the process of label detection, and the detection limit can only reach 10 when the fluorescence brightness is used for detecting indexes -9 g/L, can be used for detecting only partial high-abundance targets.
If a 2,6-di (N-pyrazole) pyridine amino carboxylic acid lanthanide complex with a large molar extinction coefficient is developed, the development of time-resolved immunofluorescence detection in a homogeneous system is greatly promoted.
Disclosure of Invention
The invention provides a lanthanide complex, a preparation method thereof, a fluorescent labeling reagent and a fluorescent labeling method, and aims to solve the problem of small molar extinction coefficient of the lanthanide complex.
In order to solve the technical problems, the invention provides a lanthanide complex, which has the following structure:
In yet another aspect, the present invention also provides a method for preparing the lanthanide complexes described above, comprising the steps of: step S1, compound 1, ethanol and [ Pd (PPh) 3 ) 2 Cl 2 ]And dry triethylamine in 15mL of toluene, and oil bath at 100 ℃ under CO bubblingHeating for 14h, removing solvent under reduced pressure, dissolving the residue in 100mL dichloromethane, washing with water several times, washing with saturated brine, and adding Na to the organic phase 2 SO 4 Drying, removing solvent under reduced pressure, and purifying the residue by column chromatography to obtain yellow oily compound 2;
step S2, dissolving the compound 2 in 20mL of methanol, adding 5mL of NaOH aqueous solution, heating in a 60 ℃ oil bath for 4h under the protection of Ar, pressurizing to remove the solvent after the reaction is finished, adding 20mL of ethanol into the residue, heating to dissolve the residue, filtering to remove the residue, dropwise adding 80mL of THF into the filtrate, precipitating a large amount of solid, centrifuging, carefully removing the supernatant, and drying the residue in vacuum to obtain a white solid compound 3;
step S3, dissolve Compound 3 in 10mL deionized water and add TdCl 3 Heating in oil bath at 60 ℃ for 4h under Ar protection, and removing the solvent under reduced pressure after the reaction is finished to obtain a white solid compound 4;
step S4, preparing an intermediate compound; and step S5, preparing a probe from the compound 4 and/or the intermediate compound, wherein the probe is the lanthanide complex.
In a third aspect, the present invention also provides a fluorescent labeling reagent comprising: a lanthanide complex as hereinbefore described.
In a fourth aspect, the present invention also provides a fluorescence labeling method, comprising: the complex formed from the rare earth metal as described above is used as a labeling agent.
The lanthanide complex, the preparation method thereof, the fluorescent labeling reagent and the fluorescent labeling method have the beneficial effects that the problem of small molar extinction coefficient of 2,6-bis (N-pyrazole) pyridine amino carboxylic acid lanthanide complex is solved by connecting a plurality of lanthanide complexes together to form a polymer, and meanwhile, the lanthanide complex prepared by the method has the advantages of good stability and high brightness.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the invention.
In order to make the aforementioned and other objects, features and advantages of the present invention comprehensible, preferred embodiments accompanied with figures are described in detail below.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing fluorescence lifetime measurements of probe 5 of a lanthanide complex of the present invention;
FIG. 2 is a plot of time-resolved fluorescence delay of 300us versus concentration for probe15 of the lanthanide complexes of the present invention.
Detailed Description
To make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some, but not all embodiments of the present invention. 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 invention.
The invention provides a lanthanide complex, which has the following structure:
In this embodiment, specifically, when a is a polyethyleneimine, the structure is as follows:
In this embodiment, specifically, when a is a polypropyleneimine, the structure is as follows:
In a second aspect, the present invention also provides a process for the preparation of a lanthanide complex as described above, comprising the steps of: step S1, compound 1 (300mg, 0.43mmol), ethanol 0.2mL and [ Pd (PPh) 3 ) 2 Cl 2 ](60.8mg, 0.09mmol) anddissolving 10mL of dry triethylamine in 15mL of toluene, heating in 100 ℃ oil bath under CO bubbling for 14h, removing the solvent under reduced pressure after the reaction is finished, adding 100mL of dichloromethane to the residue to dissolve, washing with water (100mL. Times.3), washing with saturated brine, and using Na as an organic phase 2 SO 4 Drying, removing the solvent under reduced pressure, and purifying the residue by column chromatography to obtain yellow oily compound 2 (0.264 g, 89%);
of said Compound 2 1 H NMR(400MHz，CDCl3):d＝8.62(d，J＝8.4Hz，2H)，8.18(d，J＝7.7Hz，2H)，7.97(s，2H)；7.50–7.26(m，4H)，4.40(s，4H)，4.19(q，J＝7.0Hz，8H)，3.67(s，8H)，1.28ppm(t，J＝7.0Hz，12H)；ESI/MS：686.3[M+H] + ；
Step S2, dissolving the compound 2 (400mg, 0.58mmol) in 20mL of methanol, adding 5mL of NaOH (147mg, 2.90mmol) aqueous solution, heating in 60 ℃ oil bath for 4h under Ar protection condition, pressurizing to remove the solvent after the reaction is finished, adding 20mL of ethanol to the residue, heating to dissolve, filtering to remove the residue, dropwise adding 80mL of THF into the filtrate, precipitating a large amount of solid, centrifuging, carefully removing the supernatant, and drying the residue in vacuum to obtain a white solid compound 3 (375mg, 98%);
of said compound 3 1 H NMR(d 4 -methanol，400MHz):δ8.66(d，J＝2.5Hz，2H)，8.14(d，J＝8.0Hz，1H)，7.73(d，J＝8.0Hz，2H)，6.74(d，J＝2.5Hz，1H)，3.95(s，4H)，3.45(s，4H)，2.71(d，J＝11.0Hz，4H)；ESI/MS：576.1[M+Na] + ；
Step S3, dissolve Compound 3 (300mg, 0.46mmol) in 10mL deionized water and add TdCl 3 (135mg, 0.51mmol) and heating in oil bath at 60 ℃ under Ar protection for 4h, and removing the solvent under reduced pressure after the reaction is finished to obtain a white solid compound 4 (350mg, 95%);
of said Compound 4 1 H NMR(D 2 O，400MHz):δ(8.45，s，2H)，7.70(d，J＝8.0Hz，2H)，6.65(s，2H)，4.09(br，s，4H)，3.66(br s，4H)，2.94(br,s,4H)；ESI/MS：806.6[M+H] + ；
Step S4, preparing an intermediate compound; and step S5, preparing a probe from the compound 4 and/or the intermediate compound, wherein the probe is the lanthanide complex.
In this embodiment, specifically, the step S4 includes: step S4a, dissolving pentaethylenehexamine (2.32g, 10mmol) in 50mL of ethanol, dropwise adding 10mL of dichloromethane solution containing di-tert-butyl dicarbonate (2.18g, 10mmol) under an ice-bath condition, returning the system to room temperature after dropwise adding, reacting for 6h under the protection of Ar, and removing the solvent under reduced pressure after the reaction is finished to obtain a colorless oily liquid compound 5 (3.32g, 100%); of said Compound 5 1 H NMR(DMSO-d 6 ，400MHz):δ4.99(s，6H)，3.18-3.24(m，20H)，1.43(s，9H)；ESI-MS(m/z):530.2[M-H] - ；
Step S4b, dissolving compound 4 (200mg, 0.25mmol) in 5mL of anhydrous DMF, adding compound 5 (11.5mg, 0.11mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (156mg, 0.3mmol) and 0.2mL of dry TEA, stirring at room temperature under the protection of Ar, removing the solvent under reduced pressure after the reaction is finished, and separating the residue by reverse phase preparative chromatography to obtain compound 6 (285mg, 69%); of said Compound 6 1 H NMR(DMSO-d 6 ，400MHz):δ8.85(m，10H)，7.71(s，10H)，6.54(m，10H)，4.99(s，2H)，3.95(s，20H)，3.49(s，40H)，3.18-3.24(m，2H)，2.65-2.81(m，18H)，1.43(s，9H)；ESI-MS(m/z):530.2[M-H] - ；
Step S4c, dissolving the compound 5 (300mg, 0.46mmol) in 10mL of anhydrous N, N-dimethylformamide, adding tris (2-aminoethylamino) amine (20mg, 0.14mmol) and 20uL of dried triethylamine, stirring at room temperature under the protection of Ar, pressurizing to remove the solvent after the reaction is finished, and purifying the residue by reverse-phase preparative chromatography to obtain a light yellow oily compound 7 (261mg, 89%); of said compound 7 1 H NMR(300MHz，D2O):d＝8.34(s，6H)，7.43(s，6H)，7.26(d，J＝7.5Hz，6H)，7.02(d，J＝7.5Hz，6H)，6.39(s，6H)，3.62(s，12H)，3.18-3.24(m，6H)；3.01(s，24H)，2.65-2.81(m，6H)；ESI-MS(m/z):2143.63.[M+Na] + ；
Step S4d, compound 8 (2g, 3.78mmol) and K 2 CO 3 (1.56g, 5.66mmol) is dissolved in 150mL of water, succinic acid monomethyl ester acyl chloride (0.62g, 3.78mmol) is slowly added under the protection of Ar, the system is returned to the room temperature after the dropwise addition, the stirring is carried out for 0.5h, the system is extracted by ethyl acetate (100 mL multiplied by 5), organic phases are combined, the mixture is washed by saturated saline solution, and the organic phase is washed by Na 2 SO 4 The product, compound 9 (8.2g, 33%) was dried and the organic solvent was removed under reduced pressure as a colorless oil; of said Compound 9 1 H NMR(400MHz，DMSO-d 6 ):3.92(q，8.4Hz，2H)，3.45(t，8.2Hz，4H)，2.85-2.78(m，4)，2.76-2.64(m，32H)，1.71-1.58(m，18H)，1.43(t，8.4Hz，3H).ESI-MS(m/z):659.6[M+H] + ；
Step S4e, dissolving the compound 6 in 10mL of anhydrous N, N-dimethylformamide, adding the compound 9 and 20mL of dried triethylamine, stirring at room temperature under the protection of Ar, pressurizing after the reaction is finished to remove the solvent, and carrying out reversed-phase preparation on the residuePurifying by chromatography to obtain light yellow solid compound 10 (121mg, 45%); of said Compound 10 1 H NMR(400MHz，DMSO-d 6 ):d＝8.34(s，2H)，7.43(s，2H)，7.26(d，J＝7.5Hz，2H)，7.02(d，J＝7.5Hz，2H)，6.39(s，2H)，3.93(q，8.4Hz，2H)，3.62(s，4H)，3.45(t，8.2Hz，4H)，3.65-3.73(m，4)，3.32(s，8H).2.96-3.12(m，32H)，1.77-1.88(m，36H)，1.43(t，8.4Hz，3H).MALDI-TOF(m/z):6606[M+Na + ] + ；
Step S4f, dissolving the compound 10 in 20mL of methanol, adding 5mL of NaOH aqueous solution, heating in a 60 ℃ oil bath for 4h under the protection of Ar, pressurizing to remove the solvent after the reaction is finished, adding 20mL of ethanol into the residue, heating to dissolve the residue, filtering to remove the residue, dropwise adding 80mL of THF into the filtrate, precipitating a large amount of solid, centrifuging, carefully removing the supernatant, and drying the residue in vacuum to obtain a compound 11 (254mg, 89%); of said Compound 11 1 H NMR(400MHz，DMSO-d 6 ):d＝8.34(s，2H)，7.43(s，2H)，7.26(d，J＝7.5Hz，2H)，7.02(d，J＝7.5Hz，2H)，6.39(s，2H)，3.62(s，4H)，3.45(t，8.2Hz，4H)，3.65-3.73(m，4)，3.32(s，8H).2.96-3.12(m，32H)，1.77-1.88(m，36H)。MALDI-TOF(m/z):6578[M+Na] + ；
In this embodiment, specifically, the step S5 includes: step S5a, dissolving compound 4 (200mg, 0.25mmol) in 5mL of anhydrous DMF, adding N-hydroxyethyl ethylenediamine (11.5mg, 0.11mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (156mg, 0.3mmol) and dried TEA, stirring at room temperature under the protection of Ar, removing the solvent under reduced pressure after the reaction is completed, and separating the residue by reverse phase preparative chromatography to obtain a white solid probe1 (283mg, 68%); of the probe1 1 H NMR(DMSO-d 6 ，400M Hz):δ8.85(d，J＝2.4Hz，4H)，7.71(s，4H)，6.54(d，J＝2.4Hz，4H)，4.21(s，2H)，4.69(s，4H)，3.95(s，8H)，3.61(t，8.2Hz，2H)，3.49(s，16H)，3.21(t，8.2Hz，6H)。ESI-MS(m/z):530.2[M-H] - ；
Step S5b, dissolving compound 4 (200mg, 0.25mmol) in 5mL of anhydrous DMF, adding diethylenetriamine, benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (156mg, 0.3mmol) and dried TEA, stirring at room temperature under the protection of Ar, removing the solvent under reduced pressure after the reaction is finished, and separating the residue by reverse phase preparative chromatography to obtain probe 2 (125mg, 62%); of said probe 2 1 H NMR(DMSO-d 6 ，400MHz):δ8.85(d，J＝2.4Hz，6H)，7.71(s，6H)，6.54(d，J＝2.4Hz，6H)，4.21(s，3H)，4.69(s，6H)，3.95(s，12H)，3.61(t，8.2Hz，2H)，3.49(s，24H)，3.21(t，8.2Hz，6H)。MALDI-TOF(m/z):2465[M+H] - ；
Step S5c, dissolving the compound 4 (200mg, 0.25mmol) in 5mL of anhydrous DMF, adding pentaethylenehexamine, benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (156mg, 0.3mmol) and dried TEA, stirring at room temperature under the protection of Ar, removing the solvent under reduced pressure after the reaction is finished, and separating the residue by reverse phase preparative chromatography to obtain a probe 3 (285mg, 69%); of said probe 3 1 H NMR(DMSO-d 6 ，400MHz):δ＝8.85(d，J＝2.4Hz，12H)，7.71(s，12H)，6.54(d，J＝2.4Hz，12H)，3.95(s，24H)，3.55-3.51(m，20H)，3.49(s，8H).MALDI-TOF(m/z):4956[M+H] + ；
Step S5d, compound 4 (200 mg,0.25 mmol) was dissolved in 5mL anhydrous DMF and nonaethylenedecaamine, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (156mg, 0.3 mmol) and dry TEA were added, stirring at room temperature under Ar protection, the solvent was removed under reduced pressure after the reaction was complete, and the residue was isolated by reverse phase preparative chromatography to give Probe 4 (185mg, 39%); of said probe 4 1 H NMR(DMSO-d 6 ，400MHz):δ＝8.85(d，J＝2.4Hz，12H)，7.71(s，12H)，6.54(d，J＝2.4Hz，12H)，3.95(s，24H)，3.55-3.51(m，20H)，3.49(s，8H).MALDI-TOF(m/z):8297[M+H] - ；
Step S5e, dissolving the compound 6 in 20mL of a solution of dichloromethane and N, N-dimethylformamide with the mass ratio of 1:1, adding 2mL of trifluoroacetic acid, stirring at room temperature for 2h under the protection of Ar, after the reaction is finished, removing the solvent under pressure, dissolving the residue in 50mL of hot methanol, adding 70mL of THF, and filtering to obtain a white probe 5; of said probe 5 1 H NMR(DMSO-d 6 ，400MHz):δ8.85(m，10H)，7.71(s，10H)，6.54(m，10H)，4.99(s，2H)，3.95(s，20H)，3.49(s，40H)，3.18-3.24(m，2H)，2.65-2.81(m，18H)，；ESI-MS(m/z):530.2[M-H] - ；
Step S5f, dissolve Compound 7 (50mg, 0.024mmol) in 2mL deionized water and add TdCl 3 (18.8mg, 0.071mmol) and heating in a 60 ℃ oil bath for 4h under the protection of Ar, and removing the solvent under reduced pressure after the reaction is finished to obtain a white solid probe 6 (60mg, 98%); of said probe 6 1 H NMR(300MHz，D2O):d＝8.35(s，6H)，7.45(s，6H)，7.27(d，J＝7.5Hz，6H)，7.08(d，J＝7.5Hz，6H)，6.39(s，6H)，3.63(s，12H)，3.18-3.25(m，6H)；3.04(s，24H)，2.65-2.84(m，6H)；MALDI-TOF(m/z):3095[M+H] + ；
Step S5g, compound 7 (50mg, 0.024mmol) dissolved in 2mL deionized water and EuCl added 3 (18.3mg, 0.071mmol) and heated in an oil bath at 60 ℃ for 4h under the protection of Ar, and after the reaction is finished, the solvent is removed under reduced pressure to obtain a white solid probe 7 (57mg, 95%); of said probe 7 1 H NMR(300MHz，D2O):d＝8.35(s，6H)，7.45(s，6H)，7.27(d，J＝7.5Hz，6H)，7.08(d，J＝7.5Hz，6H)，6.39(s，6H)，3.63(s，12H)，3.18-3.25(m，6H)；3.04(s，24H)，2.65-2.84(m，6H)；MALDI-TOF(m/z):3073[M+H] + ；
Step S5h, dissolving the compound 4 (200mg, 0.25mmol) in 5mL of anhydrous DMF, adding dipropylene triamine, benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (156mg, 0.3mmol) and dried TEA, stirring at room temperature under the protection of Ar, removing the solvent under reduced pressure after the reaction is finished, and separating the residue by reverse phase preparative chromatography to obtain a probe 8 (285mg, 69%); of said probe 8 1 H NMR(400MHz，DMSO-d 6 ):8.85(d，J＝2.4Hz，2H)，7.71(s，2H)，6.54(d，J＝2.4Hz，2H)，3.95(s，4H)，3.49(s，8H)，2.85-2.78(m，4)，2.76-2.64(m，4H)，1.71-1.58(m，4H)，1.43(s，2H).MALDI-TOF(m/z):2494[M+H] + ；
Step S5i, dissolving the compound 4 (200mg, 0.25mmol) in 5mL of anhydrous DMF, adding the compound 8, benzotriazole-1-yl-oxytriazolidinyl phosphorus hexafluorophosphate (156mg, 0.3mmol) and dry TEA, stirring at room temperature under the protection of Ar, removing the solvent under reduced pressure after the reaction is finished, and separating the residue by reverse phase preparative chromatography to obtain a probe 9 (285mg, 69%); of said probe 9 1 H NMR(400MHz，DMSO-d 6 ):δ＝8.85(d，J＝2.4Hz，16H)，7.71(s，16H)，6.54(d，J＝2.4Hz，16H)，3.95(s，4H)，3.49(s，48H)，2.85-2.78(m，4H，)，2.96-3.08(m，12H)，1.58-1.71(m，14H)，1.43(s，7H).MALDI-TOF(m/z):6715[M+H] + ；
Step S5j, dissolving the compound 4 in 5mL of anhydrous DMF, adding the compound 9, benzotriazole-1-yl-oxy tripyrrolidinophosphonium hexafluorophosphate and dry TEA, stirring at room temperature under the protection of Ar, removing the solvent under reduced pressure after the reaction is finished, and separating the residue by reverse phase preparative chromatography to obtain a probe 10 (285mg, 69%); of said probe 10 1 H NMR(400MHz，DMSO-d 6 ):δ＝8.85(d， J＝2.4Hz，2H)，7.71(s，2H)，6.54(d， J＝2.4Hz，2H)，3.95(m，6H)，3.49(m，12H)，2.85-2.78(m，4)，2.76-2.64(m，32H)，1.71-1.58(m，4H)，1.43(s，3H).MALDI-TOF(m/z):7744[M+H] + ；
Step S5k, dissolve Compound 10 in 2mL deionized water and add TdCl 3 (18.8mg, 0.071mmol) and heated in an oil bath at 60 ℃ for 4h under the protection of Ar, and after the reaction is finished, the solvent is removed under reduced pressure to obtain a white solid probe 11 (285mg, 69%); of said probe 11 1 H NMR(400MHz，DMSO-d 6 ):d＝8.35(s，2H)，7.44(s，2H)，7.27(d，J＝7.5Hz，2H)，7.02(d,J＝7.5Hz,2H)，6.38(s，2H)，3.94(q，8.4Hz，2H)，3.63(s，4H)，3.45(t，8.2Hz，4H)，3.65-3.73(m，4)，3.32(s，8H).2.96-3.12(m，32H)，1.77-1.88(m，36H)，1.43(t，8.4Hz，3H).MALDI-TOF(m/z):8014[M+H] + ；
Step S5l, mixingCompound 10 was dissolved in 2mL deionized water and EuCl was added 3 (18.3mg, 0.071mmol) and heated in an oil bath at 60 ℃ for 4h under the protection of Ar, and after the reaction is finished, the solvent is removed under reduced pressure to obtain a probe 12 (285mg, 69%); of said probe 12 1 H NMR(400MHz，DMSO-d 6 )：d＝8.35(s，2H)，7.44(s，2H)，7.27(d，J＝7.5Hz，2H)，7.02(d，J＝7.5Hz，2H)，6.38(s，2H)，3.94(q，8.4Hz，2H)，3.63(s，4H)，3.45(t，8.2Hz，4H)，3.65-3.73(m，4)，3.32(s，8H).2.96-3.12(m，32H)，1.77-1.88(m，36H)，1.43(t，8.4Hz，3H)。MALDI-TOF(m/z):7952[M+H] + ；
Step S5m, dissolve Compound 11 in 2mL deionized water and add TdCl 3 (18.8mg, 0.071mmol) and heating in an oil bath at 60 ℃ for 4h under the protection of Ar, and removing the solvent under reduced pressure after the reaction is finished to obtain a probe 13 (54mg, 89%); of said probe 13 1 H NMR(400MHz，DMSO-d 6 ):d＝8.34(s，2H)，7.43(s，2H)，7.26(d，J＝7.5Hz，2H)，7.02(d，J＝7.5Hz，2H)，6.39(s，2H)，3.62(s，4H)，3.45(t，8.2Hz，4H)，3.65-3.73(m，4)，3.32(s，8H).2.96-3.12(m，32H)，1.77-1.88(m，36H)。MALDI-TOF(m/z):7987[M+H] + ；
Step S5n, dissolve Compound 11 in 2mL deionized water and add EuCl 3 (18.3mg, 0.071mmol) and heating in an oil bath at 60 ℃ under the protection of Ar for 4h, and removing the solvent under reduced pressure after the reaction is finished to obtain a probe 14 (54mg, 89%); of said probe 14 1 H NMR(400MHz，DMSO-d 6 ):d＝8.34(s，2H)，7.43(s，2H)，7.26(d，J＝7.5Hz，2H)，7.02(d，J＝7.5Hz，2H)，6.39(s，2H)，3.62(s，4H)，3.45(t，8.2Hz，4H)，3.65-3.73(m，4)，3.32(s，8H).2.96-3.12(m，32H)，1.77-1.88(m，36H)。MALDI-TOF(m/z):7924[M+H] + ；
Step S5o, dissolving the probe 13 (100mg, 0.012mmol) in 3mL of dry N, N-dimethylformamide, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (3mg, 0.014mmol) and N-hydroxysuccinimide (2mg, 0.17mmol), stirring at room temperature for 2h under the protection of Ar, adding 6mL of tetrahydrofuran after the reaction is finished, precipitating a large amount of solid, centrifuging, carefully removing the supernatant, and drying the residue in vacuum to obtain a white solid probe15 (92mg, 91%); of said probe15 1 H NMR(400MHz，DMSO-d 6 ):d＝8.34(s，2H)，7.43(s，2H)，7.26(d，J＝7.5Hz，2H)，7.02(d，J＝7.5Hz，2H)，6.39(s，2H)，3.62(s，4H)，3.45(t，8.2Hz，4H)，3.65-3.73(m，4)，3.32(s，8H).2.96-3.12(m，32H)，1.77-1.88(m，36H)。MALDI-TOF(m/z):8020[M+H] + 。
Example 1
Determination of optical Properties:
accurately weighing about 1-15 mg of the probe, dissolving the probe in anhydrous dimethyl sulfoxide to prepare 10mmol of mother liquor, adding 3uL of the mother liquor into 3mL of deionized water by using a liquid gun, repeatedly blowing and uniformly mixing the solution by using the liquid gun, transferring the solution into a 1cm quartz cuvette, and measuring the optical properties by using an Agilent ultraviolet visible absorption spectrometer and a fluorescence spectrophotometer, wherein the results are shown in the table I.
TABLE-optical Properties of probes 1 to 15
| Name (R) | Maximum absorption wavelength | Maximum absorption wavelength | Molar extinction coefficient | Quantum yield |
| Probe1 | 275nm/325nm | 545nm | 40000M -1 cm -1 | 95 |
| Probe | ||||
| 2 | 275nm/325nm | 545nm | 59000M -1 cm -1 | 95％ |
| Probe 3 | 275nm/325nm | 545nm | 190000M -1 cm -1 | 92％ |
| Probe 4 | 275nm/325nm | 545nm | 98000M -1 cm -1 | 95 |
| Probe | ||||
| 5 | 275nm/325nm | 545nm | 98000M -1 cm -1 | 93％ |
| Probe 6 | 275nm/325nm | 545nm | 66000M -1 cm -1 | 90 |
| Probe | ||||
| 7 | 275nm/325nm | 615nm | 66000M -1 |
15％ |
| Probe 8 | 275nm/325nm | 545nm | 60000M -1 cm -1 | 89％ |
| Probe 9 | 275nm/325nm | 545nm | 15600M -1 cm -1 | 92 |
| Probe | ||||
| 10 | 275nm/325nm | 545nm | 181000M -1 cm -1 | 78％ |
| Probe 11 | 275nm/325nm | 545nm | 191000M -1 cm -1 | 85％ |
| Probe 12 | 275nm/325nm | 615nm | 191000M -1 cm -1 | 17％ |
| Probe 13 | 275nm/325nm | 545nm | 192000M -1 cm -1 | 77％ |
| Probe 14 | 275nm/325nm | 615nm | 190000M -1 cm -1 | 87％ |
| Probe15 | 275nm/325nm | 545nm | 191000M -1 cm -1 | 90％ |
Example 2
And (3) measuring the fluorescence lifetime: accurately weighing 5mg of probe 5, dissolving in anhydrous dimethyl sulfoxide to obtain 10mmol of mother liquor, taking 3uL of the mother liquor and 3mL of deionized water, repeatedly and uniformly blowing and stirring by a liquid transfer gun, transferring the solution into a 1cm quartz cuvette, and detecting the fluorescence life of a sample by using a steady-state transient fluorescence spectrometer of Edinburghlnstruments. UK, wherein the fluorescence life of the compound is 2.497ms as shown in figure 1.
Example 3
Stability in aqueous solution test: accurately weighing about 5mg of probe1, probe 2, probe 3, probe 4, probe 5, probe 7, probe 9, probe 12, probe 13 and probe 14, dissolving in anhydrous dimethyl sulfoxide to prepare a mother solution of 10mmol, adding 3uL of the mother solution into 3mL of deionized water or 100uM of EDTA aqueous solution respectively by a pipette, repeatedly and uniformly blowing the solution by the pipette, transferring the solution into a 1cm quartz cuvette, and performing optical performance measurement at 0h, 4h, 8h, 16h, 24h and 48h by using an Agilent fluorescence spectrophotometer respectively, wherein the fluorescence intensity of the probe is kept above 80% within 48h no matter in the aqueous solution or in the EDTA aqueous solution, which indicates that the probe has better stability.
TABLE II stability in aqueous solution of Probe1, 2, 3, 4, 5, 7, 9, 12, 13, 14
Example 4
Time-resolved immunoassay for CRP: dissolving the Probe15 in dimethyl sulfoxide to prepare a mother solution with the concentration of 10mmol/L, adding 1uL of a 30uL monoclonal antibody (Mab 1, cat # A5811,4.9mg/mL, bioSpacitic Inc.) for resisting CRP into the system, shaking overnight at 4 ℃, after the reaction is finished, dialyzing the system in a 10K dialysis belt for 2 days, replacing a dialysate HEPES buffer solution for 5 times, centrifugally concentrating the system after the dialysis is finished to obtain Mab1-Probe15, spraying the Mab1-Probe15 (3 mg/mL) onto glass fibers, and drying for 24 hours at 37 ℃.
CRP Mab (Mab 2, cat # A5804,2.3mg/mL, bioSpacitic Inc.) and polylysine (10 mg/mL) in water were streaked with a T-line and a C-line, respectively, and oven-dried at 37 ℃ for 24h. The strips were assembled and different concentrations of CRP standard were dissolved in 2% tween 20, 0.1% bsa, 1% sucrose solution for lateral chromatography with a fluorescence delay of 300us for signal detection as shown in fig. 2 using background signal +3SD (standard deviation as detection sensitivity limit, detection limit in this method is 1.56pg/mL.
In conclusion, the lanthanide complex, the preparation method thereof, the fluorescent labeling reagent and the fluorescent labeling method solve the problem of small molar extinction coefficient of 2,6-di (N-pyrazole) pyridine amino carboxylic acid lanthanide complexes by connecting a plurality of lanthanide complexes together to form a polymer, and meanwhile, the lanthanide complex prepared by the method has the advantages of good stability and high brightness.
In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
Claims (9)
1. A lanthanide complex characterized by the structure:
A is any one of polyethyleneimine and polypropyleneimine;
f is 2,6-bis (N-pyrazole) pyridinecarboxylic acids, comprising the following structure:
R 4 Is ester group, amido group, aryl with active substitution, heterocyclic group, alkynyl or with active substitutionAny one of heterocyclic groups;
ln is a lanthanide metal, including Td 3+ 、Eu 3+ 。
2. The lanthanide complex of claim 1,
the structure of the polyethylene imine A is as follows:
R 1 Is any one of hydrogen, hydroxyl, sulfydryl, carboxyl, amino, isocyanate, isothiocyanate, ester group and active ester group;
m is an integer of 1 to 10;
R 2 is any one of hydrogen, methyl, ethyl, hydroxyethyl, methylene carboxyl, formyl and acetyl.
3. The lanthanide complex of claim 1,
the structure of the polypropylene imine is as follows:
R 1 Is any one of hydrogen, hydroxyl, sulfydryl, carboxyl, amino, isocyanate, isothiocyanate, ester group and active ester group;
m is an integer of 1 to 10;
R 2 is any one of hydrogen, methyl, ethyl, hydroxyethyl, methylene carboxyl, formyl and acetyl.
4. A process for preparing a lanthanide complex as defined in claim 1, which comprises the steps of:
step S1, compound 1, ethanol and [ Pd (PPh) 3 ) 2 Cl 2 ]And dry triethylamine dissolved inHeating in 15mL toluene in 100 deg.C oil bath under bubbling CO for 14h, removing solvent under reduced pressure, dissolving residue in 100mL dichloromethane, washing with water for several times, washing with saturated saline, and adding Na to organic phase 2 SO 4 Drying, removing the solvent under reduced pressure, and purifying the residue by column chromatography to obtain yellow oily compound 2;
step S2, dissolving the compound 2 in 20mL of methanol, adding 5mL of NaOH aqueous solution, heating in a 60 ℃ oil bath for 4h under the protection of Ar, pressurizing to remove the solvent after the reaction is finished, adding 20mL of ethanol into the residue, heating to dissolve the residue, filtering to remove the residue, dropwise adding 80mL of THF into the filtrate, precipitating a large amount of solid, centrifuging, carefully removing the supernatant, and drying the residue in vacuum to obtain a white solid compound 3;
step S3, dissolve Compound 3 in 10mL deionized water and add TdCl 3 Heating in oil bath at 60 ℃ for 4h under Ar protection, and removing the solvent under reduced pressure after the reaction is finished to obtain a white solid compound 4;
step S4, preparing an intermediate compound;
and step S5, preparing a probe from the compound 4 and/or the intermediate compound, wherein the probe is the lanthanide complex.
5. The process for preparing a lanthanide complex as defined in claim 4,
the step S4 includes:
step S4a, dissolving pentaethylenehexamine in 50mL of ethanol, dropwise adding 10mL of dichloromethane solution containing di-tert-butyl dicarbonate under an ice bath condition, returning the system to room temperature after dropwise adding, reacting for 6 hours under the protection of Ar, and removing the solvent under reduced pressure after the reaction is finished to obtain a colorless oily liquid compound 5;
step S4b, dissolving the compound 4 in 5mL of anhydrous DMF, adding the compound 5, benzotriazole-1-yl-oxy tripyrrolidinophosphonium hexafluorophosphate and dry TEA, stirring at room temperature under the protection of Ar, reducing pressure to remove the solvent after the reaction is finished, and separating the residue by reversed-phase preparative chromatography to obtain a compound 6;
step S4c, dissolving the compound 5 in 10mL of anhydrous N, N-dimethylformamide, adding tri (2-amine ethylamine) amine and 20uL of dry triethylamine, stirring at room temperature under the protection of Ar, pressurizing after the reaction is finished, removing the solvent, and purifying the residue by reversed-phase preparative chromatography to obtain a light yellow oily compound 7;
step S4d, reacting Compound 8 with K 2 CO 3 Dissolving in 150mL of water, slowly adding succinic acid monomethyl ester acyl chloride under Ar protection, returning the system to room temperature after dropwise addition, stirring for 0.5h, extracting the system with ethyl acetate for several times, combining organic phases, washing with saturated saline water, and washing the organic phase with Na 2 SO 4 Drying, and removing the colorless oily product compound 9 of the organic solvent under reduced pressure;
step S4e, dissolving the compound 6 in 10mL of anhydrous N, N-dimethylformamide, adding the compound 9 and 20mL of dried triethylamine, stirring at room temperature under the protection of Ar, pressurizing after the reaction is finished, removing the solvent, and purifying the residue by reverse phase preparative chromatography to obtain a light yellow solid compound 10;
step S4f, dissolving the compound 10 in 20mL of methanol, adding 5mL of NaOH aqueous solution, heating in a 60 ℃ oil bath for 4h under the protection of Ar, pressurizing to remove the solvent after the reaction is finished, adding 20mL of ethanol into the residue, heating to dissolve the residue, filtering to remove the residue, dropwise adding 80mL of THF into the filtrate, precipitating a large amount of solid, centrifuging, carefully removing the supernatant, and drying the residue in vacuum to obtain a compound 11;
6. the process for preparing a lanthanide complex as defined in claim 4,
the step S5 includes:
step S5a, dissolving the compound 4 in 5mL of anhydrous DMF, adding N-hydroxyethyl ethylenediamine, benzotriazole-1-yl-oxy tripyrrolidinyl phosphorus hexafluorophosphate and dry TEA, stirring at room temperature under the protection of Ar, reducing pressure to remove the solvent after the reaction is finished, and separating the residue by reversed-phase preparative chromatography to obtain a white solid probe 1;
step S5b, dissolving the compound 4 in 5mL of anhydrous DMF, adding diethylenetriamine, benzotriazole-1-yl-oxy tripyrrolidinyl phosphorus hexafluorophosphate and dry TEA, stirring at room temperature under the protection of Ar, reducing pressure to remove the solvent after the reaction is finished, and separating the residue by reversed-phase preparative chromatography to obtain a probe 2;
step S5c, dissolving the compound 4 in 5mL of anhydrous DMF, adding pentaethylenehexamine, benzotriazole-1-yl-oxy tripyrrolidinophosphonium hexafluorophosphate and dry TEA, stirring at room temperature under the protection of Ar, reducing pressure to remove the solvent after the reaction is finished, and separating the residue by reversed-phase preparative chromatography to obtain a probe 3;
step S5d, dissolving the compound 4 in 5mL of anhydrous DMF, adding nonaethylene decaamine, benzotriazole-1-yl-oxy tripyrrolidinophosphonium hexafluorophosphate and dry TEA, stirring at room temperature under the protection of Ar, reducing pressure to remove the solvent after the reaction is finished, and separating the residue by reversed-phase preparative chromatography to obtain a probe 4;
step S5e, dissolving the compound 6 in 20mL of a solution of dichloromethane and N, N-dimethylformamide with the mass ratio of 1:1, adding 2mL of trifluoroacetic acid, stirring at room temperature for 2h under the protection of Ar, after the reaction is finished, removing the solvent under pressure, dissolving the residue in 50mL of hot methanol, adding 70mL of THF, and filtering to obtain a white probe 5;
step S5f, dissolve Compound 7 in 2mL deionized water and add TdCl 3 Heating in oil bath at 60 ℃ for 4h under Ar protection, and removing the solvent under reduced pressure after the reaction is finished to obtain a white solid probe 6;
step S5g, dissolve Compound 7 in 2mL deionized water and add EuCl 3 Heating in oil bath at 60 ℃ for 4h under Ar protection, and removing the solvent under reduced pressure after the reaction is finished to obtain a white solid probe 7;
step S5h, dissolving the compound 4 in 5mL of anhydrous DMF, adding dipropylene triamine, benzotriazole-1-yl-oxy tripyrrolidinyl phosphorus hexafluorophosphate and dry TEA, stirring at room temperature under the protection of Ar, reducing pressure to remove the solvent after the reaction is finished, and separating residues by reversed-phase preparative chromatography to obtain a probe 8;
step S5i, dissolving the compound 4 in 5mL of anhydrous DMF, adding the compound 8, benzotriazole-1-yl-oxy tripyrrolidinophosphonium hexafluorophosphate and dry TEA, stirring at room temperature under the protection of Ar, reducing pressure to remove the solvent after the reaction is finished, and separating the residue by reversed-phase preparative chromatography to obtain a probe 9;
step S5j, dissolving the compound 4 in 5mL of anhydrous DMF, adding the compound 9, benzotriazole-1-yl-oxy tripyrrolidinophosphonium hexafluorophosphate and dry TEA, stirring at room temperature under the protection of Ar, reducing pressure to remove the solvent after the reaction is finished, and separating the residue by reversed-phase preparative chromatography to obtain a probe 10;
step S5k, dissolve Compound 10 in 2mL deionized water and add TdCl 3 Heating in oil bath at 60 ℃ for 4h under the protection of Ar, and removing the solvent under reduced pressure after the reaction is finished to obtain a white solid probe 11;
step S5l, dissolve Compound 10 in 2mL deionized water and add EuCl 3 Heating in oil bath at 60 ℃ for 4h under the protection of Ar, and removing the solvent under reduced pressure after the reaction is finished to obtain a probe 12;
step S5m, dissolve Compound 11 in 2mL deionized water and add TdCl 3 Heating in oil bath at 60 ℃ for 4h under Ar protection, and removing the solvent under reduced pressure after the reaction is finished to obtain a probe 13;
step S5n, dissolve Compound 11 in 2mL deionized water and add EuCl 3 Heating in oil bath at 60 ℃ for 4h under Ar protection, and removing the solvent under reduced pressure after the reaction is finished to obtain a probe 14;
step S5o, dissolving the probe 13 in 3mL of dry N, N-dimethylformamide, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, stirring at room temperature for 2h under the protection of Ar, after the reaction is finished, adding 6mL of tetrahydrofuran, precipitating a large amount of solid, centrifuging, carefully removing a supernatant, and drying the residue in vacuum to obtain a white solid probe 15;
7. a fluorescent labeling reagent, comprising:
the lanthanide complex of any one of claims 1-6.
8. The fluorescent labeling reagent of claim 7,
the rare earth metal ions in the fluorescent labeling reagent are complexes of trivalent europium ions and trivalent terbium ions.
9. A method of fluorescent labeling, comprising:
a complex comprising the rare earth metal according to any one of claims 1 to 6 is used as a labeling agent.
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| US5859215A (en) * | 1995-10-25 | 1999-01-12 | Wallac Oy | Biospecific binding reactants labelled with luminescent lanthanide chelates and their use |
| JP2001335574A (en) * | 2000-05-26 | 2001-12-04 | Kazuko Matsumoto | New labeled compound |
| CN1685232A (en) * | 2002-03-08 | 2005-10-19 | 松本和子 | Novel fluorescent label compounds |
| CN101221169A (en) * | 2007-12-27 | 2008-07-16 | 中国科学院长春应用化学研究所 | Homogeneous phase time discrimination fluorescence immunity analysis chelating agent and its preparing method |
| FR2935973A1 (en) * | 2008-09-18 | 2010-03-19 | Centre Nat Rech Scient | New dipyrazol-1-yl-pyridine compounds useful for preparing complex, which is useful for e.g. the detection of metal cations in solutions, as a luminescent marker for the absorption of two-photons, and the recognition of e.g. neuravidin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5859215A (en) * | 1995-10-25 | 1999-01-12 | Wallac Oy | Biospecific binding reactants labelled with luminescent lanthanide chelates and their use |
| JP2001335574A (en) * | 2000-05-26 | 2001-12-04 | Kazuko Matsumoto | New labeled compound |
| CN1685232A (en) * | 2002-03-08 | 2005-10-19 | 松本和子 | Novel fluorescent label compounds |
| CN101221169A (en) * | 2007-12-27 | 2008-07-16 | 中国科学院长春应用化学研究所 | Homogeneous phase time discrimination fluorescence immunity analysis chelating agent and its preparing method |
| FR2935973A1 (en) * | 2008-09-18 | 2010-03-19 | Centre Nat Rech Scient | New dipyrazol-1-yl-pyridine compounds useful for preparing complex, which is useful for e.g. the detection of metal cations in solutions, as a luminescent marker for the absorption of two-photons, and the recognition of e.g. neuravidin |
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