CN111675737A - Electroneutral homoleptic cyclometalated iridium complexes for biomarkers - Google Patents
Electroneutral homoleptic cyclometalated iridium complexes for biomarkers Download PDFInfo
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- iridium complex
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- 239000000090 biomarker Substances 0.000 title claims description 15
- 150000002503 iridium Chemical class 0.000 title description 2
- 229910052741 iridium Inorganic materials 0.000 claims abstract description 47
- GKOZUEZYRPOHIO-UHFFFAOYSA-N iridium atom Chemical compound [Ir] GKOZUEZYRPOHIO-UHFFFAOYSA-N 0.000 claims abstract description 45
- 238000002372 labelling Methods 0.000 claims abstract description 29
- 230000007935 neutral effect Effects 0.000 claims abstract description 22
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 7
- 238000006243 chemical reaction Methods 0.000 claims description 24
- 239000003446 ligand Substances 0.000 claims description 20
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 18
- 239000002243 precursor Substances 0.000 claims description 15
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical compound O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 claims description 15
- 150000002148 esters Chemical class 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- 238000010992 reflux Methods 0.000 claims description 11
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- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 10
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 10
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- 230000003301 hydrolyzing effect Effects 0.000 claims description 7
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- WZJYKHNJTSNBHV-UHFFFAOYSA-N benzo[h]quinoline Chemical compound C1=CN=C2C3=CC=CC=C3C=CC2=C1 WZJYKHNJTSNBHV-UHFFFAOYSA-N 0.000 claims description 4
- 239000011261 inert gas Substances 0.000 claims description 4
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- 230000002378 acidificating effect Effects 0.000 claims description 3
- 150000002500 ions Chemical class 0.000 claims description 3
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- 150000005360 2-phenylpyridines Chemical class 0.000 claims description 2
- 108091023037 Aptamer Proteins 0.000 claims description 2
- 230000009471 action Effects 0.000 claims description 2
- VQGHOUODWALEFC-UHFFFAOYSA-N alpha-Phenylpyridine Natural products C1=CC=CC=C1C1=CC=CC=N1 VQGHOUODWALEFC-UHFFFAOYSA-N 0.000 claims description 2
- 150000001413 amino acids Chemical class 0.000 claims description 2
- 239000000427 antigen Substances 0.000 claims description 2
- 108091007433 antigens Proteins 0.000 claims description 2
- 102000036639 antigens Human genes 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 125000001072 heteroaryl group Chemical group 0.000 claims description 2
- 239000001257 hydrogen Substances 0.000 claims description 2
- 229910052739 hydrogen Inorganic materials 0.000 claims description 2
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 2
- 125000005647 linker group Chemical group 0.000 claims description 2
- 229910052751 metal Inorganic materials 0.000 claims description 2
- 239000002184 metal Substances 0.000 claims description 2
- 229910052757 nitrogen Inorganic materials 0.000 claims description 2
- 125000004433 nitrogen atom Chemical group N* 0.000 claims description 2
- 108020004707 nucleic acids Proteins 0.000 claims description 2
- 150000007523 nucleic acids Chemical class 0.000 claims description 2
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- 125000003835 nucleoside group Chemical group 0.000 claims description 2
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- -1 iridium metal complex Chemical class 0.000 claims 2
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- 150000001875 compounds Chemical class 0.000 description 22
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene chloride Substances ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 20
- 229940125904 compound 1 Drugs 0.000 description 14
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 13
- 229940098773 bovine serum albumin Drugs 0.000 description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 229940125782 compound 2 Drugs 0.000 description 8
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 8
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- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- MSDMPJCOOXURQD-UHFFFAOYSA-N C545T Chemical compound C1=CC=C2SC(C3=CC=4C=C5C6=C(C=4OC3=O)C(C)(C)CCN6CCC5(C)C)=NC2=C1 MSDMPJCOOXURQD-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
- C07F15/0033—Iridium compounds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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Abstract
The invention relates to an electroneutral homoleptic cyclometalated iridium complex and a synthesis method thereof. The invention also relates to application of the electroneutral homoleptic cyclometalated iridium complex as a phosphorescent marker labeled biomolecule and a labeling method. Compared with a non-neutral cyclometalated iridium complex, the electroneutral complex has more excellent chemical stability and higher luminous efficiency, and has relatively low influence on a labeled species as a marker. The neutral phosphorescent cyclometalated iridium complex labeled molecules can meet the requirements of a biological analysis method (such as protein labeling) with high sensitivity and high reproducibility by taking a luminescent signal as a detection mode.
Description
Technical Field
The invention relates to the technical field of biological labeling, in particular to an electroneutral homoleptic cyclometalated iridium complex and application thereof as a novel labeling molecule in the field of biological analysis.
Background
Bioanalytical techniques based on the affinity between biological macromolecules (e.g., immunoassays, DNA detection techniques, etc.) play an important role in the fields of modern life sciences, clinical medicine, and food and environmental analysis applications. The core and key of such bioanalytical techniques are biomarkers, labeling techniques, and detection of signals generated from labeled molecules, and the biomarkers are important substances for studying information transfer of bioactive molecules, interactions between biomolecules, and interactions between biomolecules and drugs.
The biomarker is generally composed of a group capable of binding to a target biomolecule and a substance capable of generating a detection signal, and commonly used biomarkers include a substance containing a radioactive element, a photoluminescent substance, an electroluminescent substance, a chemiluminescent substance, and a biochemical luminescent substance. The biomarker comprises one or more signal generating units and one or more reactive groups capable of binding to the target biomolecule. The reactive group capable of binding to the target biomolecule is susceptible to forming a covalent bond with the biomolecule to be labeled. The labeling process is to combine one or more labeling molecules with the biologically active substance to form a complex that retains its biological affinity for the particular substance to be detected.
Research finds that compared with an ionic cyclometalated iridium complex, the neutral cyclometalated iridium complex has higher environmental stability and more excellent luminescence performance, and the charge state of a labeled biomolecule cannot be changed by an electrically neutral luminescent marker, so that the influence of the electrically neutral marker on the biological activity of the to-be-labeled biomolecule is minimal, however, a synthetic method for introducing a reactive bioconjugate group into the neutral cyclometalated iridium complex metal complex is not established so far, and the work of the related neutral cyclometalated iridium complex marker is not reported in the prior art, so that the development of the neutral cyclometalated iridium complex-based biomarker is beneficial to the development of novel high-performance biomarker molecules, and the development of a high-sensitivity biological analysis technology is promoted.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a charge-neutral homoleptic cyclometalated iridium complex, which has more excellent chemical stability and higher luminous efficiency compared with a non-neutral cyclometalated iridium complex, and has relatively low influence on a labeled species when used as a marker.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a charge neutral homoleptic cyclometalated iridium complex, which has a structure shown in the following general formula (1):
the above general formula (1) is abbreviated Ir [ L ]c∧n]3X, wherein:
Lc∧nrepresents by an sp2Hybridized carbon atom and one sp2A heteroaromatic ring carbon nitrogen ligand with a hybridized nitrogen atom coordinated to an ir (iii) ion;
said Lc∧nIn the ligand, at least one Lc∧nThe ligand is connected with a biological coupling connecting group X: carboxylic acid (-COOH) or NHS ester of carboxylic acid
In the present invention, Lc∧nThe ligand is a monoanionic carbon-nitrogen bidentate ligand, is combined with an iridium trivalent positive ion central atom through a covalent bond and a coordination bond to form a neutral stable structure complex, has the phosphorescence luminescent property, and a connecting group capable of being biologically coupled is connected with a phosphorescence luminophor through a flexible carbon chain.
Compared with the existing non-neutral (ionic) cyclometalated iridium complex, the homoleptic cyclometalated iridium complex has more excellent chemical stability and higher luminous efficiency. In addition, when the non-neutral cyclometalated iridium complex is used as a biomarker, the charge of the non-neutral cyclometalated iridium complex can influence the charge property of biomolecules, so that the specific binding between organisms is influenced. The homoleptic cyclometalated iridium complex is neutral in electricity and has relatively low influence on the marked species when being used as a biomarker.
Further, said Lc∧nThe ligand is selected from:
substituted or unsubstituted 2-phenylpyridine, 2-phenylquinoline, benzo [ h ] quinoline, 2-phenylbenzo [ b ] thiophene; and derivatives thereof.
Further, L to which a bioconjugateable linking group is attachedc∧nThe ligand is selected from one of the following structures:
wherein X is-COOH or
T is C1-10Y is hydrogen or an uncharged substituent group.
Through the above aspects, the phosphorescent label may be used to label a biomacromolecule.
Further, the charge neutral homoleptic cyclometalated iridium complex is selected from one of the following structures:
in a second aspect, the present invention provides a method for synthesizing the electroneutral homoleptic cyclometalated iridium complex according to the first aspect, including:
(1) under the protection of inert gas, carrying out reflux reaction on the ligand 1 and the ligand 2 in a solvent, and separating to obtain a precursor;
(2) refluxing and hydrolyzing the precursor under an acidic condition, and extracting a product to obtain an electroneutral homoleptic cyclometalated iridium complex with a-COOH connecting group;
wherein ligand 1 is Lc∧nCN, ligand 2 is selected from (L)c∧n)2Ir(acac)、(Lc∧n)Ir(acac)2、Ir(acac)3One kind of (1).
Further, the synthesis method comprises the following steps:
the electroneutral homoleptic cyclometalated iridium complex with the-COOH connecting group, N-hydroxysuccinimide active ester (HOSu) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/dicyclohexylcarbodiimide are mixed(DCC) reacts in solvent, the reaction liquid is dropped into ether for sedimentation, and the product is obtained after filtration, washing, drying and spin-drying and separation
The electroneutrality of the connecting group is matched with the cyclometalated iridium complex.
In a third aspect, the invention provides the use of the charge-neutral homoleptic cyclometalated iridium complex as described in the first aspect as a phosphorescent label for labeling a target biomolecule. After the complex is used to mark biomolecule, the complex can form { Ir [ L ]c∧n]3A labeled biomolecule complex of structure-Z, wherein the target biomolecule Z includes but is not limited to biological molecules such as haptens, amino acids, nucleic acids, nucleosides, nucleotides, proteins, aptamers, antibodies and antigens.
In a fourth aspect of the present invention, there is provided a method for biomarker of the electroneutral homoleptic cyclometalated iridium complex as described in the first aspect, wherein the method for biomarker comprises:
in a proper solution system, active ester with succinimide
The electroneutrality of the connecting group is uniformly matched with the cyclometalated iridium complex to be mixed with target biomolecules, and the labeling reaction is directly carried out.
Or the marking method comprises the following steps:
the preparation method comprises the following steps of (1) reacting an electroneutral cyclohomoleptic metal iridium complex with a carboxylic acid (-COOH) connecting group with N-hydroxysuccinimide active ester (HOSu) under the action of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or Dicyclohexylcarbodiimide (DCC) to generate the succinimide active ester in situ;
in a proper solution system, active ester of succinimide generated in situ is mixed with target biological molecules for labeling reaction. It is pointed out that the active ester of succinimide does not need to be separated after in situ generation, and can be directly mixed with target biological molecules for labeling reaction.
In the above labeling method, the appropriate solution system is a conventional buffer system, such as Phosphate Buffered Saline (PBS), Tris buffer, Bis-Tris buffer, MES buffer, HEPES buffer, etc.
Compared with the prior art, the invention has the beneficial effects that:
the phosphorescence marker of the invention adopts carboxylic acid group which can be indirectly combined with target biological molecules or active ester group of succinimide which can be directly combined as a reaction group, and the central cyclometalated iridium complex as a phosphorescence signal body, so that the phosphorescence-marked biological molecule compound after efficiently marking biological molecules has high luminous efficiency and high stability, keeps high biological activity, and improves the sensitivity and the reproducibility of a biological analysis method.
Compared with a non-neutral cyclometalated iridium complex, the electroneutral complex has more excellent chemical stability and higher luminous efficiency, and has relatively low influence on a labeled species as a marker. The neutral phosphorescent cyclometalated iridium complex labeled molecule can meet the requirements of a biological analysis method (such as protein labeling) with high sensitivity and high reproducibility by taking a luminescent signal as a detection mode.
Drawings
FIG. 1 shows labeling of a precursor of Compound 11H NMR spectrum;
FIG. 2 is a schematic representation of the labeling of Compound 11H NMR spectrum;
FIG. 3 is a mass spectrum of labeled Compound 1;
FIG. 4 is a diagram of labeling Compound 21H NMR spectrum;
FIG. 5 is a photoluminescence spectrum of Bovine Serum Albumin (BSA) labeled with labeled Compound 2;
FIG. 6 is a photoluminescence spectrum of marker compound 4;
FIG. 7 is an absorption spectrum of a precursor of the labeled compound 1;
FIG. 8 is an absorption spectrum of the labeled compound 8.
Detailed Description
The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
In each of the following examples, the structures of the synthesized labeled compounds are as follows:
example 1: synthesis of labeled Compound 1
The reaction formula is as follows:
will (ppy)2Ir (acac) and LppyRefluxing CN in glycerol under inert gas protection, tracking reaction by TLC, removing solvent by spinning, and subjecting the obtained residue to silica gel column chromatography, preferably with mobile phase of CH2Cl2MeOH system, the precursor of the labelled compound 1 was isolated, yield: 60 percent.
Labelling of precursors of Compound 11H NMR is shown in FIG. 1: (CDCl3,400MHz)7.87(s,1H),7.85(s,1H),7.63(m,4H),7.51(m,4H),7.39(m,2H),6.83(m,11H),6.70(d,1H),2.67(m,2H),2.41(m,2H),1.99(m, 2H).
The absorption spectrum of the precursor of the labeled compound 1 is shown in FIG. 7.
Refluxing and hydrolyzing the precursor of the labeled compound 1 under an acidic condition, tracking by TLC to complete the reaction, extracting by dichloromethane to obtain the labeled compound 1, wherein the yield is as follows: 100 percent.
Of the labelled Compound 11H NMR is shown in FIG. 2: (CDCl3,400MHz)7.85(m,2H),7.63(m,4H),7.52(m,4H),7.39(d,1H),6.83(m,11H),6.70(d,1H),2.67(m,2H),2.41(m,2H),1.97(m, 2H).
Mass spectrometric characterization of labeled Compound 1 is shown in FIG. 3, consistent with theoretical values.
Example 2: synthesis of labeled Compound 2
The reaction formula is as follows:
weighing a mixture with a molar ratio of 1: 2: 2 in 10mL of DMF solvent, filtering off Dicyclohexylurea (DCU), adding the filtrate dropwise into ether for settling, filtering, washing with ether, drying and spin-drying, and performing silica gel column chromatography to preferably select the mobile phase as CH2Cl2MeOH system, isolated and purified to afford the title compound 2, yield: 80 percent.
Of the labelled Compound 21H NMR is shown in FIG. 4: (CDCl3,400MHz)7.85(m,2H),7.71(s,1H),7.63(d,3H),7.51(m,4H),7.40(d,1H),6.83(m,11H),6.72(m,1H),2.84(d,4H),2.75(m,2H),2.65(m,2H),2.07(m, 2H).
Elemental analysis: c, 56.70%; h, 4.01%; n, 6.69% (found); c, 58.77%; h, 3.97%; n, 6.69% (C41H33IrN4O4 theoretical).
The labeled compound 2 can be used directly for the next step of labeling proteins.
Example 3: synthesis of labeled Compounds 3 and 4
The reaction formula is as follows:
ir (acac)3And LppyRefluxing CN in glycerol under inert gas protection, tracking reaction by TLC, spin-drying solvent, subjecting the obtained residue to silica gel column chromatography, preferably with mobile phase CH2Cl2MeOH system, the precursor of the labelled compound 3 was isolated, yield: 65 percent.
And (3) refluxing and hydrolyzing the precursor of the labeled compound 3 under the condition of hydrochloric acid, tracking by TLC (thin layer chromatography) to completely react, and extracting by using dichloromethane to obtain the labeled compound 3 quantitatively. Weighing a mixture with a molar ratio of 1: 2: 2, dissolving the labeled compound 3, HOSu and DCC in 10mL of DMF solvent, reacting at room temperature overnight, filtering to remove Dicyclohexylurea (DCU), adding the filtrate dropwise into ether for settling, filtering, washing with ether, drying, spinning, passing through silica gel columnThe preferred mobile phase for chromatography is CH2Cl2MeOH system, isolated and purified to afford the title compound 4, yield: 75 percent.
Elemental analysis of labeled Compound 3: c, 60.60%; h, 4.61%; n, 3.09% (found); c, 60.64%; h, 4.65%; n, 3.07% (C46H42IrN2O6 theoretical).
Elemental analysis of labeled Compound 4: c, 56.83%; h, 4.24%; n, 6.96% (found); c, 56.85%; h, 4.27%; n, 6.98% (C57H51IrN6O12 theoretical).
The labeled compound 3 can be used for the next step of labeling proteins without isolation by in situ formation of NHS ester, and the labeled compound 4 can be used for labeling proteins directly. The photoluminescence pattern of marker compound 4 is shown in figure 6.
Example 4: synthesis of labeled Compound 5
The reaction formula is as follows:
refluxing and hydrolyzing a precursor of the labeled compound 5 under the condition of hydrochloric acid, tracking the reaction by TLC completely, extracting and purifying by dichloromethane to obtain the labeled compound 5, wherein the separation yield is as follows: 85 percent.
Weighing a mixture with a molar ratio of 1: 2: 2 in 10mL of DMF solvent, reacting at room temperature overnight, filtering off Dicyclohexylurea (DCU), adding the filtrate dropwise into diethyl ether for settling, filtering, washing with diethyl ether, drying, and performing silica gel column chromatography to obtain the final product with a preferable mobile phase of CH2Cl2MeOH system, isolated and purified to afford the title compound 8, yield: 75 percent.
Elemental analysis: c, 59.51%; h, 4.40%; n, 5.06% (found); c, 59.55%; h, 4.39%; n, 5.08% (C41H36IrN3O4 theoretical). The absorption spectrum of labeled compound 8 is shown in FIG. 8.
Example 5: synthesis of labeled Compound 6
The reaction formula is as follows:
refluxing and hydrolyzing a precursor of the labeled compound 6 under the condition of hydrochloric acid, tracking the reaction by TLC completely, extracting and purifying by dichloromethane to obtain the labeled compound 6, wherein the separation yield is as follows: 81 percent.
Elemental analysis: c, 56.84%; h, 3.30%; n, 4.66% (found); c, 56.81%; h, 3.33%; n, 4.62% (C43H30IrN3O2S3 theoretical).
Example 6: synthesis of labeled Compound 7
The reaction formula is as follows:
refluxing and hydrolyzing a precursor of the labeled compound 7 under the condition of hydrochloric acid, tracking the reaction by TLC completely, extracting and purifying by dichloromethane to obtain the labeled compound 7, wherein the separation yield is as follows: 83 percent.
Elemental analysis: c, 64.37%; h, 4.50%; n, 3.96% (found); c, 64.39%; h, 4.55%; n, 3.95% (C57H48IrN3O6 theoretical).
Example 7: labeling Compound 1 Bovine Serum Albumin (BSA) labeling with EDC and HOSu
The reaction formula is as follows:
to a PBS buffer (pH 7.4) of the labeled compound 1((1 molar equivalent)), housu ((2 molar equivalent)) and EDC ((2 molar equivalent)) were added, and the mixture was reacted at room temperature for 30 minutes, followed by addition of BSA and stirring at room temperature overnight. The resulting mixture was purified by dialysis in PBS for three days using dialysis bags with a cut-off of 10000Da to remove the non-crosslinked compounds, i.e.to obtain crosslinked labeled BSA which emits a green light under UV excitation (see FIG. 5).
Example 8: direct labeling of BSA with a succinimide-active ester-containing labeling Compound 2
The reaction formula is as follows:
the labeled compound 2 containing succinimide active ester was dissolved in anhydrous DMF, and the resulting solution was mixed with PBS buffer solution (pH 7.4) of BSA, and stirred at room temperature for 4 h. The resulting mixture was purified by dialysis in PBS for three days using dialysis bags with a cut-off of 10000Da to remove the non-crosslinked compounds, i.e.to obtain crosslinked labeled BSA which emits a green light under UV excitation.
Example 9: labeling Compound 6 Bovine Serum Albumin (BSA) labeling with EDC and HOSu
The reaction formula is as follows:
to a PBS buffer solution (pH 7.4) of labeled compound 6(1 molar equivalent), HOSu (2 molar equivalents) and EDC (2 molar equivalents) were added, and the mixture was reacted at room temperature for 30 minutes, followed by addition of BSA and stirring at room temperature overnight. The resulting mixture was purified by dialysis in PBS for three days using dialysis bags with a cut-off of 10000Da to remove the non-crosslinked compounds, i.e.to obtain crosslinked labeled BSA which emitted orange-red light under UV excitation.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. An electroneutral homoleptic cyclometalated iridium complex characterized by having a structure represented by the following general formula (1):
the above general formula (1) is abbreviated Ir [ L ]c∧n]3X, wherein:
Lc∧nrepresents by an sp2Hybridized carbon atom and one sp2A heteroaromatic ring carbon nitrogen ligand with a hybridized nitrogen atom coordinated to an ir (iii) ion;
said Lc∧nIn the ligand, at least one Lc∧nThe ligand is connected with a connecting group X which can be coupled biologically, and the connecting group X is-COOH or
2. The charge-neutral homoleptic cyclometalated iridium complex as claimed in claim 1 wherein L isc∧nThe ligand is selected from:
substituted or unsubstituted 2-phenylpyridine, 2-phenylquinoline, benzo [ h ] quinoline, 2-phenylbenzo [ b ] thiophene; and derivatives thereof.
3. The charge neutral homoleptic cyclometalated iridium complex as claimed in claim 2 wherein L to which the bioconjugateable linking group X is attachedc∧nThe ligand is selected from one of the following structures:
wherein X is-COOH or
T is C1-10Y is hydrogen or an uncharged substituent group.
4. The charge neutral homoleptic cyclometalated iridium complex according to claim 1, wherein the charge neutral homoleptic cyclometalated iridium complex is selected from one of the following structures:
5. the method for synthesizing an electroneutral homoleptic cyclometalated iridium complex as claimed in claim 1, characterized in that it comprises the following steps:
(1) under the protection of inert gas, carrying out reflux reaction on the ligand 1 and the ligand 2 in a solvent, and separating to obtain a precursor;
(2) refluxing and hydrolyzing the precursor under an acidic condition, and extracting a product to obtain an electroneutral homoleptic cyclometalated iridium complex with a-COOH connecting group;
wherein ligand 1 is Lc∧nCN, ligand 2 is selected from (L)c∧n)2Ir(acac)、(Lc∧n)Ir(acac)2、Ir(acac)3One kind of (1).
6. The method of synthesizing an electroneutral homoleptic cyclometalated iridium complex as claimed in claim 5, comprising:
the preparation method comprises the steps of reacting an electroneutral homoleptic cyclometalated iridium complex with a-COOH connecting group, N-hydroxysuccinimide active ester and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride/dicyclohexylcarbodiimide in a solvent, dripping the reaction solution into ether for sedimentation, filtering, washing, drying, spin-drying and separating to obtain the iridium complex with the function of-COOH
The electroneutrality of the connecting group is matched with the cyclometalated iridium complex.
7. Use of the charge-neutral homoleptic cyclometalated iridium complex as claimed in claim 1 as a phosphorescent label for labeling target biomolecules.
8. The use according to claim 7, wherein the target biomolecule comprises haptens, amino acids, nucleic acids, nucleosides, nucleotides, proteins, aptamers, antibodies and antigens.
9. A method for biomarker of the electroneutral homoleptic cyclometalated iridium complex according to claim 1, wherein the method comprises:
in a suitable solution system, will have
The electric neutrality of the connecting group is matched with the cyclometalated iridium metal complex to be mixed with target biological molecules for labeling reaction.
10. A method for biomarker of the electroneutral homoleptic cyclometalated iridium complex of claim 1, comprising:
reacting an electric neutral ring metal iridium metal complex with a-COOH connecting group with N-hydroxysuccinimide active ester under the action of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride or dicyclohexylcarbodiimide to generate the succinimide active ester in situ;
in a proper solution system, active ester of succinimide generated in situ is mixed with target biological molecules for labeling reaction.
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