Coding molecule for marking biomolecule and preparing method thereof

Abstract

The invention relates to a coding molecule for marking biomolecules and a preparing method thereof. The coding molecule has a chain or dendritic shaped main skeleton which is provided with one or more than two luminescence groups along the length direction of the main skeleton at different positions, and the end of which is provided with a function group able to connect with bioactive molecules. The luminescence group consists of cyclometalated iridium coordination compound, cyclometalated ruthenium coordination compound, and rare earth metal coordination compound. The preparing method comprises: taking the luminescence group to be connected to the compound having more than two reaction sites to form a luminescence unit; taking a plurality of luminescence units to form the coding molecule having a main skeleton; or taking compound having more than two reaction sites for reacting to form the main skeleton of the coding molecule, and then connecting the luminescence group to the main skeleton to form coding molecule via the reacting of the active groups distributed upon the branched chain of the main skeleton with the corresponding active groups upon the luminescence groups. The invention allows selective coding over biomolecules.

Description

technical field

[0001] The invention particularly relates to a coding molecule containing functional groups such as carboxyl groups, which can be used for fluorescently labeling biologically active substances such as antibodies, antigens, enzymes, polypeptides, proteins, and amino-modified nucleic acids, as well as its preparation method and application. Background technique

[0002] One-dimensional and two-dimensional barcode technology is widely used in computer-based identification process management. The use of barcodes is limited when smaller items need to be encoded and read. Barcodes can usually only be printed on the outer packaging. When the "item" to be marked or coded is as small as a biomacromolecule, the traditional method is to use microspheres of fluorescent substances with different emission wavelengths to code different biomolecules. This technique has been successfully applied to Luminex's high-throughput bioanalysis systems. Later, Nie et al. also devel...

Examples

Embodiment 1

[0033] Example 1: Synthesis of encoding molecules whose backbone is linear

[0034]

[0035] The synthetic steps of compound 3: get compound 1 (1mmol), HOSu (1mmol) in a 50 milliliter flask, add 20 milliliters of dry DMF, add DCC (1mmol), stir at room temperature for 20 hours, add Boc-lysine (2mmol ), DIEA (1mmol), stirred at room temperature for 24 hours, the reaction was completed, the solvent was spun off, and the column was passed to obtain the product. Elemental analysis: C, 49.42%; H, 3.82%; N, 7.04% (found value); C, 49.39%; H, 3.8 / 4%; N, 7.02% (C 41 h 38 f 4 IrN 5 o 8 theoretical value).

[0036] The synthesis steps of compound 4: take compound 2 (1mmol), Boc-lysine methyl ester (2mmol), DMAP (1mmol) in a 100 ml flask, add 20 ml of toluene, add DCC (1mmol), stir at room temperature for 2 hours After the reaction was completed, the solvent was spun off, 60 milliliters of dichloromethane was added, trifluoroacetic acid (6 mmol) was added, and stirred at room tem...

Embodiment 2

[0038] Example 2: Synthesis of Encoding Molecules with Dendritic Main Backbone

[0039]

[0040] Compound 10

[0041] The synthetic steps of compound 9: get compound 6 (1mmol), DMAP (1mmol) in a 100 milliliter flask, add 20 milliliters of DMF, get compound 8 (1mmol) and make DMF solution, under magnetic stirring, the solution that is made is dripped Add it to the reaction bottle, after the dropwise addition is completed, stir at room temperature for 24 hours. After the reaction is complete, spin off the solvent and pass through the column to obtain the product. Elemental analysis: C, 63.86%; H, 4.95%; N, 6.05% (found value); C, 63.84%; H, 4.92%; N, 6.08% (C 49 h 45 EuN 4 o 5 theoretical value).

[0042] The synthetic steps of compound 10: take compound 7 (1mmol) in a 100 milliliter flask, add 20 milliliters of DMA, take compound 9 (1mmol) and add 10 milliliters of DMA to form a solution, and add the solution dropwise to the reaction flask under stirring , the dropwise a...

Embodiment 3

[0043] Example 3 Compound 5 labels BSA (bovine serum albumin) under the effects of EDC and NHS

[0044] NHS (1.05 mmol) and EDC (1 mmol) were added to compound 5 (1 mmol) in PBS buffer solution (pH=7.4), reacted at room temperature for 30 minutes, then added BSA, and stirred overnight at room temperature. The resulting mixture was purified by dialysis in PBS solution with a molecular weight cut-off of 10000 Da for three days to remove uncrosslinked compounds, that is, to obtain BSA crosslinked with the labeled compound.

technical field

[0001] The invention particularly relates to a coding molecule containing functional groups such as carboxyl groups, which can be used for fluorescently labeling biologically active substances such as antibodies, antigens, enzymes, polypeptides, proteins, and amino-modified nucleic acids, as well as its preparation method and application. Background technique

[0002] One-dimensional and two-dimensional barcode technology is widely used in computer-based identification process management. The use of barcodes is limited when smaller items need to be encoded and read. Barcodes can usually only be printed on the outer packaging. When the "item" to be marked or coded is as small as a biomacromolecule, the traditional method is to use microspheres of fluorescent substances with different emission wavelengths to code different biomolecules. This technique has been successfully applied to Luminex's high-throughput bioanalysis systems. Later, Nie et al. also devel...

Examples

Embodiment 1

[0033] Example 1: Synthesis of encoding molecules whose backbone is linear

[0034]

[0035] The synthetic steps of compound 3: get compound 1 (1mmol), HOSu (1mmol) in a 50 milliliter flask, add 20 milliliters of dry DMF, add DCC (1mmol), stir at room temperature for 20 hours, add Boc-lysine (2mmol ), DIEA (1mmol), stirred at room temperature for 24 hours, the reaction was completed, the solvent was spun off, and the column was passed to obtain the product. Elemental analysis: C, 49.42%; H, 3.82%; N, 7.04% (found value); C, 49.39%; H, 3.8 / 4%; N, 7.02% (C 41 h 38 f 4 IrN 5 o 8 theoretical value).

[0036] The synthesis steps of compound 4: take compound 2 (1mmol), Boc-lysine methyl ester (2mmol), DMAP (1mmol) in a 100 ml flask, add 20 ml of toluene, add DCC (1mmol), stir at room temperature for 2 hours After the reaction was completed, the solvent was spun off, 60 milliliters of dichloromethane was added, trifluoroacetic acid (6 mmol) was added, and stirred at room tem...

Embodiment 2

[0038] Example 2: Synthesis of Encoding Molecules with Dendritic Main Backbone

[0039]

[0040] Compound 10

[0041] The synthetic steps of compound 9: get compound 6 (1mmol), DMAP (1mmol) in a 100 milliliter flask, add 20 milliliters of DMF, get compound 8 (1mmol) and make DMF solution, under magnetic stirring, the solution that is made is dripped Add it to the reaction bottle, after the dropwise addition is completed, stir at room temperature for 24 hours. After the reaction is complete, spin off the solvent and pass through the column to obtain the product. Elemental analysis: C, 63.86%; H, 4.95%; N, 6.05% (found value); C, 63.84%; H, 4.92%; N, 6.08% (C 49 h 45 EuN 4 o 5 theoretical value).

[0042] The synthetic steps of compound 10: take compound 7 (1mmol) in a 100 milliliter flask, add 20 milliliters of DMA, take compound 9 (1mmol) and add 10 milliliters of DMA to form a solution, and add the solution dropwise to the reaction flask under stirring , the dropwise a...

Embodiment 3

[0043] Example 3 Compound 5 labels BSA (bovine serum albumin) under the effects of EDC and NHS

[0044] NHS (1.05 mmol) and EDC (1 mmol) were added to compound 5 (1 mmol) in PBS buffer solution (pH=7.4), reacted at room temperature for 30 minutes, then added BSA, and stirred overnight at room temperature. The resulting mixture was purified by dialysis in PBS solution with a molecular weight cut-off of 10000 Da for three days to remove uncrosslinked compounds, that is, to obtain BSA crosslinked with the labeled compound.

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