CN113735916B - Method for converting terminal alkyne into amide and application of method in construction of gene coding library - Google Patents

Method for converting terminal alkyne into amide and application of method in construction of gene coding library Download PDF

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CN113735916B
CN113735916B CN202111094475.8A CN202111094475A CN113735916B CN 113735916 B CN113735916 B CN 113735916B CN 202111094475 A CN202111094475 A CN 202111094475A CN 113735916 B CN113735916 B CN 113735916B
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terminal alkyne
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mmol
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oligonucleotide
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CN113735916A (en
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胡允金
杨珂新
杨少光
孙兆美
曹红丽
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Kanglong Beijing New Drug Technology Ltd By Share Ltd
Kanglong Huacheng Ningbo Technology Development Co ltd
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Kanglong Beijing New Drug Technology Ltd By Share Ltd
Kanglong Huacheng Ningbo Technology Development Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/08—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention provides a method for converting terminal alkyne into amide on oligonucleotide, which has good universality, milder conditions, convenient operation and high yield, and is suitable for synthesizing gene coding compound libraries in porous plates. The invention increases the reaction types which can be successfully applied in the gene coding library technology, and particularly provides a method for directly carrying out amide coupling reaction on a primary or secondary amine micromolecule compound with free carboxylic acid and unprotected. Thus, the synthesis steps of the gene coding compound library are simplified, and the synthesis efficiency of the gene coding compound library and the quality of the oligonucleotide product are improved.

Description

Method for converting terminal alkyne into amide and application of method in construction of gene coding library
Technical Field
The invention belongs to the field of biochemistry, and particularly relates to a method for converting terminal alkyne into amide and application thereof in construction of a gene coding compound library.
Background
The gene coding chemical library (DEL) was originally proposed by Brenner and Lerner in 1992 [1] . In DEL, each compound is bound to a unique gene tag whose oligonucleotide gene sequence represents its chemical structure. All library compounds were mixed together and simultaneously bioscreened against the target protein. The conjugate after binding to the target protein may be amplified using Polymerase Chain Reaction (PCR) to decode its corresponding chemical structure. DEL can contain billions or even trillions of libraries of compounds, and biological screening can be accomplished in a matter of hours [2-4] 。
Today, the gene-encoded compound library technology (DELT) has become a powerful active compound discovery technology in biomedical research [5] An increasing number of pharmaceutical companies have employed DELT to discover small organic molecules of biological or pharmaceutical interest [6] . The discovery of these small molecule compounds capable of binding to target proteins has driven the progress of new drug development. In recent years, the method of screening for living cells DEL has emerged in this field as an attractive achievement [7] It represents a compound that can be screened for cellular biological activity by the DELT platform. DEL live cell screening would eliminate the need for purified target proteins, nor modification of the proteins. This not only simplifies the biological screening process, but also better maintains the original ecological structure of the protein [8] From the slaveAnd the pharmacist can find a better lead compound on this platform.
In most cases, DEL synthesis involves an amide coupling reaction [10] . This is often done between the free amino groups of the linked oligonucleotides and the carboxylic acid small molecule substrate. Dario Neri and its co-researchers found that amide coupling reactions of high yields and better versatility could be obtained by using a combination of coupling reagents of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, 1-hydroxy-7-azabenzotriazole, and N, N' -diisopropylethylamine (EDCI/HOAt/DIPEA) [9] . These reaction conditions are applicable to a variety of primary and secondary amines and a variety of types of free amino-containing oligonucleotide substrates, and to the synthesis of DEL.
The amide bond can be established by amide coupling of inexpensive and diverse carboxylic acids with various organic amine compounds. However, most amide reactions are achieved by oligomeric nucleic acid-free amine substrates with small molecule carboxylic acid reactants. However, the amide coupling reaction of oligomeric nucleic acid-free carboxylic acid substrates with small molecule amines is still very inefficient at present [11] . Michael J. Waring recently reported a method for amide coupling using surfactant micelles [12] . Their method must use long fatty alkanes as the linkers between the oligonucleotides and the small molecule library. That very long fatty alkane linker can help form micelles using surfactants, thus allowing the reaction on the oligonucleotide to proceed smoothly. However, that very long fatty alkane linker may interfere with subsequent bioscreening experiments.
Thus, the present invention reports a novel method for synthesizing amide coupling by reacting terminal alkynes with free ammonia-based small molecules for successful application on oligonucleotides. It is suitable for establishing a coupling product from a carboxylic acid to an amide. Moreover, unlike conventional amide coupling reactions, small molecule substrates with both free amino groups and carboxylic acids can directly participate in chemical reactions without protecting groups to form amides. Each step of synthetic chemical reaction performed on the oligonucleotide can cause more or less damage to the nucleotide sequence, and the synthesis efficiency and quality of the gene coding library can be improved by using as few chemical reactions as possible and as mild reaction conditions as possible. Therefore, the invention has higher synthesis efficiency and better quality of the oligonucleotide than the traditional method.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for converting terminal alkyne into amide and application of the method in construction of gene coding compound libraries. Specifically, an oligonucleotide terminal alkyne compound is used as a substrate, and reacts with a small molecular compound containing free amino groups in the presence of a copper catalyst and a nitrone reagent to convert the terminal alkyne into amide. And based on the reaction, a gene coding library is established.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a method for converting terminal alkyne into amide in construction of a gene coding compound library, which specifically takes an oligomeric nucleic acid terminal alkyne compound as a substrate, and reacts with a small molecule compound containing free amino groups in the presence of a copper catalyst and a nitrone reagent to form the amide compound. The specific reaction equation is as follows:
preferably, the terminal alkyne compound of the oligonucleotide is taken as a substrate, and reacts with the small molecular compound containing free amino groups at the temperature of 0-90 ℃ for 1-24 hours in the presence of a copper catalyst and a nitrone reagent to form the amide compound.
Wherein the structural formula of the oligonucleic acid terminal alkyne compound isIs formed by connecting chemical groups with terminal alkynyl by oligonucleotide; the structural formula of the prepared amide compound is +.>Is composed of oligonucleotide connectorThe chemical groups having amide bonds, R being hydrogen, halogen, amino, nitro, cyano, hydroxy, mercapto, aryl ketone, alkyl ketone, C 1 -C 12 Alkyl, C 2 -C 6 Alkylene, C 2 -C 6 Alkynyl, C 3 -C 8 Cycloalkyl, C 1 -C 6 Alkyl oxygen, C 4 -C 12 Aryl, C 4 -C 12 Any one to more of the heterocyclic aryl groups or any combination thereof;
wherein the oligonucleotide is a single-stranded or double-stranded oligonucleotide strand obtained by polymerization of an artificially modified and/or unmodified oligonucleotide monomer;
wherein the structural formula of the small molecular compound containing free amino is R 1 -NH-R 2 Can be primary or secondary amine compounds, including aromatic compounds, aliphatic compounds, carbocycle compounds, heteroatom-containing ring compounds, amino acid compounds, and free amino compounds with other protecting groups, R 1 、R 2 Is carboxylic acid, hydrogen, amino, nitro, cyano, hydroxyl, mercapto, aryl ketone, alkyl ketone, C 1 -C 12 Alkyl, C 2 -C 6 Alkylene, C 2 -C 6 Alkynyl, C 3 -C 8 Cycloalkyl, C 1 -C 6 Alkyl oxygen, C 4 -C 12 Aryl, C 4 -C 12 Any one to more of the heteroaryl groups or any combination thereof;
wherein the copper catalyst is copper acetate, copper sulfate, copper chloride, copper nitrate, copper carbonate, copper iodide, copper-beta-cyclodextrin complex, copper bis (2, 4-pentanedionate), copper acetylacetonate, copper tetra (acetonitrile) tetrafluoroborate, copper dichloro (1, 10-phenanthroline), copper bis (8-hydroxyquinoline), copper trifluoromethane sulfonate, copper bis (trifluoro-2, 4-pentanedione), copper perchlorate, copper tetra (acetonitrile) hexafluorophosphate, copper acetate, copper bromide, copper fluoride, copper bromide, copper chloride-bis (lithium chloride) complex, copper dimethyl sulfide bromide; preferably, the copper catalyst is cuprous iodide;
wherein the nitrone knotIs constructed asR in the structural formula 3 、R 4 、R 5 Is hydrogen, halogen, amino, nitro, cyano, hydroxy, mercapto, aryl ketone, alkyl ketone, C 1 -C 12 Alkyl, C 2 -C 6 Alkylene, C 2 -C 6 Alkynyl, C 3 -C 8 Cycloalkyl, C 1 -C 6 Alkyl oxygen, C 4 -C 12 Aromatic group, C 4 -C 12 Any one to more of heterocyclic aromatic groups or any combination thereof, wherein R 5 Not hydrogen; preferably, the nitrone structure is +.>
Wherein the alkali is one or a mixture of several of cesium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, lithium carbonate, lithium hydroxide, potassium hydroxide, sodium hydroxide, cesium hydroxide, sodium borate, potassium dihydrogen phosphate, sodium acetate, sodium fluoride, potassium fluoride, cesium fluoride, trimethylamine, triethylamine, isobutylamine, isopropylamine, 4-dimethylaminopyridine, N, N-diisopropylethylamine, 1, 8-diazabicyclo [5.4.0] undec-7-ene, N, N, N ', N' -tetramethyl ethylenediamine, tetramethyl guanidine, pyridine, N-methyl dicyclohexylamine or dicyclohexylamine; preferably, the base is sodium borate;
wherein the reaction solvent is water, methanol, ethanol, propanol, isopropanol, N-butanol, isobutanol, tert-butanol, pentanol, cyclohexanol, 2-fluoroethanol, 2-difluoroethanol, 2-trifluoroethanol, hexafluoroisopropanol, benzyl alcohol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, glycerol, diethyl ether, propylene oxide, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydropyran, 1, 4-dioxane, anisole, dimethyl sulfide, diethyl sulfide, ethylene glycol dimethyl ether, any one or a mixture of solvents of ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N-dimethylformamide, N-dimethylacetamide, dimethylsulfoxide, acetonitrile, acetone, cyclohexanone, methylene chloride, chloroform, chlorobenzene, 1, 2-dichloroethane, ethyl acetate, N-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methoxypyridine, toluene, xylene, an inorganic salt buffer, and an organic base buffer; preferably, the reaction solvent is a mixed solvent of an inorganic salt buffer solution and dimethyl sulfoxide; more preferably, the reaction solvent is a mixed solvent of sodium borate buffer solution having ph=9.5 and dimethyl sulfoxide.
Preferably, 5 nanomolar oligonucleotide terminal alkyne compounds are dissolved in 5 microliters of sodium borate buffer (ph=9.5, 250 millimoles/liter, 1 equivalent), and 20 microliters of dimethyl sulfoxide, 20 equivalents of cuprous iodide (20 millimoles/liter), 200 equivalents of small molecule compounds containing free amino groups (200 millimoles/liter), and 100 equivalents of nitrone (200 millimoles/liter) are added and reacted with shaking at 50 ℃ for 3 hours.
In one embodiment, the molar concentration of the oligomeric nucleic acid terminal alkyne compound is 0.1 to 2 millimoles per liter; preferably, the molar concentration of the oligonucleic acid terminal alkyne compound is 0.5 to 1.5 mmoles/liter; more preferably, the molar concentration of the oligonucleic acid terminal alkyne compound is 1.0 mmoles/liter.
In one embodiment, the equivalent weight of the copper catalyst is from 1 to 100 equivalents; preferably, the equivalent of the copper catalyst is 5 to 35 equivalents; more preferably, the equivalent of the copper catalyst is 20 equivalents.
In one embodiment, the equivalent weight of the nitrone agent is from 5 to 500 equivalents; preferably, the equivalent of nitrone reagent is 50 to 150 equivalents; more preferably, the equivalent of nitrone reagent is 100 equivalents.
In one embodiment, the free amino group-containing small molecule compound has an equivalent weight of 5 to 500; preferably, the equivalent weight of the small molecular compound containing free amino groups is 100-300; more preferably, the equivalent of the small molecular compound containing a free amino group is 200 equivalents.
In one embodiment, the temperature of the reaction is from 0 to 90 ℃; preferably, the temperature of the reaction is 30-70 ℃; more preferably, the temperature of the reaction is 50 ℃.
In one embodiment, the reaction time is from 0 to 24 hours; preferably, the reaction time is 2 to 5 hours; more preferably, the reaction time is 3 hours.
The invention provides a method for converting terminal alkyne into amide in construction of a gene coding compound library, and provides a novel method for forming the gene coding compound library amide. The method provided by the invention has the advantages of good universality, convenient operation and high yield, and is suitable for synthesizing the gene coding compound library by a porous plate. The method has the advantages that reagents different from conventional carboxylic acids can be used for carrying out the amide coupling reaction, so that the source diversity of reaction substrates is increased, and particularly, the method for directly carrying out the amide coupling on the small-molecule compound simultaneously carrying the free carboxylic acid and the unprotected primary or secondary amine is provided; the application range of the reaction substrate is enlarged; the number of reaction steps in the synthesis of the gene-encoded compound library is reduced; improves the synthesis efficiency and quality of the gene coding compound library. The method provided by the invention is applicable to conventional aliphatic and aromatic alkynes, and the types of substrates applicable to the reaction are listed in fig. 11. The amine substrates of the present invention are suitable for use with free primary amines (as set forth in FIG. 21), or secondary amines as set forth in FIG. 21), and also with substrates of both free amines and carboxylic acids (as set forth in FIG. 21).
Drawings
FIG. 1 shows the results of liquid chromatography mass spectrometry detection of terminal alkyne compounds 1-2 of the oligonucleotide of the invention.
FIG. 2 shows the results of liquid chromatography mass spectrometry detection of terminal alkyne compounds 5-5 of the oligonucleotide of the invention.
FIG. 3 shows the results of liquid chromatography mass spectrometry detection of amide formation with 1,3, 5-trimethyl-pyrazole-4-methylamine in the presence of copper catalyst and nitrone reagent using oligo-nucleic acid terminal alkyne compounds 1-2 as substrates according to the invention.
FIG. 4 shows the results of liquid chromatography mass spectrometry detection of amide formation with 1,3, 5-trimethyl-pyrazole-4-methylamine in the presence of copper catalyst and nitrone reagent using oligo-nucleic acid terminal alkyne compounds 1-3 as substrates according to the invention.
FIG. 5 shows the results of liquid chromatography mass spectrometry detection of amide formation with 1,3, 5-trimethyl-pyrazole-4-methylamine in the presence of copper catalyst and nitrone reagent using oligo-nucleic acid terminal alkyne compounds 1-6 as substrates according to the invention.
FIG. 6 shows the results of liquid chromatography mass spectrometry detection of amide formation with 1,3, 5-trimethyl-pyrazole-4-methylamine in the presence of copper catalyst and nitrone reagent using oligo-nucleic acid terminal alkyne compound 2-2 as substrate in the present invention.
FIG. 7 shows the results of liquid chromatography mass spectrometry detection of amide formation with 1,3, 5-trimethyl-pyrazole-4-methanamine in the presence of copper catalyst and nitrone reagent using oligo-nucleic acid terminal alkyne compound 3-3 as substrate in the present invention.
FIG. 8 shows the results of liquid chromatography mass spectrometry detection of amide formation with 1,3, 5-trimethyl-pyrazole-4-methanamine in the presence of copper catalyst and nitrone reagent using oligo-nucleic acid terminal alkyne compounds 5-4 as substrates according to the present invention.
FIG. 9 shows the results of liquid chromatography mass spectrometry detection of amide formation with 1,3, 5-trimethyl-pyrazole-4-methanamine in the presence of copper catalyst and nitrone reagent using oligo-nucleic acid terminal alkyne compounds 5-5 as substrates according to the present invention.
FIG. 10 shows the results of liquid chromatography mass spectrometry detection of amide formation with 1,3, 5-trimethyl-pyrazole-4-methanamine in the presence of copper catalyst and nitrone reagent using oligo-nucleic acid terminal alkyne compounds 5-6 as substrates according to the present invention.
FIG. 11 is a schematic representation of the terminal alkyne compound moiety of an oligonucleotide of the invention.
FIG. 12 shows the results of liquid chromatography mass spectrometry detection of amide formation with (S) -3-aminotetrahydrofuran hydrochloride in the presence of copper catalyst and nitrone reagent using oligo-nucleic acid terminal alkyne compounds 1-2 as substrates according to the present invention.
FIG. 13 is a liquid chromatography mass spectrometry detection result of amide formation with decahydroisoquinoline in the presence of copper catalyst and nitrone reagent using the terminal alkyne compound 1-2 of the oligonucleotide as a substrate in the present invention.
FIG. 14 shows the results of liquid chromatography mass spectrometry detection of amide formation with 2-amino-4-pyridinemethylamine in the presence of copper catalyst and nitrone reagent using oligo-nucleic acid terminal alkyne compounds 1-2 as substrates according to the invention.
FIG. 15 shows the results of liquid chromatography mass spectrometry detection of amide formation with trans-4- (aminomethyl) cyclohexanecarboxylic acid in the presence of copper catalyst and nitrone reagent using oligo-nucleic acid terminal alkyne compounds 1-2 as substrates according to the present invention.
FIG. 16 is a liquid chromatography mass spectrometry detection result of amide formation with 3-aminomethylbenzoic acid hydrochloride in the presence of copper catalyst and nitrone reagent using oligo-nucleic acid terminal alkyne compound 1-2 as substrate according to the present invention.
FIG. 17 shows the results of liquid chromatography mass spectrometry detection of amide formation with 1- (2-aminophenyl) ethylamine salt in the presence of copper catalyst and nitrone reagent using the terminal alkyne compound 1-2 of the oligonucleotide of the present invention as a substrate.
FIG. 18 shows the results of liquid chromatography mass spectrometry detection of amide formation with dicyclohexylamine in the presence of copper catalyst and nitrone reagent using oligo-nucleic acid terminal alkyne compounds 1-2 as substrates according to the present invention.
FIG. 19 shows the results of liquid chromatography mass spectrometry detection of amide formation with 3-aminocyclohexanecarboxylic acid in the presence of copper catalyst and nitrone reagent using oligomeric nucleic acid terminal alkyne compounds 1-2 as substrates according to the invention.
FIG. 20 shows the results of liquid chromatography mass spectrometry detection of amide formation with 4-hydroxybenzylamine in the presence of copper catalyst and nitrone reagent using oligo-nucleic acid terminal alkyne compounds 1-2 as substrates according to the present invention.
FIG. 21 is a schematic representation of the reaction of an oligomeric nucleic acid terminal alkyne compound containing free amino small molecule compounds according to the invention.
Detailed Description
The technical scheme of the invention will be clearly and completely described below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. All the oligonucleotide materials in the embodiments of the invention are substrates of double-stranded or single-stranded oligonucleotides. The synthesis of the substrate for the terminal alkyne compound of the oligonucleotide is quite similar. The general synthetic method can be accomplished with reference to example 1 or example 2.
Example 1 Synthesis of polynucleic acid terminal alkyne Compounds 1-2
10 nanomolar oligonucleotide was dissolved in 10 microliters of sodium borate buffer (ph=9.5, 250 mmol/liter, 1 eq), 80 eq of 5-hexynoic acid (80 mmol/liter) was added to the oligonucleotide solution at room temperature, and 50 eq of 2- (7-azobenzotriazole) -N, N' -tetramethylurea hexafluorophosphate (50 mmol/liter) and 200 eq of N, N-diisopropylethylamine (200 mmol/liter) were added to react for 1 hour at room temperature. After the reaction, 4. Mu.l of 5 mol/l sodium chloride aqueous solution, 100. Mu.l of absolute ethyl alcohol was added to the reaction mixture, followed by shaking and mixing. After being frozen in a refrigerator at-80 ℃ for 10-30 minutes, the mixture is subjected to high-speed freezing and centrifugal separation (at 4 ℃ for 12000 revolutions per minute for 5 minutes) to obtain the terminal alkyne compound 1-2 of the oligonucleotide. The molecular weight of the product was measured by liquid chromatography mass spectrometry and the yield was confirmed, and the results are shown in FIG. 1. The molecular weight of the product was 16284.6 and the yield of the product was 95.0%.
The specific reaction equation is as follows:
example 2 Synthesis of oligo-nucleic acid terminal alkyne Compound 5-5
10 nanomolar oligonucleotide was dissolved in 10 microliters of sodium borate buffer (ph=9.5, 250 mmol/liter, 1 eq), 30 eq of 4-alkynyl-2-fluoroaniline (30 mmol/liter) was added to the oligonucleotide solution at room temperature, and 20 eq of 2- (7-azobenzotriazole) -N, N' -tetramethylurea hexafluorophosphate (20 mmol/liter) and 20 eq of N, N-diisopropylethylamine (20 mmol/liter) were added to react for 1 hour at room temperature. After the reaction, 4. Mu.l of 5 mol/l sodium chloride aqueous solution, 100. Mu.l of absolute ethyl alcohol was added to the reaction mixture, followed by shaking and mixing. After being frozen in a refrigerator at the temperature of minus 80 ℃ for 10 to 30 minutes, the mixture is subjected to high-speed freezing and centrifugal separation (at the temperature of 4 ℃ for 12000 revolutions per minute for 5 minutes) to obtain the terminal alkyne compound 5 to 5 of the substrate oligonucleotide. The molecular weight of the product was measured by liquid chromatography mass spectrometry and the yield was confirmed, and the results are shown in FIG. 2. The molecular weight of the product was 16347.9 and the yield of the product was 93.0%.
The specific reaction equation is as follows:
EXAMPLE 3 Synthesis of nitrone
0.08 mmol of aromatic nitro compound was dissolved in 700. Mu.l of acetonitrile, 0.16 mmol of aromatic aldehyde compound, 0.096 mmol of ammonium chloride, 0.08 mmol of zinc powder and 100. Mu.l of water were added, and the reaction was stirred under nitrogen at 40℃for 12 hours. After the completion of the reaction, the supernatant was centrifuged and used directly for the next reaction.
The nitrones used in the examples below are all nitrones prepared in example 3.
Example 4 conversion of an oligomeric nucleic acid terminal alkyne compound 1-2 (preparation method as in example 1) with 1,3, 5-trimethyl-pyrazole-4-methanamine to an amide
5 nanomolar oligonucleotide terminal alkyne compounds 1-2 were dissolved in 5 μl sodium borate buffer (ph=9.5, 250 mmol/liter, 1 eq) and 20 μl dimethyl sulfoxide, 20 eq cuprous iodide (20 mmol/liter), 200 eq 1,3, 5-trimethyl-pyrazole-4-methylamine (200 mmol/liter) and 100 eq nitrone (200 mmol/liter) were added and reacted at 50 ℃ for 3 hours. After the completion of the reaction, 50 equivalents of an aqueous solution of sodium diethyldithiocarbamate (100 mmol/L) was added to the reaction mixture, and the mixture was stirred and mixed uniformly, and reacted at 50℃for 10 minutes. High-speed freezing centrifugal separation (4 ℃,12000 rpm, 5 minutes), adding 3.5 microliter of 5 mol/liter sodium chloride aqueous solution into the upper layer solution, 87.5 microliter of absolute ethyl alcohol, shaking and mixing uniformly, and placing into a refrigerator at the temperature of minus 80 ℃ for freezing for 10 to 30 minutes. High-speed refrigerated centrifugation (4 ℃,12000 rpm, 5 min) gave a product, which was checked for molecular weight and yield by liquid chromatography mass spectrometry, and the results are shown in fig. 3. The molecular weight of the product was 16436.9 and the yield of the product was 76.2%.
Example 5 conversion of an oligomeric nucleic acid terminal alkyne compound 1-3 (preparation method as in example 1) with 1,3, 5-trimethyl-pyrazole-4-methanamine to an amide
5 nanomolar oligonucleotide terminal alkyne compounds 1-3 were dissolved in 5 μl sodium borate buffer (ph=9.5, 250 mmol/liter, 1 eq) and 20 μl dimethyl sulfoxide, 20 eq cuprous iodide (20 mmol/liter), 200 eq 1,3, 5-trimethyl-pyrazole-4-methylamine (200 mmol/liter) and 100 eq nitrone (200 mmol/liter) were added and reacted at 50 ℃ for 3 hours. After the completion of the reaction, 50 equivalents of an aqueous solution of sodium diethyldithiocarbamate (100 mmol/L) was added to the reaction mixture, and the mixture was stirred and mixed uniformly, and reacted at 50℃for 10 minutes. High-speed freezing centrifugal separation (4 ℃,12000 rpm, 5 minutes), adding 3.5 microliter of 5 mol/liter sodium chloride aqueous solution into the upper layer solution, 87.5 microliter of absolute ethyl alcohol, shaking and mixing uniformly, and placing into a refrigerator at the temperature of minus 80 ℃ for freezing for 10 to 30 minutes. The product was obtained by high-speed refrigerated centrifugation (4 ℃,12000 rpm, 5 minutes), and the molecular weight of the product was measured by liquid chromatography mass spectrometry to confirm the yield, and the result was shown in FIG. 4. The molecular weight of the product was 16422.6 and the yield of the product was 76.6%.
Example 6 conversion of an oligomeric nucleic acid terminal alkyne compound 1-6 (preparation method as in example 1) with 1,3, 5-trimethyl-pyrazole-4-methanamine to an amide
5 nanomolar oligonucleotide terminal alkyne compounds 1-6 were dissolved in 5 μl sodium borate buffer (ph=9.5, 250 mmol/liter, 1 eq) and 20 μl dimethyl sulfoxide, 20 eq cuprous iodide (20 mmol/liter), 200 eq 1,3, 5-trimethyl-pyrazole-4-methylamine (200 mmol/liter) and 100 eq nitrone (200 mmol/liter) were added and reacted at 50 ℃ for 3 hours. After the completion of the reaction, 50 equivalents of an aqueous solution of sodium diethyldithiocarbamate (100 mmol/L) was added to the reaction mixture, and the mixture was stirred and mixed uniformly, and reacted at 50℃for 10 minutes. High-speed freezing centrifugal separation (4 ℃,12000 rpm, 5 minutes), adding 3.5 microliter of 5 mol/liter sodium chloride aqueous solution into the upper layer solution, 87.5 microliter of absolute ethyl alcohol, shaking and mixing uniformly, and placing into a refrigerator at the temperature of minus 80 ℃ for freezing for 10 to 30 minutes. The product was obtained by high-speed refrigerated centrifugation (4 ℃,12000 rpm, 5 minutes), and the molecular weight of the product was measured by liquid chromatography mass spectrometry to confirm the yield, and the result was shown in FIG. 5. The molecular weight of the product was 16487.0 and the yield of the product was 58.2%.
EXAMPLE 7 conversion of an oligomeric nucleic acid terminal alkyne compound 2-2 (prepared in the same manner as in example 1) with 1,3, 5-trimethyl-pyrazole-4-methanamine to an amide
5 nanomolar oligonucleotide terminal alkyne compound 2-2 was dissolved in 5 μl sodium borate buffer (ph=9.5, 250 mmol/liter, 1 eq) and 20 μl dimethyl sulfoxide, 20 eq cuprous iodide (20 mmol/liter), 200 eq 1,3, 5-trimethyl-pyrazole-4-methylamine (200 mmol/liter) and 100 eq nitrone (200 mmol/liter) were added and reacted at 50 ℃ for 3 hours. After the completion of the reaction, 50 equivalents of an aqueous solution of sodium diethyldithiocarbamate (100 mmol/L) was added to the reaction mixture, and the mixture was stirred and mixed uniformly, and reacted at 50℃for 10 minutes. High-speed freezing centrifugal separation (4 ℃,12000 rpm, 5 minutes), adding 3.5 microliter of 5 mol/liter sodium chloride aqueous solution into the upper layer solution, 87.5 microliter of absolute ethyl alcohol, shaking and mixing uniformly, and placing into a refrigerator at the temperature of minus 80 ℃ for freezing for 10 to 30 minutes. The product was obtained by high-speed refrigerated centrifugation (4 ℃,12000 rpm, 5 minutes), and the molecular weight of the product was measured by liquid chromatography mass spectrometry to confirm the yield, and the result was shown in FIG. 6. The molecular weight of the product was 16485.3 and the yield of the product was 60.9%.
Example 8 conversion of an oligomeric nucleic acid terminal alkyne compound 3-3 (prepared in the same manner as in example 2) with 1,3, 5-trimethyl-pyrazole-4-methanamine to an amide
5 nanomolar oligonucleotide terminal alkyne compound 3-3 was dissolved in 5 μl sodium borate buffer (ph=9.5, 250 mmol/liter, 1 eq) and 20 μl dimethyl sulfoxide, 20 eq cuprous iodide (20 mmol/liter), 200 eq 1,3, 5-trimethyl-pyrazole-4-methylamine (200 mmol/liter) and 100 eq nitrone (200 mmol/liter) were added and reacted at 50 ℃ for 3 hours. After the completion of the reaction, 50 equivalents of an aqueous solution of sodium diethyldithiocarbamate (100 mmol/L) was added to the reaction mixture, and the mixture was stirred and mixed uniformly, and reacted at 50℃for 10 minutes. High-speed freezing centrifugal separation (4 ℃,12000 rpm, 5 minutes), adding 3.5 microliter of 5 mol/liter sodium chloride aqueous solution into the upper layer solution, 87.5 microliter of absolute ethyl alcohol, shaking and mixing uniformly, and placing into a refrigerator at the temperature of minus 80 ℃ for freezing for 10 to 30 minutes. The product was obtained by high-speed refrigerated centrifugation (4 ℃,12000 rpm, 5 minutes), and the molecular weight of the product was measured by liquid chromatography mass spectrometry to confirm the yield, and the result was shown in FIG. 7. The molecular weight of the product was 16434.9 and the yield of the product was 62.1%.
Example 9 conversion of an oligomeric nucleic acid terminal alkyne compound 5-4 (preparation method as in example 2) with 1,3, 5-trimethyl-pyrazole-4-methanamine to an amide
5 nanomolar oligonucleotide terminal alkyne compounds 5-4 were dissolved in 5 μl sodium borate buffer (ph=9.5, 250 mmol/liter, 1 eq) and 20 μl dimethyl sulfoxide, 20 eq cuprous iodide (20 mmol/liter), 200 eq 1,3, 5-trimethyl-pyrazole-4-methylamine (200 mmol/liter) and 100 eq nitrone (200 mmol/liter) were added and reacted at 50 ℃ for 3 hours. After the completion of the reaction, 50 equivalents of an aqueous solution of sodium diethyldithiocarbamate (100 mmol/L) was added to the reaction mixture, and the mixture was stirred and mixed uniformly, and reacted at 50℃for 10 minutes. High-speed freezing centrifugal separation (4 ℃,12000 rpm, 5 minutes), adding 3.5 microliter of 5 mol/liter sodium chloride aqueous solution into the upper layer solution, 87.5 microliter of absolute ethyl alcohol, shaking and mixing uniformly, and placing into a refrigerator at the temperature of minus 80 ℃ for freezing for 10 to 30 minutes. The product was obtained by high-speed refrigerated centrifugation (4 ℃,12000 rpm, 5 minutes), and the molecular weight of the product was measured by liquid chromatography mass spectrometry to confirm the yield, and the result was shown in FIG. 8. The molecular weight of the product was 16500.5 and the yield of the product was 58.7%.
Example 10 conversion of an oligo-nucleic acid terminal alkyne compound 5-5 (preparation method as in example 2) to an amide by reaction with 1,3, 5-trimethyl-pyrazole-4-methanamine
5 nanomolar oligonucleotide terminal alkyne compounds 5-5 were dissolved in 5 μl sodium borate buffer (ph=9.5, 250 mmol/liter, 1 eq) and 20 μl dimethyl sulfoxide, 20 eq cuprous iodide (20 mmol/liter), 200 eq 1,3, 5-trimethyl-pyrazole-4-methylamine (200 mmol/liter) and 100 eq nitrone (200 mmol/liter) were added and reacted at 50 ℃ for 3 hours. After the completion of the reaction, 50 equivalents of an aqueous solution of sodium diethyldithiocarbamate (100 mmol/L) was added to the reaction mixture, and the mixture was stirred and mixed uniformly, and reacted at 50℃for 10 minutes. High-speed freezing centrifugal separation (4 ℃,12000 rpm, 5 minutes), adding 3.5 microliter of 5 mol/liter sodium chloride aqueous solution into the upper layer solution, 87.5 microliter of absolute ethyl alcohol, shaking and mixing uniformly, and placing into a refrigerator at the temperature of minus 80 ℃ for freezing for 10 to 30 minutes. The product was obtained by high-speed refrigerated centrifugation (4 ℃,12000 rpm, 5 minutes), and the molecular weight of the product was measured by liquid chromatography mass spectrometry to confirm the yield, and the result was shown in FIG. 9. The molecular weight of the product was 16504.2 and the yield of the product was 58.9%.
EXAMPLE 11 conversion of an oligomeric nucleic acid terminal alkyne compound 5-6 (preparation method same as in example 2) with 1,3, 5-trimethyl-pyrazole-4-methanamine to an amide
5 nanomolar oligonucleotide terminal alkyne compounds 5-6 were dissolved in 5 μl sodium borate buffer (ph=9.5, 250 mmol/liter, 1 eq) and 20 μl dimethyl sulfoxide, 20 eq cuprous iodide (20 mmol/liter), 200 eq 1,3, 5-trimethyl-pyrazole-4-methylamine (200 mmol/liter) and 100 eq nitrone (200 mmol/liter) were added and reacted at 50 ℃ for 3 hours. After the completion of the reaction, 50 equivalents of an aqueous solution of sodium diethyldithiocarbamate (100 mmol/L) was added to the reaction mixture, and the mixture was stirred and mixed uniformly, and reacted at 50℃for 10 minutes. High-speed freezing centrifugal separation (4 ℃,12000 rpm, 5 minutes), adding 3.5 microliter of 5 mol/liter sodium chloride aqueous solution into the upper layer solution, 87.5 microliter of absolute ethyl alcohol, shaking and mixing uniformly, and placing into a refrigerator at the temperature of minus 80 ℃ for freezing for 10 to 30 minutes. The product was obtained by high-speed refrigerated centrifugation (4 ℃,12000 rpm, 5 minutes), and the molecular weight of the product was measured by liquid chromatography mass spectrometry to confirm the yield, and the result was shown in FIG. 10. The molecular weight of the product was 16488.1 and the yield of the product was 61.7%.
We have validated the conversion of terminal alkynes of different oligonucleotides to amides, with part of the alkyne compounds representing the structural formula shown in FIG. 11. Through a liquid chromatography mass spectrogram, we confirm that the terminal alkyne of the oligonucleotide is converted into the amide, and the method has the advantages of mild reaction conditions, high yield and good substrate universality of the reaction.
Example 12 conversion of an oligomeric nucleic acid terminal alkyne compound 1-2 with (S) -3-aminotetrahydrofuran hydrochloride to an amide
5 nanomolar oligonucleotide terminal alkyne compounds 1-2 were dissolved in 5 μl sodium borate buffer (ph=9.5, 250 mmol/liter, 1 eq.) and 20 μl dimethyl sulfoxide, 20 eq cuprous iodide (20 mmol/liter), 200 eq (S) -3-aminotetrahydrofuran hydrochloride (200 mmol/liter) and 100 eq nitrone (200 mmol/liter) were added and reacted at 50 ℃ for 3 hours. After the completion of the reaction, 50 equivalents of an aqueous solution of sodium diethyldithiocarbamate (100 mmol/L) was added to the reaction mixture, and the mixture was stirred and mixed uniformly, and reacted at 50℃for 10 minutes. High-speed freezing centrifugal separation (4 ℃,12000 rpm, 5 minutes), adding 3.5 microliter of 5 mol/liter sodium chloride aqueous solution into the upper layer solution, 87.5 microliter of absolute ethyl alcohol, shaking and mixing uniformly, and placing into a refrigerator at the temperature of minus 80 ℃ for freezing for 10 to 30 minutes. The product was obtained by high-speed refrigerated centrifugation (4 ℃,12000 rpm, 5 minutes), and the molecular weight of the product was measured by liquid chromatography mass spectrometry to confirm the yield, and the result was shown in FIG. 12. The molecular weight of the product was 16384.1 and the yield of the product was 72.5%.
Example 13 conversion of an oligomeric nucleic acid terminal alkyne compound 1-2 with decahydroisoquinoline to an amide
5 nanomolar oligonucleotide terminal alkyne compounds 1-2 were dissolved in 5 μl sodium borate buffer (ph=9.5, 250 mmol/liter, 1 eq) and reacted at 50 ℃ for 3 hours with the addition of 20 μl dimethyl sulfoxide, 20 eq cuprous iodide (20 mmol/liter), 200 eq decahydroisoquinoline (200 mmol/liter) and 100 eq nitrone (200 mmol/liter). After the completion of the reaction, 50 equivalents of an aqueous solution of sodium diethyldithiocarbamate (100 mmol/L) was added to the reaction mixture, and the mixture was stirred and mixed uniformly, and reacted at 50℃for 10 minutes. High-speed freezing centrifugal separation (4 ℃,12000 rpm, 5 minutes), adding 3.5 microliter of 5 mol/liter sodium chloride aqueous solution into the upper layer solution, 87.5 microliter of absolute ethyl alcohol, shaking and mixing uniformly, and placing into a refrigerator at the temperature of minus 80 ℃ for freezing for 10 to 30 minutes. The product was obtained by high-speed refrigerated centrifugation (4 ℃,12000 rpm, 5 minutes), and the molecular weight of the product was measured by liquid chromatography mass spectrometry to confirm the yield, and the result was shown in FIG. 13. The molecular weight of the product was 16438.4 and the yield of the product was 52.9%.
EXAMPLE 14 conversion of an oligomeric nucleic acid terminal alkyne compound 1-2 with 2-amino-4-pyridinemethylamine to an amide
5 nanomolar oligonucleotide terminal alkyne compounds 1-2 were dissolved in 5 μl sodium borate buffer (ph=9.5, 250 mmol/liter, 1 eq.) and reacted at 50 ℃ for 3 hours with the addition of 20 μl dimethyl sulfoxide, 20 eq cuprous iodide (20 mmol/liter), 200 eq 2-amino-4-pyridinemethylamine (200 mmol/liter) and 100 eq nitrone (200 mmol/liter). After the completion of the reaction, 50 equivalents of an aqueous solution of sodium diethyldithiocarbamate (100 mmol/L) was added to the reaction mixture, and the mixture was stirred and mixed uniformly, and reacted at 50℃for 10 minutes. High-speed freezing centrifugal separation (4 ℃,12000 rpm, 5 minutes), adding 3.5 microliter of 5 mol/liter sodium chloride aqueous solution into the upper layer solution, 87.5 microliter of absolute ethyl alcohol, shaking and mixing uniformly, and placing into a refrigerator at the temperature of minus 80 ℃ for freezing for 10 to 30 minutes. The product was obtained by high-speed refrigerated centrifugation (4 ℃,12000 rpm, 5 minutes), and the molecular weight of the product was measured by liquid chromatography mass spectrometry to confirm the yield, and the result was shown in FIG. 14. The molecular weight of the product was 16420.5 and the yield of the product was 79.5%.
Example 15 conversion of an oligomeric nucleic acid terminal alkyne compound 1-2 with trans-4- (aminomethyl) cyclohexyl carboxylic acid to an amide
5 nanomolar oligonucleotide terminal alkyne compounds 1-2 were dissolved in 5 μl sodium borate buffer (ph=9.5, 250 mmol/liter, 1 eq.) and 20 μl dimethyl sulfoxide, 20 eq. cuprous iodide (20 mmol/liter), 200 eq. trans-4- (aminomethyl) cyclohexyl carboxylic acid (200 mmol/liter) and 100 eq. nitrone (200 mmol/liter) were added and reacted at 50 ℃ for 3 hours. After the completion of the reaction, 50 equivalents of an aqueous solution of sodium diethyldithiocarbamate (100 mmol/L) was added to the reaction mixture, and the mixture was stirred and mixed uniformly, and reacted at 50℃for 10 minutes. High-speed freezing centrifugal separation (4 ℃,12000 rpm, 5 minutes), adding 3.5 microliter of 5 mol/liter sodium chloride aqueous solution into the upper layer solution, 87.5 microliter of absolute ethyl alcohol, shaking and mixing uniformly, and placing into a refrigerator at the temperature of minus 80 ℃ for freezing for 10 to 30 minutes. The product was obtained by high-speed refrigerated centrifugation (4 ℃,12000 rpm, 5 minutes), and the molecular weight of the product was measured by liquid chromatography mass spectrometry to confirm the yield, and the result was shown in FIG. 15. The molecular weight of the product was 16454.2 and the yield of the product was 72.3%.
EXAMPLE 16 conversion of oligomeric nucleic acid terminal alkyne compounds 1-2 with 3-aminomethylbenzoic acid hydrochloride to amides
5 nanomolar oligonucleotide terminal alkyne compounds 1-2 were dissolved in 5 μl sodium borate buffer (ph=9.5, 250 mmol/liter, 1 eq.) and reacted at 50 ℃ for 3 hours with the addition of 20 μl dimethyl sulfoxide, 20 eq. cuprous iodide (20 mmol/liter), 200 eq. 3-aminomethylbenzoate (200 mmol/liter) and 100 eq. nitrone (200 mmol/liter). After the completion of the reaction, 50 equivalents of an aqueous solution of sodium diethyldithiocarbamate (100 mmol/L) was added to the reaction mixture, and the mixture was stirred and mixed uniformly, and reacted at 50℃for 10 minutes. High-speed freezing centrifugal separation (4 ℃,12000 rpm, 5 minutes), adding 3.5 microliter of 5 mol/liter sodium chloride aqueous solution into the upper layer solution, 87.5 microliter of absolute ethyl alcohol, shaking and mixing uniformly, and placing into a refrigerator at the temperature of minus 80 ℃ for freezing for 10 to 30 minutes. The product was obtained by high-speed refrigerated centrifugation (4 ℃,12000 rpm, 5 minutes), and the molecular weight of the product was measured by liquid chromatography mass spectrometry to confirm the yield, and the result was shown in FIG. 16. The molecular weight of the product was 16449.2 and the yield of the product was 73.8%.
Example 17 conversion of an oligomeric nucleic acid terminal alkyne compound 1-2 with 1- (2-aminophenyl) ethylamine to an amide
5 nanomolar oligonucleotide terminal alkyne compounds 1-2 were dissolved in 5 μl sodium borate buffer (ph=9.5, 250 mmol/liter, 1 eq.) and reacted at 50 ℃ for 3 hours with the addition of 20 μl dimethyl sulfoxide, 20 eq cuprous iodide (20 mmol/liter), 200 eq 1- (2-aminophenyl) ethylamine (200 mmol/liter) and 100 eq nitrone (200 mmol/liter). After the completion of the reaction, 50 equivalents of an aqueous solution of sodium diethyldithiocarbamate (100 mmol/L) was added to the reaction mixture, and the mixture was stirred and mixed uniformly, and reacted at 50℃for 10 minutes. High-speed freezing centrifugal separation (4 ℃,12000 rpm, 5 minutes), adding 3.5 microliter of 5 mol/liter sodium chloride aqueous solution into the upper layer solution, 87.5 microliter of absolute ethyl alcohol, shaking and mixing uniformly, and placing into a refrigerator at the temperature of minus 80 ℃ for freezing for 10 to 30 minutes. The product was obtained by high-speed refrigerated centrifugation (4 ℃,12000 rpm, 5 minutes), and the molecular weight of the product was measured by liquid chromatography mass spectrometry to confirm the yield, and the result was shown in FIG. 17. The molecular weight of the product was 16433.1 and the yield of the product was 70.5%.
Example 18 conversion of an oligomeric nucleic acid terminal alkyne compound 1-2 with dicyclohexylamine to an amide
5 nanomolar oligonucleotide terminal alkyne compounds 1-2 were dissolved in 5 μl sodium borate buffer (ph=9.5, 250 mmol/liter, 1 eq.) and reacted at 50 ℃ for 3 hours with the addition of 20 μl dimethyl sulfoxide, 20 eq. cuprous iodide (20 mmol/liter), 200 eq. dicyclohexylamine (200 mmol/liter) and 100 eq. nitrone (200 mmol/liter). After the completion of the reaction, 50 equivalents of an aqueous solution of sodium diethyldithiocarbamate (100 mmol/L) was added to the reaction mixture, and the mixture was stirred and mixed uniformly, and reacted at 50℃for 10 minutes. High-speed freezing centrifugal separation (4 ℃,12000 rpm, 5 minutes), adding 3.5 microliter of 5 mol/liter sodium chloride aqueous solution into the upper layer solution, 87.5 microliter of absolute ethyl alcohol, shaking and mixing uniformly, and placing into a refrigerator at the temperature of minus 80 ℃ for freezing for 10 to 30 minutes. The product was obtained by high-speed refrigerated centrifugation (4 ℃,12000 rpm, 5 minutes), and the molecular weight of the product was measured by liquid chromatography mass spectrometry to confirm the yield, and the result was shown in FIG. 18. The molecular weight of the product was 16476.8 and the yield of the product was 51.8%.
Example 19 conversion of an oligomeric nucleic acid terminal alkyne compound 1-2 with 3-aminocyclohexanecarboxylic acid to an amide
5 nanomolar oligonucleotide terminal alkyne compounds 1-2 were dissolved in 5 μl sodium borate buffer (ph=9.5, 250 mmol/liter, 1 eq.) and reacted at 50 ℃ for 3 hours with the addition of 20 μl dimethyl sulfoxide, 20 eq. cuprous iodide (20 mmol/liter), 200 eq. 3-aminocyclohexanecarboxylic acid (200 mmol/liter) and 100 eq. nitrone (200 mmol/liter). After the completion of the reaction, 50 equivalents of an aqueous solution of sodium diethyldithiocarbamate (100 mmol/L) was added to the reaction mixture, and the mixture was stirred and mixed uniformly, and reacted at 50℃for 10 minutes. High-speed freezing centrifugal separation (4 ℃,12000 rpm, 5 minutes), adding 3.5 microliter of 5 mol/liter sodium chloride aqueous solution into the upper layer solution, 87.5 microliter of absolute ethyl alcohol, shaking and mixing uniformly, and placing into a refrigerator at the temperature of minus 80 ℃ for freezing for 10 to 30 minutes. The product was obtained by high-speed refrigerated centrifugation (4 ℃,12000 rpm, 5 minutes), and the molecular weight of the product was measured by liquid chromatography mass spectrometry to confirm the yield, and the result was shown in FIG. 19. The molecular weight of the product was 16440.7 and the yield of the product was 65.4%.
Example 20 conversion of an oligomeric nucleic acid terminal alkyne compound 1-2 with 4-hydroxybenzylamine to an amide
5 nanomolar oligonucleotide terminal alkyne compounds 1-2 were dissolved in 5 μl sodium borate buffer (ph=9.5, 250 mmol/liter, 1 eq.) and reacted at 50 ℃ for 3 hours with the addition of 20 μl dimethyl sulfoxide, 20 eq. cuprous iodide (20 mmol/liter), 200 eq. 4-hydroxybenzylamine (200 mmol/liter) and 100 eq. nitrone (200 mmol/liter). After the completion of the reaction, 50 equivalents of an aqueous solution of sodium diethyldithiocarbamate (100 mmol/L) was added to the reaction mixture, and the mixture was stirred and mixed uniformly, and reacted at 50℃for 10 minutes. High-speed freezing centrifugal separation (4 ℃,12000 rpm, 5 minutes), adding 3.5 microliter of 5 mol/liter sodium chloride aqueous solution into the upper layer solution, 87.5 microliter of absolute ethyl alcohol, shaking and mixing uniformly, and placing into a refrigerator at the temperature of minus 80 ℃ for freezing for 10 to 30 minutes. The product was obtained by high-speed refrigerated centrifugation (4 ℃,12000 rpm, 5 minutes), and the molecular weight of the product was measured by liquid chromatography mass spectrometry to confirm the yield, and the result was shown in FIG. 20. The molecular weight of the product was 16420.0 and the yield of the product was 77.7%.
The invention verifies the reaction of the terminal alkyne of the oligonucleotide and different free amino micromolecular compounds to be converted into amide, and the partial representative compound structural formula is shown in figure 21. According to the invention, through a liquid chromatography mass spectrogram, the reaction of converting the alkyne at the tail end of the oligonucleotide into the amide has the advantages of mild reaction conditions, high yield and good universality of a reaction substrate.
In summary, the foregoing embodiments and drawings are merely illustrative of the general principles of the present invention, and the invention is not limited to the specific principles of the invention, but can be modified or practiced in different among the specific details.
Reference to the literature
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Claims (19)

1. The method for converting terminal alkyne into amide in the construction of gene coding compound library uses the terminal alkyne compound of oligonucleotide as substrate, and reacts with small molecule compound containing free amino group in the presence of copper catalyst and nitrone to form amide compound, and the specific reaction equation is as follows:
wherein the structural formula of the oligonucleic acid terminal alkyne compound isIs formed by connecting chemical groups with alkynyl by oligonucleotide; the structural formula of the prepared amide compound is +.>Is formed by connecting chemical groups with amide bonds with oligonucleotide, R is C 1 -C 12 Alkyl or C 4 -C 12 An aromatic group;
wherein the oligonucleotide is a single-stranded or double-stranded oligonucleotide strand obtained by polymerization of an artificially modified and/or unmodified oligonucleotide monomer;
wherein the structural formula of the small molecular compound containing free amino is R 1 -NH-R 2 ，R 1 、R 2 optionally-COOH, hydrogen, amino, hydroxy, C 1 -C 12 Alkyl, C 3 -C 8 Cycloalkyl, C 4 -C 12 Aromatic group, C 4 -C 12 Any one to more of the aromatic heterocyclic groups or any combination thereof;
wherein the copper catalyst is cuprous iodide;
wherein the nitrone has the structure of
Wherein the alkali is sodium borate;
wherein the solvent for the reaction is a mixed solvent of sodium borate buffer solution with pH=9.5 and dimethyl sulfoxide.
2. The method according to claim 1, wherein the molar concentration of the oligonucleic acid terminal alkyne compound is 0.1 to 2 mmoles/liter.
3. The method according to claim 2, characterized in that the molar concentration of the oligonucleic acid terminal alkyne compound is between 0.5 and 1.5 mmoles/liter.
4. The method according to claim 3, wherein the molar concentration of the oligonucleic acid terminal alkyne compound is 1.0 mmol/l.
5. The process according to claim 1, characterized in that the equivalent of the copper catalyst is 1 to 100 equivalents.
6. The process according to claim 5, wherein the equivalent of the copper catalyst is 5 to 35 equivalents.
7. The process according to claim 6, wherein the equivalent of the copper catalyst is 20 equivalents.
8. The method according to claim 1, characterized in that the equivalent weight of the nitrone is 5 to 500.
9. The method according to claim 8, wherein the equivalent weight of the nitrone is 50 to 150.
10. A process according to claim 9, characterized in that the equivalent of nitrone is 100 equivalents.
11. The method according to claim 1, wherein the equivalent weight of the small molecular compound containing a free amino group is 5 to 500.
12. The method according to claim 11, wherein the equivalent weight of the small molecular compound containing a free amino group is 100 to 300.
13. The method according to claim 12, wherein the equivalent weight of the small molecular compound containing a free amino group is 200.
14. The process according to claim 1, wherein the temperature of the reaction is from 0 to 90 ℃.
15. The process according to claim 14, characterized in that the temperature of the reaction is 30-70 ℃.
16. The method of claim 15, wherein the temperature of the reaction is 50 ℃.
17. The process according to claim 1, characterized in that the reaction time is 0 to 24 hours.
18. The process according to claim 17, characterized in that the reaction time is 2 to 5 hours.
19. The method of claim 18, wherein the reaction time is 3 hours.
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