CN114195842A - Terminal alkynyl deoxyribonucleic acid and self-coupling method thereof - Google Patents
Terminal alkynyl deoxyribonucleic acid and self-coupling method thereof Download PDFInfo
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- CN114195842A CN114195842A CN202111556377.1A CN202111556377A CN114195842A CN 114195842 A CN114195842 A CN 114195842A CN 202111556377 A CN202111556377 A CN 202111556377A CN 114195842 A CN114195842 A CN 114195842A
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- 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
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Abstract
The invention provides terminal alkynyl deoxyribonucleic acid with a structure shown as a formula I and long-chain deoxyribonucleic acid coupled with the terminal alkynyl deoxyribonucleic acid, wherein the terminal DNA of the terminal alkynyl deoxyribonucleic acid cannot be damaged in the reaction process and is consistent with a preset DNA chain, and the terminal alkynyl deoxyribonucleic acid can be used as a DNA brick or a DNA folding module to construct a precise and complex DNA nano structure and has a wide application prospect. Meanwhile, the invention also provides a method for chemically coupling the terminal alkynyl deoxyribonucleic acid, which does not damage the activity of DNA, has high yield and has good popularization and application prospects.
Description
Technical Field
The invention belongs to the technical field of DNA nanometer, and particularly relates to terminal alkynyl deoxyribonucleic acid and a self-coupling method thereof.
Background
Deoxyribonucleic acid (DNA) is a polymer linearly formed by a plurality of deoxynucleotides, and comprises four deoxynucleotides with different basic groups, namely adenine (A), guanine (G), cytosine (C) and thymine (T), wherein the A, T, G and the C are strictly complemented according to the Watson-Crick base pairing principle, and have good primary structure stability; meanwhile, single-stranded and double-stranded DNA have different mechanical properties, the single-stranded DNA has stronger flexibility and excellent double-stranded stability, and the DNA can be self-assembled to form various and stable nano structures by local alternative use, so the DNA is very suitable for serving as a nano technical raw material. The DNA nanotechnology is a technology that uses DNA as a raw material and uses stable branched DNA motifs to construct various target structures and functional devices, such as DNA Tile self-assembly, DNA origami, and the like, and based on the base complementary pairing principle, highly programmable nanostructures are assembled into arbitrary sizes and shapes to realize various complex functions, and has potential application value in the fields of nanomaterials, information carriers, biosensors, artificial molecular machines, and the like (chenxin, wang, new creation, bodawn, DNA programmable nanofabrication research progress [ J ]. guangzhou university press (nature science edition), 2020,19(06): 13-22.).
Although there are many reports on different nanostructures and nanometer devices constructed by DNA nanotechnology, both DNA "bricks" (DNA tiles) and DNA folding modules (various nanometer shapes formed by folding long-chain DNA with the help of small short chains) rely on Watson-Crick base pairing to realize connection and construct DNA nanostructures. However, the nanostructures constructed by the base-complementary pairing method are easy to denature under the condition of heating or the presence of a denaturant, so that the nanostructures can only be studied under the non-denaturing condition, which makes the characterization and purification of the nanostructures complicated, and limits the application of the nanostructures as complicated DNA nanostructure construction modules. Meanwhile, the source of the long-chain DNA template serving as the DNA folding module is limited, the existing method for synthesizing the long-chain DNA by solid phase has low synthesis efficiency, great difficulty and high cost, and the usually synthesized deoxyribonucleic acid chain does not exceed 200 oligonucleides, so that the DNA folding module with a large and complex structure is difficult to form.
The chemical construction of DNA modules (bricks or folding modules) provides a solution to the above problems, but the DNA chemistry is different from the common chemical reaction, and many common small molecule chemical reaction conditions are harsh, so that DNA denaturation and DNA activity damage are easily caused, and the method is also one of the reasons for hindering the development of DNA nanostructures. At present, the work of implementing DNA-DNA coupling reaction by chemical reaction is very little, i.e. the kind of DNA module by chemical ligation is very limited, and further development is urgently needed. The alkynyl is an unsaturated group, the triple bond of unsaturated carbon and carbon has addition and oxidation reaction activity, and alkyne hydrogen connected to the triple bond carbon also has reaction activity and has the potential of chemical coupling. The alkynyl coupling reaction is a classical name reaction in small molecule chemistry: the Glaser reaction, however, has not been reported to couple DNA strands via alkynyl groups due to the problems described above.
Therefore, the method has important significance for enriching the types of DNA modules and widening the technical field of DNA nanometer by exploring new chemically modified DNA for constructing the DNA nanometer structure module and researching the coupling reaction of the DNA.
Disclosure of Invention
The invention provides a terminal alkynyl deoxyribonucleic acid, which has the following structure:
wherein R is
R1、R2Is a linker, R0Is H or C1-C6 alkyl, R3H or C1-C5 alkyl; or R2And R3Linked to form a saturated nitrogen-containing heterocycle or nitrogen-containing spiro ring with the common N;
DNA is either a single-stranded or double-stranded deoxyribonucleotide, which may or may not be artificially modified.
The invention also provides alkyne-coupled deoxyribonucleic acid, which has the following structure:
wherein R isa、RbAre each independently selected from
R1、R2Is a linker, R0Is H or C1-C6 alkyl, R3H or C1-C5 alkyl; or R2And R3Connecting to form a saturated nitrogen-containing heterocycle or a nitrogen-containing spiro ring with the shared N;
DNA1、DNA2is artificially modified or unmodified deoxyribonucleotide single-stranded or double-stranded DNA1And DNA2Are the same or different, when RaAnd RbAre the same as
When being structured, RaR in (1)0And RbR in (1)0May be of different structures as described below, RaR in (1)1And RbR in (1)1Different configurations are possible as described below. When R isaAnd RbAre the same as
When being structured, RaR in (1)2And RbR in (1)2May be of different structures as described below, RaR in (1)3And RbR in (1)3Also can be used forA different configuration is described below.
Further, in the above-mentioned terminal alkynyl deoxyribonucleic acid or alkyne-conjugated deoxyribonucleic acid, R0H or C1-C3 alkyl;
R1is substituted or unsubstituted
Wherein A is nothing, C1-C3 alkyl chain, aromatic ring or aromatic heterocycle, LaIs a C1-C4 alkyl chain,
Or C3-C6 naphthene, n is an integer of 1-3; the substituted substituent is halogen or C1-C3 alkyl;
R2is substituted or unsubstituted
Wherein, L 'is alkyl chain of none or C1-C4, B is none, aromatic ring, aromatic heterocycle, saturated heterocycle or amido bond, L' is alkyl chain of none, C1-C4,
m and p are respectively and independently selected from integers of 1-3; the substituted substituent is halogen, hydroxyl, C1-C3 alkyl, hydroxyl substituted or C1-C3 alkoxy substituted C1-C3 alkyl, C1-C3 naphthenic base, aryl, halogen substituted or hydroxyl substituted aryl;
R3h or C1-C6 alkyl;
or R2And R3Connecting to form a 2-6 membered saturated nitrogen-containing heterocycle or a 5-11 membered nitrogen-containing spiro ring with the shared N.
Further, R0Is a compound of formula (I) in the formula (H),
R1is substituted or unsubstituted
Wherein A is,
Benzene or thiophene ring, LaIs a C1-C4 alkyl chain,
Or cyclopropane; the substituted substituent is F, Br or methyl;
R2is substituted or unsubstituted
Wherein, L 'is alkyl chain of none or C1-C4, B is none, benzene ring, pyridine ring, saturated nitrogen-containing heterocycle or amido bond, L' is alkyl chain of none, C1-C4,
Or
m and p are respectively and independently selected from 1 or 2; the substituted substituent is F, Cl, hydroxyl, methyl, hydroxyl-substituted or methoxy-substituted methyl, cyclopropyl, phenyl, Br-substituted, Cl-substituted or hydroxyl-substituted phenyl;
R3h or C1-C6 alkyl;
or R2And R3And connecting to form a 4-6 membered saturated nitrogen-containing heterocycle or a 7-10 membered nitrogen-containing spiro ring with the shared N.
Further, in the present invention,
R1is absent,
R2Is composed of
Or R2And R3Connected, N in common therewith forms:
furthermore, the above-mentioned deoxyribonucleic acid terminal alkynyl deoxyribonucleic acid has any of the following structures:
further, the alkyne-conjugated deoxyribonucleic acid has any one of the following structures:
wherein, the DNA1And DNA2The same or different.
The invention also provides a method for self-coupling of terminal alkynyl deoxyribonucleic acid, which comprises the following steps:
dissolving one or more terminal alkynyl deoxyribonucleic acids of any one of claims 1 and 3-6 in a solvent, and adding a copper catalyst, a base and a bromine source for reaction;
the reaction formula is as follows:
wherein, DNA 'and DNA "are the same or different, and R' and R" are the same or different.
Further, the solvent is water, methanol, ethanol, propanol, isopropanol, N-butanol, isobutanol, t-butanol, pentanol, cyclohexanol, 2-fluoroethanol, 2, 2-difluoroethanol, 2,2, 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, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, acetone, cyclohexanone, dichloromethane, chloroform, methanol, ethanol, isopropanol, 2-fluoroethanol, 2-trifluoroethanol, hexafluoroisopropanol, benzyl alcohol, ethylene glycol monomethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, N-methylpyrrolidone, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, acetone, cyclohexanone, dichloromethane, chloroform, water, Chlorobenzene, 1, 2-dichloroethane, ethyl acetate, n-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methoxypyridine, toluene, xylene, inorganic salt buffer solution and organic base buffer solution, preferably, the solvent for the reaction is the mixture of water and acetonitrile; more preferably, the solvent of the reaction is a mixture of water and acetonitrile and the total content of acetonitrile is not less than 50%;
the copper catalyst is copper acetate, copper sulfate, copper chloride, copper nitrate, copper carbonate, cuprous iodide, a copper-beta-cyclodextrin compound, copper bis (2, 4-pentanedionate), copper acetylacetonate, copper tetra (acetonitrile) tetrafluoroborate, copper dichloro (1, 10-phenanthroline), copper bis (8-hydroxyquinoline), copper trifluoromethanesulfonate, copper bis (trifluoro-2, 4-pentanedionate), copper perchlorate, copper tetra (acetonitrile) hexafluorophosphate, cuprous acetate, copper bromide, copper fluoride, cuprous bromide, cuprous chloride-bis (lithium chloride) complex, cuprous bromide dimethyl sulfide complex; preferably, the copper catalyst is cuprous iodide, copper tetrakis (acetonitrile) hexafluorophosphate, cuprous chloride-bis (lithium chloride) complex; more preferably, the copper catalyst is cuprous iodide;
the alkali is 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, methylamine, ethylamine, propylamine, isopropylamine, N, one or more of N-diethylamine, triethylamine, N-butylamine, isobutylamine, 4-dimethylaminopyridine, N, N-diisopropylethylamine, 1, 5-diazabicyclo [4.3.0] non-5-ene, 1, 8-diazabicyclo [5.4.0] undec-7-ene, N, N, N ', N' -tetramethylethylenediamine, tetramethylguanidine, pyridine, piperidine, pyrrole, tetrahydropyrrole, N-methyldicyclohexylamine or dicyclohexylamine; preferably, the alkali is one or a mixture of piperidine, tetrahydropyrrole, N-methyl dicyclohexylamine or dicyclohexylamine; more preferably, the base is piperidine.
The bromine source is one or a mixture of more of 1,3, 5-tribromo-1, 3, 5-thiazinane-2, 4, 6-trione, N-bromosuccinimide, N-bromophthalimide, dibromohydantoin, N-dibromobenzenesulfonamide, tetrabromocyclohexadiene-1-one, bromine simple substance, phosphorus tribromide, cyanogen bromide, trichlorobromomethane, sodium bromide, potassium bromide and cuprous bromide; preferably, the bromine source is one or a mixture of more of 1,3, 5-tribromo-1, 3, 5-thiazinane-2, 4, 6-trione, N-bromosuccinimide, N-bromophthalimide and bromine simple substance; more preferably, the bromine source is 1,3, 5-tribromo-1, 3, 5-thiazinan-2, 4, 6-trione.
Further, the concentration of the alkynyl-terminated deoxyribonucleic acid is 0.1 to 2 mmol/l; preferably, the concentration of the oligonucleic acid-terminal alkynyl compound is 0.5 to 1.5 mmol/l; more preferably, the concentration of the oligonucleic acid-terminal alkynyl compound is 1.0 mmol/l;
the molar ratio of the alkynyl deoxyribonucleic acid to the copper catalyst, the alkali and the bromine source is 1 (0.1-100): 1-500), preferably 1 (0.1-50): 10-200): 1-100, more preferably 1:10:100: 40.
Further, the reaction temperature is 0-90 ℃, and the reaction time is 1-48 hours; preferably, the reaction temperature is 0-50 ℃, and the reaction time is 24-48 hours; more preferably, the reaction temperature is 25 ℃ and the reaction time is 36 hours.
The terminal alkynyl deoxyribonucleic acid and the coupled long-chain deoxyribonucleic acid provided by the invention have the advantages that the terminal DNA cannot be damaged in the reaction process and is consistent with a preset DNA chain, the terminal alkynyl deoxyribonucleic acid and the coupled long-chain deoxyribonucleic acid can be applied to a DNA nanotechnology, for example, the terminal alkynyl deoxyribonucleic acid and the coupled long-chain deoxyribonucleic acid can be used as a DNA brick or a DNA folding module to construct a precise and complex DNA nanostructure, and the terminal alkynyl deoxyribonucleic acid and the coupled long-chain deoxyribonucleic acid have the potential of constructing a DNA compound library and have very good application prospects.
Meanwhile, the method for chemically coupling the DNA for synthesizing the terminal alkynyl deoxyribonucleic acid and the coupled long-chain deoxyribonucleic acid does not damage the activity of the DNA, and has high yield which can reach 95 percent.
The deoxyribonucleic acid is a single-stranded or double-stranded deoxyribonucleotide chain obtained by polymerizing artificially modified or unmodified deoxyribonucleotide monomers, and the length of the chain is not limited.
The 3-6 membered saturated nitrogen-containing heterocycle refers to 3-6 atoms constituting a ring skeleton, wherein at least one of the atoms is a nitrogen atom; the "5-11 membered nitrogen-containing spiro ring" means that the number of atoms constituting the spiro ring skeleton is 5-11, and at least one of them is a nitrogen atom, and so on.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 shows the detection result of liquid chromatography mass spectrometry for preparing oligo-nucleic acid-diacetylene compound P1 by reacting oligo-nucleic acid-terminal alkynyl compound S1 with a bromine source.
FIG. 2 shows the detection result of liquid chromatography mass spectrometry for preparing oligo-nucleic acid-diacetylene compound P2 by reacting oligo-nucleic acid-terminal alkynyl compound S2 with a bromine source.
FIG. 3 shows the detection result of liquid chromatography mass spectrometry for preparing oligo-nucleic acid-diacetylene compound P3 by reacting oligo-nucleic acid-terminal alkynyl compound S3 with a bromine source.
FIG. 4 shows the detection result of liquid chromatography mass spectrometry for preparing oligo-nucleic acid-diacetylene compound P4 by reacting oligo-nucleic acid-terminal alkynyl compound S4 with a bromine source.
FIG. 5 shows the detection result of liquid chromatography mass spectrometry for preparing oligo-nucleic acid-diacetylene compound P5 by reacting oligo-nucleic acid-terminal alkynyl compound S5 with a bromine source.
FIG. 6 shows the detection result of liquid chromatography mass spectrometry for preparing oligo-nucleic acid-diacetylene compound P6 by reacting oligo-nucleic acid-terminal alkynyl compound S6 with a bromine source.
FIG. 7 shows the detection result of liquid chromatography mass spectrometry for preparing oligo-nucleic acid-diacetylene compound P7 by reacting oligo-nucleic acid-terminal alkynyl compound S7 with a bromine source.
FIG. 8 shows the detection result of liquid chromatography mass spectrometry for preparing oligo-nucleic acid-diacetylene compound P8 by reacting oligo-nucleic acid-terminal alkynyl compound S8 with a bromine source.
FIG. 9 shows the detection result of liquid chromatography mass spectrometry for preparing oligo-nucleic acid-diacetylene compound P9 by reacting oligo-nucleic acid-terminal alkynyl compound S9 with a bromine source.
FIG. 10 shows the detection result of liquid chromatography mass spectrometry for preparing oligo-nucleic acid-diacetylene compound P10 by reacting oligo-nucleic acid-terminal alkynyl compound S10 with a bromine source.
FIG. 11 shows the detection result of liquid chromatography mass spectrometry for preparing oligo-nucleic acid-diacetylene compound P11 by reacting oligo-nucleic acid-terminal alkynyl compound S11 with a bromine source.
FIG. 12 shows the detection result of liquid chromatography mass spectrometry for preparing oligo-nucleic acid-diacetylene compound P12 by reacting oligo-nucleic acid-terminal alkynyl compound S12 with a bromine source.
FIG. 13 shows the detection result of liquid chromatography mass spectrometry for preparing oligo-nucleic acid-diacetylene compound P13 by reacting oligo-nucleic acid-terminal alkynyl compound S13 with a bromine source.
FIG. 14 shows the detection result of liquid chromatography mass spectrometry for preparing oligo-nucleic acid-diacetylene compound P14 by reacting oligo-nucleic acid-terminal alkynyl compound S14 with a bromine source.
FIG. 15 shows the detection result of liquid chromatography mass spectrometry for preparing oligo-nucleic acid-diacetylene compound P15 by reacting oligo-nucleic acid-terminal alkynyl compound S15 with a bromine source.
FIG. 16 shows the detection result of liquid chromatography mass spectrometry for preparing oligo-nucleic acid-diacetylene compound P16 by reacting oligo-nucleic acid-terminal alkynyl compound S16 with a bromine source.
FIG. 17 shows the detection result of liquid chromatography mass spectrometry for preparing oligo-nucleic acid-diacetylene compound P17 by reacting oligo-nucleic acid-terminal alkynyl compound S17 with a bromine source.
FIG. 18 shows the detection result of liquid chromatography mass spectrometry for preparing oligo-nucleic acid-diacetylene compound P18 by reacting oligo-nucleic acid-terminal alkynyl compound S18 with a bromine source.
FIG. 19 shows the detection result of liquid chromatography mass spectrometry for preparing oligo-nucleic acid-diacetylene compound P19 by reacting oligo-nucleic acid-terminal alkynyl compound S19 with a bromine source.
FIG. 20 shows the results of liquid chromatography mass spectrometry detection of S1-1 on the reaction raw material S1 and the product before and after the ethynylation reaction, wherein (A) is the result spectrum of S1 and (B) is the result spectrum of S1-1.
FIG. 21 is a liquid chromatography mass spectrometry result of P1-1 of the reaction raw material P1 and the product before and after the detection of alkynyl coupling, wherein (A) is a liquid chromatography mass spectrometry result spectrogram of P1, and (B) is a liquid chromatography mass spectrometry result spectrogram of P1-1.
"P" in all figures represents the reaction product.
Detailed Description
The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.
The structures of two kinds of DNA linked with initiation heads used in the examples of the present invention are shown as follows:
wherein HP has the following chemical structure:
however, it should be specifically noted that the method of the present invention is not limited to the specific DNA strand of the examples of the present invention, and may be a single-stranded or double-stranded deoxyribonucleotide chain obtained by polymerizing other artificially modified or unmodified deoxyribonucleotide monomers, and the length of the chain is not limited.
The technical scheme of the invention is clearly and completely described below with reference to the accompanying drawings. It is to be understood that the described embodiments are merely a subset of the embodiments of the invention and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. All oligonucleotide starting materials in the embodiments of the present invention are double-stranded or single-stranded substrates for the oligonucleotides. The synthesis of the oligo-nucleic acid-terminal alkynyl compound substrate is very similar and the general synthesis can be performed as described in example 1.
Example 1 oligonucleic acid-NH2Synthesis of starting materials
1) Oligo-nucleic acid-NHFmoc starting material was synthesized according to the following reaction:
wherein HP has the following structural formula:
100 nanomoles of HP described above were dissolved in deionized water to make a 1 millimole/liter solution (100. mu.L, 100nmol, 1 eq). 40 equivalents of the starting headpiece compound SM1(200 mmol/l DMSO solution, 40 equivalents), 250 equivalents of sodium tetraborate (Na) pH 9.472B4O7250 mmol/l aqueous solution, 250 equivalents) of buffer, 40 equivalents of 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (DMT-MM, 200 mmol/l aqueous solution, 40 equivalents) were mixed. After the mixture was sufficiently mixed by a vortex shaker, it was added to a solution of HP and mixed well, and then reacted at 4 ℃ for 1 hour. After completion of the reaction, a 5 mol/l sodium chloride solution was added to the reaction mixture in an amount of 10% by volume. Then, absolute ethyl alcohol with the volume 3 times of the total volume is continuously added, after uniform oscillation, the reaction is frozen in a refrigerator with the temperature of minus 80 ℃ for 2 hours. After that, the mixture was centrifuged at 4000rpm for half an hour, and the supernatant was decanted. Dissolving the rest precipitate with deionized water to obtain the oligo-nucleic acid-NHFmoc.
2) Synthesis of oligo-nucleic acid-NH according to the following reaction scheme2Raw materials:
100nmol of the above DNA-NHFmoc was dissolved in deionized water to prepare a 1 mmol/L solution (100. mu.L, 100nmol, 1 eq), 36. mu.L of a 10% piperidine (piperidine) aqueous solution was added thereto, and the two solutions were mixed uniformly and reacted at room temperature for 1 hour. After completion of the reaction, a 5 mol/l sodium chloride solution was added to the reaction mixture in an amount of 10% by volume. Then, absolute ethyl alcohol with the volume 3 times of the total volume is continuously added, after uniform oscillation, the reaction is frozen in a refrigerator with the temperature of minus 80 ℃ for 2 hours. After that, the mixture was centrifuged at 4000rpm for half an hour, and the supernatant was decanted. Dissolving the rest precipitate with deionized water to obtain oligo-nucleic acid-NH2。
Example 2 Synthesis of oligo-nucleic acid-COOH starting Material:
oligo-nucleic acid-COOH starting material was synthesized according to the following reaction:
wherein HP has the following structural formula:
100 nanomolar HP was dissolved in deionized water to make a 1 millimolar/liter solution (100. mu.L, 100nmol, 1 eq). 100 equivalents of the starting headpiece compound SM2(200 mmol/l DMSO solution, 100 equivalents), 250 equivalents of sodium tetraborate (Na) pH 9.472B4O7250 mmol/l aqueous solution, 250 equivalents) of buffer, 50 equivalents of 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (DMT-MM, 200 mmol/l aqueous solution, 50 equivalents) were mixed and the mixture was thoroughly mixed with a vortex shaker. The mixture was added to a solution of HP, mixed well and reacted at room temperature for 1 hour. After completion of the reaction, a 5 mol/l sodium chloride solution was added to the reaction mixture in an amount of 10% by volume. Then, absolute ethyl alcohol with the volume 3 times of the total volume is continuously added, after uniform oscillation, the reaction is frozen in a refrigerator with the temperature of minus 80 ℃ for 2 hours. After that, the mixture was centrifuged at 4000rpm for half an hour, and the supernatant was decanted. Dissolving the rest precipitate with deionized water to obtain oligo-nucleic acid-COOH.
EXAMPLE 3 Synthesis of oligo-nucleic acid-terminal alkynyl Compound starting Material
1) The synthesis method 1:
compound B is any one of:
the product obtained
The structures of (A) are respectively as follows:
10 nanomolar oligo-nucleic acid-NH2(Compound A) was dissolved in deionized water to make a 1 mmol/L solution (10. mu.L, 10nmol, 1 eq). To this was added 80 equivalents of compound B bearing carboxyl and alkynyl groups (200 mmol/l DMSO solution, 80 equivalents), 200 equivalents of N, N-diisopropylethylamine (200 mmol/l DMSO solution, 200 equivalents), 50 equivalents of 2- (7-azobenzotriazol) -N, N' -tetramethyluronium hexafluorophosphate (200 mmol/l DMSO solution, 50 equivalents). The mixture was mixed well by vortexing. Adding the mixture into oligo-nucleic acid-NH2After mixing uniformly, the mixture was reacted at room temperature for 1 hour. After the reaction is finished, adding a sodium chloride solution of 5 mol/L with the total volume of 10 percent into the reaction solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times, after uniform oscillation, placing the reaction in a refrigerator with the temperature of minus 80 ℃ for freezing for 2 hours. After that, the mixture was centrifuged at 4000rpm for half an hour, and the supernatant was decanted. Dissolving the rest precipitate with deionized water, and purifying by high performance liquid chromatography to obtain the oligo-nucleic acid-terminal alkynyl compound.
2) The synthesis method 2 comprises the following steps:
compound B' is any one of:
the product obtained
The structures of (A) are respectively as follows:
10nmol of oligo-nucleic acid-COOH (Compound A') was dissolved in deionized water to make a 1 mmol/L solution (10. mu.L, 10nmol, 1 eq). To this was added 50 equivalents of 2- (7-azobenzotriazol) -N, N ' -tetramethyluronium hexafluorophosphate (200 mmol/l DMSO solution, 50 equivalents), 50 equivalents of N, N-diisopropylethylamine (200 mmol/l DMSO solution, 50 equivalents), 50 equivalents of compound B ', compound B ' containing an amino or imino group (200 mmol/l DMSO solution, 50 equivalents). Then, the above solutions were mixed well and reacted at room temperature for 1 hour. After the reaction is finished, adding a sodium chloride solution of 5 mol/L with the total volume of 10 percent into the reaction solution, then continuously adding absolute ethyl alcohol with the volume of 3 times of the total volume, after uniform oscillation, placing the reaction in a refrigerator with the temperature of minus 80 ℃ for freezing for 2 hours. After that, the mixture was centrifuged at 4000rpm for half an hour, and the supernatant was decanted. Dissolving the rest precipitate with deionized water, and purifying by high performance liquid chromatography to obtain the oligo-nucleic acid-terminal alkynyl compound.
Example 4 Synthesis of oligo-nucleic acid-diacetylene Compounds
1nmol of any one or more of the oligonucleic acid-terminal alkynyl compounds prepared in example 1 was dissolved in deionized water to prepare a 1 mmol/L solution (1. mu.L, 1nmol, 1 equivalent), and 100 equivalents of piperidine (50 mmol/L acetonitrile solution, 100 equivalents), 40 equivalents of 1,3, 5-tribromo-1, 3, 5-thiazinan-2, 4, 6-trione (20 mmol/L aqueous solution, 40 equivalents), 10 equivalents of cuprous iodide (20 mmol/L acetonitrile solution, 10 equivalents) were added thereto. Then, the above solutions were mixed well and reacted at 25 ℃ for 36 hours.
After completion of the reaction, 100 equivalents of an aqueous solution of sodium diethyldithiocarbamate (100 mmol/l, 100 equivalents) was added to the reaction solution, and reacted at 70 ℃ for 15 minutes to remove copper. After the copper removal, 5 mol/L sodium chloride solution with the total volume of 10 percent is added into the reaction solution, and then absolute ethyl alcohol with the volume of 3 times of the total volume is added. After shaking to homogeneity, the reaction was frozen in a freezer at-80 ℃ for 2 hours, then centrifuged at 4000rpm for half an hour and the supernatant decanted. And dissolving the rest precipitate with deionized water to obtain the oligo-nucleic acid-diacetylene compound. And (3) confirming the molecular weight of the product through liquid chromatography mass spectrometry detection, and carrying out quantitative analysis on the ultraviolet-visible absorption spectrum on a liquid chromatography mass spectrogram to obtain the reaction yield.
Example 5 Synthesis of oligo-nucleic acid-diacetylene Compound P1
1) Preparation of oligo-nucleic acid-terminal alkynyl Compound S1 (Compound 1-1 of example 1)
The preparation method is the same as the synthesis method 1 in example 1.
2) Synthesis of oligo-nucleic acid-diacetylene compound P1
The procedure is as in example 2. Wherein the molecular weight of the P1 product is 32630. The corresponding product molecular weight was detected by liquid chromatography mass spectrometry, and the reaction yield was 95% by quantitative analysis of the uv-vis absorption spectrum on the mass spectrum of the liquid chromatography, the results are shown in fig. 1.
Example 6 Synthesis of oligo-nucleic acid-diacetylene Compound P2
1) Preparation of oligo-nucleic acid-terminal alkynyl Compound S2 (Compound 1-2 of example 1)
The preparation method is the same as the synthesis method 1 in example 1.
2) Synthesis of oligo-nucleic acid-diacetylene compound P2
The procedure is as in example 2. Wherein the molecular weight of the P2 product is 32630. The corresponding product molecular weight was detected by liquid chromatography mass spectrometry, and the reaction yield was 93% by quantitative analysis of the uv-vis absorption spectrum on the mass spectrogram of liquid chromatography, the results are shown in fig. 2.
Example 7 Synthesis of oligo-nucleic acid-diacetylene Compound P3
1) Preparation of oligo-nucleic acid-terminal alkynyl Compound S3 (Compounds 1-4 of example 1)
The preparation method is the same as the synthesis method 1 in example 1.
2) Synthesis of oligo-nucleic acid-diacetylene compound P3
The procedure is as in example 2. Wherein the molecular weight of the P3 product is 32656. The corresponding product molecular weight was detected by liquid chromatography mass spectrometry, and the reaction yield was 89% by quantitative analysis of the uv-vis absorption spectrum on the mass spectrogram of liquid chromatography, the results are shown in fig. 3.
Example 8 Synthesis of oligo-nucleic acid-diacetylene Compound P4
1) Preparation of oligo-nucleic acid-terminal alkynyl Compound S4 (Compounds 1-18 of example 1)
The preparation method is the same as the synthesis method 2 in example 1.
2) Synthesis of oligo-nucleic acid-diacetylene compound P4
The procedure is as in example 2. Wherein the molecular weight of the P4 product is 32696. The corresponding product molecular weight was detected by liquid chromatography mass spectrometry, and the reaction yield was 77% by quantitative analysis of the uv-vis absorption spectrum on the mass spectrum of liquid chromatography, the results are shown in fig. 4.
Example 9 Synthesis of oligo-nucleic acid-diacetylene Compound P5
1) Preparation of oligo-nucleic acid-terminal alkynyl Compound S5 (Compounds 1-3 of example 1)
The preparation method is the same as the synthesis method 1 in example 1.
2) Synthesis of oligo-nucleic acid-diacetylene compound P5
The procedure is as in example 2. Wherein the molecular weight of the P5 product is 32656. The corresponding product molecular weight was detected by liquid chromatography mass spectrometry, and the reaction yield was 73% by quantitative analysis of the uv-vis absorption spectrum on the mass spectrum of the liquid chromatography, the results are shown in fig. 5.
Example 10 Synthesis of oligo-nucleic acid-diacetylene Compound P6
1) Preparation of oligo-nucleic acid-terminal alkynyl Compound S6 (Compounds 1-17 of example 1)
The preparation method is the same as the synthesis method 2 in example 1.
2) Synthesis of oligo-nucleic acid-diacetylene compound P6
The procedure is as in example 2. The molecular weight of the P6 product is 32660. The corresponding product molecular weight was detected by liquid chromatography mass spectrometry, and the reaction yield was 86% by quantitative analysis of the uv-vis absorption spectrum on the mass spectrum of the liquid chromatography, the results are shown in fig. 6.
Example 11 Synthesis of oligo-nucleic acid-diacetylene Compound P7
1) Preparation of oligo-nucleic acid-terminal alkynyl Compound S7 (Compounds 1-5 of example 1)
The preparation method is the same as the synthesis method 1 in example 1.
2) Synthesis of oligo-nucleic acid-diacetylene compound P7
The procedure is as in example 2. Wherein the molecular weight of the P7 product is 32664. The corresponding product molecular weight was detected by liquid chromatography mass spectrometry, and the reaction yield was 90% by quantitative analysis of the uv-vis absorption spectrum on the mass spectrum of liquid chromatography, the results are shown in fig. 7.
Example 12 Synthesis of oligo-nucleic acid-diacetylene Compound P8
1) Preparation of oligo-nucleic acid-terminal alkynyl Compound S8 (Compounds 1-19 of example 1)
The preparation method is the same as the synthesis method 2 in example 1.
2) Synthesis of oligo-nucleic acid-diacetylene compound P8
The procedure is as in example 2. Wherein the molecular weight of the P8 product is 32688. The corresponding product molecular weight was detected by liquid chromatography mass spectrometry, and the reaction yield was 82% by quantitative analysis of the uv-vis absorption spectrum on the mass spectrum of liquid chromatography, the results are shown in fig. 8.
Example 13 Synthesis of oligo-nucleic acid-diacetylene Compound P9
1) Preparation of oligo-nucleic acid-terminal alkynyl Compound S9 (Compounds 1-11 of example 1)
The preparation method is the same as the synthesis method 1 in example 1.
2) Synthesis of oligo-nucleic acid-diacetylene compound P9
The procedure is as in example 2. Wherein, the molecular weight of the P9 product is 32590. The corresponding product molecular weight was detected by liquid chromatography mass spectrometry, and the reaction yield was 88% by quantitative analysis of the uv-vis absorption spectrum on the mass spectrum of liquid chromatography, the results are shown in fig. 9.
Example 14 Synthesis of oligo-nucleic acid-diacetylene Compound P10
1) Preparation of oligo-nucleic acid-terminal alkynyl Compound S10 (Compounds 1-52 of example 1)
The preparation method is the same as the synthesis method 2 in example 1.
2) Synthesis of oligo-nucleic acid-diacetylene compound P10
The procedure is as in example 2. Wherein the molecular weight of the P10 product is 32594. The corresponding product molecular weight was detected by liquid chromatography mass spectrometry, and the reaction yield was 55% by quantitative analysis of the uv-vis absorption spectrum on the mass spectrum of liquid chromatography, the results are shown in fig. 10.
Example 15 Synthesis of oligo-nucleic acid-diacetylene Compound P11
1) Preparation of oligo-nucleic acid-terminal alkynyl Compound S11 (Compounds 1-12 of example 1)
The preparation method is the same as the synthesis method 1 in example 1.
2) Synthesis of oligo-nucleic acid-diacetylene compound P11
The procedure is as in example 2. Wherein the molecular weight of the P11 product is 32560. The corresponding product molecular weight was detected by liquid chromatography mass spectrometry, and the reaction yield was 85% by quantitative analysis of the uv-vis absorption spectrum on the mass spectrum of liquid chromatography, the results are shown in fig. 11.
Example 16 Synthesis of oligo-nucleic acid-diacetylene Compound P12
1) Preparation of oligo-nucleic acid-terminal alkynyl Compound S12 (Compounds 1-10 of example 1)
The preparation method is the same as the synthesis method 1 in example 1.
2) Synthesis of oligo-nucleic acid-diacetylene compound P12
The procedure is as in example 2. Wherein the molecular weight of the P12 product is 32640. The corresponding product molecular weight was detected by liquid chromatography mass spectrometry, and the reaction yield was 84% by quantitative analysis of the uv-vis absorption spectrum on the mass spectrum of the liquid chromatography, the results are shown in fig. 12.
Example 17 Synthesis of oligo-nucleic acid-diacetylene Compound P13
1) Preparation of oligo-nucleic acid-terminal alkynyl Compound S13 (Compounds 1-54 of example 1)
The preparation method is the same as the synthesis method 2 in example 1.
2) Synthesis of oligo-nucleic acid-diacetylene compound P13
The procedure is as in example 2. Wherein the molecular weight of the P13 product is 32564. The corresponding product molecular weight was detected by liquid chromatography mass spectrometry, and the reaction yield was 70% by quantitative analysis of the uv-vis absorption spectrum on the mass spectrum of liquid chromatography, the results are shown in fig. 13.
Example 18 Synthesis of oligo-nucleic acid-diacetylene Compound P14
1) Preparation of oligo-nucleic acid-terminal alkynyl Compound S14 (Compounds 1-62 of example 1)
The preparation method is the same as the synthesis method 2 in example 1.
2) Synthesis of oligo-nucleic acid-diacetylene compound P14
The procedure is as in example 2. Wherein the molecular weight of the P14 product is 32536. The corresponding product molecular weight was detected by liquid chromatography mass spectrometry, and the reaction yield was 77% by quantitative analysis of the uv-vis absorption spectrum on the mass spectrum of liquid chromatography, the results are shown in fig. 14.
Example 19 Synthesis of oligo-nucleic acid-diacetylene Compound P15
1) Preparation of oligo-nucleic acid-terminal alkynyl Compound S15 (Compounds 1-64 of example 1)
The preparation method is the same as the synthesis method 1 in example 1.
2) Synthesis of oligo-nucleic acid-diacetylene compound P15
The procedure is as in example 2. Wherein the molecular weight of the P15 product is 32592. The corresponding product molecular weight was detected by liquid chromatography mass spectrometry, and the reaction yield was 67% by quantitative analysis of the uv-vis absorption spectrum on the mass spectrum of the liquid chromatography, the results are shown in fig. 15.
Example 20 Synthesis of oligo-nucleic acid-diacetylene Compound P16
1) Preparation of oligo-nucleic acid-terminal alkynyl Compound S16 (Compounds 1-34 of example 1)
The preparation method is the same as the synthesis method 2 in example 1.
2) Synthesis of oligo-nucleic acid-diacetylene compound P16
The procedure is as in example 2. Wherein the molecular weight of the P16 product is 32616. The corresponding product molecular weight was detected by liquid chromatography mass spectrometry, and the reaction yield was 84% by quantitative analysis of the uv-vis absorption spectrum on the liquid chromatography mass spectrogram, the results are shown in fig. 16.
Example 21 Synthesis of oligo-nucleic acid-diacetylene Compound P17
1) Preparation of oligo-nucleic acid-terminal alkynyl Compound S17 (Compounds 1-45 of example 1)
The preparation method is the same as the synthesis method 2 in example 1.
2) Synthesis of oligo-nucleic acid-diacetylene compound P17
The procedure is as in example 2. Wherein the molecular weight of the P17 product is 32674. The corresponding product molecular weight was detected by liquid chromatography mass spectrometry, and the reaction yield was 66% by quantitative analysis of the uv-vis absorption spectrum on the mass spectrum of the liquid chromatography, the results are shown in fig. 17.
Example 22 Synthesis of oligo-nucleic acid-diacetylene Compound P18
1) Preparation of oligo-nucleic acid-terminal alkynyl Compound S18 (Compounds 1-31 of example 1)
The preparation method is the same as the synthesis method 2 in example 1.
2) Synthesis of oligo-nucleic acid-diacetylene compound P18
The procedure is as in example 2. Wherein, the molecular weight of the P18 product is 32720. The corresponding product molecular weight was detected by liquid chromatography mass spectrometry, and the reaction yield was 88% by quantitative analysis of the uv-vis absorption spectrum on the mass spectrum of liquid chromatography, the results are shown in fig. 18.
Example 23 Synthesis of oligo-nucleic acid-diacetylene Compound P19
1) Preparation of oligo-nucleic acid-terminal alkynyl Compound S19 (Compounds 1-13 of example 1)
The preparation method is the same as the synthesis method 1 in example 1.
2) Synthesis of oligo-nucleic acid-diacetylene compound P19
The procedure is as in example 2. Wherein the molecular weight of the P19 product is 32532. The corresponding product molecular weight was detected by liquid chromatography mass spectrometry, and the reaction yield was 84% by quantitative analysis of the uv-vis absorption spectrum on the mass spectrum of liquid chromatography, the results are shown in fig. 19.
The following experimental examples demonstrate the advantageous effects of the present invention.
Experimental example 1 screening of reaction reagents and reaction conditions
Taking a synthesis example of P11, the types and the amounts of the catalyst, the alkali and the bromine source additive used in the reaction and the reaction temperature conditions were screened, and the yields of P11 prepared under different conditions are shown in Table 1.
TABLE 1 screening of optimization conditionsa
a、S11(1nmol，1mmol/L in H2O), dissolving the rest reagents by using acetonitrile, and reacting for 24 hours.
b. The reaction was carried out for 36 hours.
It should be specifically noted that the screening protocol in table 1 is only a part of the screening protocol performed by the inventors during the development process. Different reagents, different amounts and reaction conditions have a great influence on the yield of each conjugate. After extensive exploration, the optimal coupling scheme was determined to be currently reacting 100 equivalents of piperidine as base, 40 equivalents of 1,3, 5-tribromo-1, 3, 5-thiazinan-2, 4, 6-trione as a bromine source and 10 equivalents of cuprous iodide at 25 ℃ for 36 hours.
Experimental example 2 in the terminal alkynyl deoxyribonucleic acid and alkyne-coupled deoxyribonucleic acid of the present invention, the DNA structure is intact and not damaged
1. On the one hand, the change in molecular weight of the DNA before and after the ethynylation reaction was determined, and on the other hand, the prepared terminal alkynyl deoxyribonucleic acid was linked to TagA (short-chain oligonucleotides, both chains having molecular weights of 3169 and 5235, respectively).
The method comprises the following steps: 1nmol of S1 was dissolved in deionized water to make a 1 mmol/L solution (1. mu.L, 1nmol, 1 eq.) and 1 eq of TagA (1 mmol/L H) was added to it2O solution, 1 eq), 1 microliter of 10XT4DNA Linked buffer solution and 0.5. mu.l of T4 DNA Linked enzyme. Then, the above solutions were mixed well and reacted at room temperature for 1 hour. After the reaction is finished, adding a sodium chloride solution of 5 mol/L with the total volume of 10 percent into the reaction solution, then continuously adding absolute ethyl alcohol with the volume of 3 times of the total volume, after uniform oscillation, placing the reaction in a refrigerator with the temperature of minus 80 ℃ for freezing for 2 hours. After that, the mixture was centrifuged at 4000rpm for half an hour, and the supernatant was decanted. And dissolving the rest precipitate with deionized water, and detecting by liquid chromatography mass spectrometry to confirm the molecular weight of the product.
No change in the molecular weight of the DNA before and after the ethynylation reaction is detected, the liquid chromatography mass spectrometry detection results of the reaction raw material S1 and the product S1-1 are shown in FIG. 20, wherein FIG. 20A is a liquid chromatography mass spectrometry result spectrogram of S1, and FIG. 20B is a liquid chromatography mass spectrometry result spectrogram of S1-1, and it can be seen that the raw material S1 is completely converted into the product S1-1, which indicates that the connection between the terminal alkynyl deoxyribonucleic acid S1 and TagA is prolonged successfully. The above results show that the DNA chain of the alkynyl-modified terminal alkynyl deoxyribonucleic acid of the invention has no chain scission phenomenon and no influence on the elongation activity, i.e. the reaction method of the invention does not damage the basic structure and activity of the DNA.
2. On one hand, the change of the molecular weight of the DNA before and after alkynyl coupling is detected, on the other hand, the alkyne-coupled deoxyribonucleic acid is linked with TagA (short-chain oligonucleotide, the molecular weight of the two chains is 3169 and 5235 respectively), and the reaction steps refer to the above.
No change in molecular weight of DNA before and after alkynyl coupling is detected, and the liquid chromatography mass spectrometry detection results of the reaction raw material P1 and the product P1-1 are shown in FIG. 21, wherein FIG. 21A is a liquid chromatography mass spectrometry result spectrogram of P1, and FIG. 21B is a liquid chromatography mass spectrometry result spectrogram of P1-1, and it can be seen that the raw material P1 has been completely converted into the product P1-1, which indicates that the successful connection of the alkyne coupled deoxyribonucleic acid P1 and TagA is prolonged. The above results show that the alkyne-coupled deoxyribonucleic acid of the present invention has no chain scission phenomenon in the DNA chain and no influence on the elongation activity, i.e., the reaction method of the present invention does not damage the basic structure and activity of DNA.
The results show that the method of the invention can ensure high yield and simultaneously can not damage DNA chains, and the prepared terminal alkynyl deoxyribonucleic acid and alkyne-coupled deoxyribonucleic acid can be further used as 'bricks' (DNA tiles) of DNA nano-structures or long-chain DNA templates in DNA origami.
In conclusion, the invention provides the deoxyribonucleic acid with the alkynyl at the tail end and the self-coupling product thereof, the yield of the self-coupling reaction of the deoxyribonucleic acid with the alkynyl at the tail end is high, DNA is not damaged, and the coupled long-chain deoxyribonucleic acid can be used as a DNA folding module and has good application potential in the construction of a DNA nano structure.
Claims (15)
1. A terminal alkynyl deoxyribonucleic acid, having the structure:
wherein R is
R1、R2Is a linker, R0Is H or C1-C6 alkyl, R3H or C1-C5 alkyl; or R2And R3Linked to form a saturated nitrogen-containing heterocycle or nitrogen-containing spiro ring with the common N;
DNA is either a single-stranded or double-stranded deoxyribonucleotide, which may or may not be artificially modified.
2. An alkyne-conjugated deoxyribonucleic acid, comprising the following structure:
wherein R isa、RbAre each independently selected from
R1、R2Is a linker, R0Is H or C1-C6 alkyl, R3H or C1-C5 alkyl; or R2And R3Linked to form a saturated nitrogen-containing heterocycle or nitrogen-containing spiro ring with the common N;
DNA1、DNA2is artificially modified or unmodified deoxyribonucleotide single-stranded or double-stranded DNA1And DNA2The same or different.
3. The terminal alkynyl deoxyribonucleic acid of claim 1 or the alkyne-conjugated deoxyribonucleic acid of claim 2, wherein R0H or C1-C3 alkyl,
R1is substituted or unsubstituted
Wherein A is nothing, C1-C3 alkyl chain, aromatic ring or aromatic heterocycle, LaIs a C1-C4 alkyl chain,
Or C3-C6 naphthene, n is an integer of 1-3; the substituted substituent is halogen or C1-C3 alkyl;
R2is substituted or unsubstituted
Wherein, L 'is alkyl chain of none or C1-C4, B is none, aromatic ring, aromatic heterocycle, saturated heterocycle or amido bond, L' is alkyl chain of none, C1-C4,
m and p are respectively and independently selected from integers of 1-3; the takingThe substituent of the substituent is halogen, hydroxyl, C1-C3 alkyl, hydroxyl-substituted or C1-C3 alkoxy-substituted C1-C3 alkyl, C1-C3 cycloalkyl, aryl, halogen-substituted or hydroxyl-substituted aryl;
R3h or C1-C6 alkyl;
or R2And R3And connecting to form a 3-6 membered saturated nitrogen-containing heterocycle or a 5-11 membered nitrogen-containing spiro ring with the shared N.
4. The deoxyribonucleic acid terminal alkynyl deoxyribonucleic acid or alkyne-conjugated deoxyribonucleic acid of claim 3, wherein R is0Is a compound of formula (I) in the formula (H),
R1is substituted or unsubstituted
Wherein A is,
Benzene or thiophene ring, LaIs a C1-C4 alkyl chain,
Or cyclopropane; the substituted substituent is F, Br or methyl;
R2is substituted or unsubstituted
Wherein, L 'is alkyl chain of none or C1-C4, B is none, benzene ring, pyridine ring, saturated nitrogen-containing heterocycle or amido bond, L' is alkyl chain of none, C1-C4,
m and p are respectively and independently selected from 1 or 2; the substituted substituent is F, Cl, hydroxyl, methyl, hydroxyl-substituted or methoxy-substituted methyl, cyclopropyl, phenyl, Br-substituted, Cl-substituted or hydroxyl-substituted phenyl;
R3h or C1-C6 alkyl;
or R2And R3And connecting to form a 4-6 membered saturated nitrogen-containing heterocycle or a 7-10 membered nitrogen-containing spiro ring with the shared N.
5. The deoxyribonucleic acid terminal alkynyl deoxyribonucleic acid or alkyne-conjugated deoxyribonucleic acid of claim 4,
R1is absent,
R2Is composed of
Or R2And R3Connected, N in common therewith forms:
6. the deoxyribonucleic acid-terminal alkynyl deoxyribonucleic acid of any one of claims 1 and 3 to 5, wherein the deoxyribonucleic acid-terminal alkynyl deoxyribonucleic acid has any one of the following structures:
7. an alkyne-conjugated deoxyribonucleic acid according to any one of claims 2 to 5, wherein the alkyne-conjugated deoxyribonucleic acid has any of the following structures:
wherein, the DNA1And DNA2The same or different.
8. A method for self-coupling of terminal alkynyl deoxyribonucleic acids, comprising the steps of:
dissolving one or more terminal alkynyl deoxyribonucleic acids of any one of claims 1 and 3-6 in a solvent, and adding a copper catalyst, a base and a bromine source for reaction;
the reaction formula is as follows:
wherein, DNA 'and DNA "are the same or different, and R' and R" are the same or different.
9. The method of claim 8, wherein the solvent is water, methanol, ethanol, propanol, isopropanol, N-butanol, isobutanol, t-butanol, pentanol, cyclohexanol, 2-fluoroethanol, 2, 2-difluoroethanol, 2,2, 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, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, methanol, ethanol, isopropanol, 2-fluoroethanol, 2-difluoroethanol, 2,2, 2-trifluoroethanol, hexafluoroisopropanol, benzyl alcohol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, or mixtures thereof, Acetone, cyclohexanone, dichloromethane, chloroform, chlorobenzene, 1, 2-dichloroethane, ethyl acetate, n-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methoxypyridine, toluene, xylene, inorganic salt buffer solution and organic alkali buffer solution;
the copper catalyst is copper acetate, copper sulfate, copper chloride, copper nitrate, copper carbonate, cuprous iodide, a copper-beta-cyclodextrin compound, copper bis (2, 4-pentanedionate), copper acetylacetonate, copper tetra (acetonitrile) tetrafluoroborate, copper dichloro (1, 10-phenanthroline), copper bis (8-hydroxyquinoline), copper trifluoromethanesulfonate, copper bis (trifluoro-2, 4-pentanedionate), copper perchlorate, copper tetra (acetonitrile) hexafluorophosphate, cuprous acetate, copper bromide, copper fluoride, cuprous bromide, cuprous chloride-bis (lithium chloride) complex, cuprous bromide dimethyl sulfide complex;
the base is one or a mixture of more of 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, methylamine, ethylamine, propylamine, isopropylamine, N, N-diethylamine, triethylamine, N-butylamine, isobutylamine, 4-dimethylaminopyridine, N, N-diisopropylethylamine, 1, 5-diazabicyclo [4.3.0] non-5-ene, 1, 8-diazabicyclo [5.4.0] undec-7-ene, N, N ', N' -tetramethylethylenediamine, tetramethylguanidine, pyridine, piperidine, pyrrole, tetrahydropyrrole, N-methyldicyclohexamine or dicyclohexylamine;
the bromine source is one or a mixture of more of 1,3, 5-tribromo-1, 3, 5-thiazinane-2, 4, 6-trione, N-bromosuccinimide, N-bromophthalimide, dibromohydantoin, N-dibromobenzenesulfonamide, tetrabromocyclohexadiene-1-one, bromine simple substance, phosphorus tribromide, cyanogen bromide, trichlorobromomethane, sodium bromide, potassium bromide and cuprous bromide.
10. The method of claim 9, wherein the solvent is a mixture of water and acetonitrile, the volume fraction of acetonitrile in the mixture of water and acetonitrile being not less than 50%.
11. The method of claim 9, wherein the copper catalyst is cuprous iodide, copper tetrakis (acetonitrile) hexafluorophosphate, or cuprous chloride-bis (lithium chloride) complex.
12. The method of claim 9, wherein the base is one or more of piperidine, tetrahydropyrrole, N-methyldicyclohexylamine, or dicyclohexylamine.
13. The method of claim 9, wherein the bromine source is one or a mixture of 1,3, 5-tribromo-1, 3, 5-thiazinan-2, 4, 6-trione, N-bromosuccinimide, N-bromophthalimide, and elemental bromine.
14. The method of claim 8, wherein the concentration of said alkynyl-terminated deoxyribonucleic acid is 0.1 to 2 mmol/l; the molar ratio of the alkynyl deoxyribonucleic acid to the copper catalyst, the alkali and the bromine source is 1 (0.1-100): 1-500).
15. The method of claim 8, wherein the reaction temperature is 0 to 90 ℃ and the reaction time is 1 to 48 hours.
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