CN112920244B - Method for synthesizing On-DNA pyrimidine compound - Google Patents

Method for synthesizing On-DNA pyrimidine compound Download PDF

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CN112920244B
CN112920244B CN202011041674.8A CN202011041674A CN112920244B CN 112920244 B CN112920244 B CN 112920244B CN 202011041674 A CN202011041674 A CN 202011041674A CN 112920244 B CN112920244 B CN 112920244B
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李进
杜甜
伍荣峰
高森
刘观赛
万金桥
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Abstract

The invention relates to a method for synthesizing an On-DNA pyrimidine compound, which is characterized in that an On-DNA alpha, beta-unsaturated carbonyl compound reacts with an amidino compound to obtain the On-DNA pyrimidine compound.

Description

Method for synthesizing On-DNA pyrimidine compound
Technical Field
The invention belongs to the technical field of coding compound libraries, and particularly relates to a method for synthesizing an On-DNA pyrimidine compound in the construction of a DNA coding compound library.
Background
In drug development, especially new drug development, high-throughput screening for biological targets is one of the main means for rapidly obtaining lead compounds. However, traditional high throughput screening based on single molecules requires long time, large equipment investment, limited number of library compounds (millions), and the building of compound libraries requires decades of accumulation, limiting the efficiency and possibility of discovery of lead compounds. The recent emergence of DNA-encoded compound library technologies (WO 2005058479, WO2018166532, CN 103882532) combined with combinatorial chemistry and molecular biology technologies, adding a DNA tag to each compound on the molecular level, and being able to synthesize up to one hundred million levels of compound libraries in a very short time, is a trend of the next generation compound library screening technology, and starts to be widely applied in pharmaceutical industry, producing many positive effects (Accounts of Chemical Research,2014,47, 1247-1255).
The DNA coding compound library can quickly generate a giant compound library through combinatorial chemistry, and can screen out a lead compound in a high-throughput manner, so that the screening of the lead compound becomes unprecedented rapidness and high efficiency. One of the challenges in constructing libraries of DNA-encoding compounds is the need to synthesize chemically diverse small molecules on DNA in high yields. Because DNA can be kept stable under certain conditions (solvent, pH, temperature and ion concentration), the On-DNA reaction applied to the construction of the DNA coding compound library also needs higher yield. Therefore, the reagent type, reaction type and reaction condition of the chemical reaction (On-DNA reaction for short) carried out On DNA directly influence the richness and selectivity of the DNA coding compound library. Therefore, the development of chemical reactions compatible with DNA is also a long-term research and research direction of the current DNA coding compound library technology, and the application and commercial value of the DNA coding compound library are directly influenced.
Pyrimidine compounds are important drug compound framework structures, however, no method for synthesizing On-DNA pyrimidine compounds through On-DNA alpha, beta-unsaturated carbonyl compounds is reported at present. Therefore, a new On-DNA pyrimidine compound synthesis method suitable for large-scale multi-well plate operation needs to be developed to increase the diversity of a DNA coding compound library and further improve the application value of the DNA coding compound library technology.
Disclosure of Invention
In order to solve the problems, a DNA coding compound synthesis method which has stable storage of raw materials, mild reaction conditions, good substrate universality and small damage to DNA and is suitable for batch operation by using a porous plate is developed, and an On-DNA alpha, beta-unsaturated carbonyl compound can be quickly converted into an On-DNA pyrimidine compound through one-step reaction.
The invention provides a method for synthesizing an On-DNA pyrimidine compound, which takes an On-DNA alpha, beta-unsaturated carbonyl compound and an amidino compound as raw materials, and reacts in the presence of alkali and an oxidant to obtain an On-DNA product; wherein the structural formula of the On-DNA alpha, beta-unsaturated carbonyl compound is shown in the specification
Figure BDA0002710536470000021
The amidino compound has the formula->
Figure BDA0002710536470000022
Wherein the DNA in the structural formula comprises a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and/or unmodified nucleotide monomers, and the nucleotide chain is connected with the rest part in the compound through one or more chemical bonds or groups; the DNA is 10 to 200 bases in length.
Wherein, the DNA and R in the structural formula 1 Or R 3 Linked by a chemical bond or multiple chemical bonds. When a chemical bond is present, it refers to DNA and R in the structural formula 1 Or R 3 Directly connecting; when multiple chemical bonds are present, they refer to DNA and R in the structural formula 1 Or R 3 Are connected with a plurality of chemical bonds at intervals, e.g. DNA and R 1 Or R 3 Through a methylene group (-CH) 2 -) are linked, i.e. linked by two chemical bonds; or DNA and R 1 Or R 3 The amino group of the DNA is connected with the amino group of the DNA through a carbonyl (-CO-) and is also connected through two chemical bonds; or DNA with R 1 Or R 3 Through a methylene carbonyl group (-CH) 2 CO-) is attached to the amino group of the DNA, again by three consecutive chemical bonds.
R 1 Selected from the group consisting of groups having a molecular weight of 1000 or less which are directly attached to the DNA and carbonyl carbon atoms or are absent;
R 2 selected from groups having a molecular weight of 1000 or less directly bonded to an alkenyl carbon atom;
R 3 selected from the group consisting of groups having a molecular weight of 1000 or less directly attached to the DNA and to an alkenyl carbon atom or are absent;
R 4 selected from groups having a molecular weight below 1000 directly attached to the carbonyl carbon atom;
R 5 selected from hydrogen or a group having a molecular weight of 1000 or less directly bonded to an alkenyl carbon atom;
R 6 selected from groups having a molecular weight of 1000 or less directly attached to an amidino carbon atom;
or R 5 Are each independently of R 1 、R 2 、R 3 Or R 4 Looping.
Preferably, R is 1 、R 2 、R 3 、R 4 Are respectively selected from alkyl, substituted alkyl, carboxyl, 5-10-membered aryl, substituted 5-10-membered aryl, 5-10-membered aromatic heterocyclic group and substituted 5-10-membered aromatic heterocyclic group; wherein the alkyl is C 1 ～C 20 Alkyl or C 3 ～C 8 A cycloalkyl group; the number of substituents of the substituted alkyl group is one or more; the substituent of the substituted alkyl is one or more independently selected from halogen, carboxyl, nitro, alkoxy, halogenated phenyl, alkyl phenyl and heterocyclic radical; the number of the substituent for substituting the 5-to 10-membered aryl is one or more, and the substituents for substituting the 5-to 10-membered aryl are independently selected from halogen, cyano, nitro, carboxyl, alkoxy and C 1 ～C 20 One or more of alkyl and trifluoromethyl; the number of the substituent(s) for substituting the 5-to 10-membered aromatic heterocyclic group is one or more, and the substituent(s) for substituting the 5-to 10-membered aromatic heterocyclic group are independently selected from the group consisting of halogen, cyano, nitro, carboxyl, alkoxy, C 1 ～C 20 One or more of alkyl and trifluoromethyl;
the R is 5 Selected from hydrogen, C 1 ～C 20 An alkyl group; the R is 6 Is selected from C 1 ～C 20 Alkyl or phenyl.
Further on;
r is as described 1 Selected from phenyl, thienyl;
said R 2 Selected from phenyl, substituted phenyl, C 1 ～C 6 Alkyl, carboxyl, pyridyl, substituted pyridyl, furyl; the substituent of the substituted phenyl is selected from carboxyl and C 1 ～C 6 Alkoxy, trifluoromethyl, C 1 ～C 6 An alkyl group; the substituent of the substituted pyridyl is selected from C 1 ～C 6 Alkyl radical, C 1 ～C 6 An alkoxy group;
r is as described 3 Selected from phenyl, thienyl;
said R 4 Selected from phenyl, substituted phenyl, C 1 ～C 6 Alkyl, carboxyl, pyridylSubstituted pyridyl, furyl; the substituent of the substituted phenyl is selected from carboxyl and C 1 ～C 6 Alkoxy, trifluoromethyl, C 1 ～C 6 An alkyl group; the substituent of the substituted pyridyl is selected from C 1 ～C 6 Alkyl radical, C 1 ～C 6 An alkoxy group;
said R is 5 Selected from hydrogen, C 1 ～C 6 An alkyl group;
said R is 6 Is selected from C 1 ～C 6 Alkyl or phenyl.
Preferably, the On-DNA alpha, beta-unsaturated carbonyl compound is specifically selected from the following compounds:
Figure BDA0002710536470000031
/>
Figure BDA0002710536470000041
amidino compounds are selected in particular from
Figure BDA0002710536470000042
A method of synthesizing an On-DNA pyrimidine compound, the method comprising the steps of: adding 10-1000 times molar equivalent of amidino compound and 10-1000 times molar equivalent of alkali into an On-DNA alpha, beta-unsaturated carbonyl compound solution with molar equivalent of 1 and molar concentration of 0.5-5mM, finally adding 10-500 times molar equivalent of oxidant, and reacting at 10-100 ℃ for 0.5-24 hours.
Further, the base is selected from sodium borate, lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, sodium phosphate, potassium phosphate, sodium hydrogen phosphate, potassium hydrogen phosphate, N-methylmorpholine, triethylamine, diisopropylethylamine, DBU (1, 8-diazabicycloundecen-7-ene), 4-dimethylaminopyridine, 2, 6-dimethylpyridine, N-methylimidazole; preferably, the base is sodium hydroxide.
Further, the oxidation is carried outThe agent is selected from oxygen, iodine simple substance, K 2 S 2 O 8 Sodium periodate, DDQ (dichlorodicyanoquinone), DTBP (di-tert-butyl peroxide), TBHP (tert-butyl hydroperoxide); preferably, the oxidant is elemental iodine.
Further, the reaction is carried out in a solvent, wherein the solvent is a water-containing mixed solvent of any one or more of water, methanol, ethanol, acetonitrile, dimethyl sulfoxide, N-dimethylformamide, N-dimethylacetamide, an inorganic salt buffer solution, an organic acid buffer solution and an organic base buffer solution; preferably, the reaction solvent contains boric acid buffer, N-dimethylacetamide.
Further, the pH value of the boric acid buffer solution is 7-11; preferably, the pH is 9.4.
Further, the reaction temperature of the reaction is 10-100 ℃; preferably, the reaction temperature is 20 ℃,30 ℃,40 ℃,50 ℃ or 60 ℃.
Further, the reaction time of the reaction is 0.5 to 24 hours; preferably, the reaction time is 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours or 16 hours.
Further, in the method, the molar equivalent of the On-DNA alpha, beta-unsaturated carbonyl compound is 1, the molar equivalent of the amidino compound is 10 to 1000, the molar equivalent of the base is 10 to 1000, and the molar equivalent of the oxidant is 10 to 500; preferably, the molar equivalents of amidino compound are 50 equivalents, 100 equivalents, 200 equivalents, 300 equivalents, 400 equivalents, 500 equivalents, 600 equivalents, 800 equivalents, 1000 equivalents; the molar equivalent of the base is 50 equivalents, 100 equivalents, 200 equivalents, 300 equivalents, 400 equivalents, 500 equivalents, 600 equivalents, 800 equivalents, 1000 equivalents; the molar equivalent of the oxidizing agent is 20 equivalents, 60 equivalents, 80 equivalents, 100 equivalents, 200 equivalents, 400 equivalents; most preferably, the molar equivalent of the amidino compound is 400, the molar equivalent of the base is 500 and the molar equivalent of the oxidizing agent is 100.
Further, the reaction is carried out in the order of adding the On-DNA alpha, beta-unsaturated carbonyl compound, the amidino compound and the base, and finally the oxidant.
Further, the above method is used for batch multi-well plate operations.
Further, the above method is used for the synthesis of libraries of DNA-encoding compounds for multi-well plates.
The method can realize the acquisition of the On-DNA pyrimidine compound by the On-DNA alpha, beta-unsaturated carbonyl compound in a DNA coding compound library, can be widely applied to various On-DNA alpha, beta-unsaturated carbonyl substrates, and can introduce various substituted amidino compounds as synthesis modules in a large scale. The method has high yield and single product, can be carried out in the mixed water phase of an organic solvent/water phase, has simple operation, does not introduce metal reagents, is environment-friendly, and is suitable for synthesizing the DNA coding compound library by using a porous plate.
Definitions of terms used in relation to the present invention: the initial definitions provided herein for a group or term apply to that group or term throughout the specification unless otherwise indicated; for terms not specifically defined herein, the meanings that would be afforded to them by a person skilled in the art, in light of the disclosure and context, should be given.
"substituted" means that a hydrogen atom in a molecule is replaced by a different atom or molecule.
The minimum and maximum values of the carbon atom content in the hydrocarbon group are indicated by a prefix, e.g. prefix (Ca-C) b ) Alkyl means any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, C 1 ～C 20 Alkyl refers to a straight or branched chain alkyl group containing 1 to 20 carbon atoms.
Alkyl is a straight or branched chain hydrocarbon radical derived from an alkane molecule by the removal of one hydrogen atom, e.g. methyl CH 3 -, ethyl CH 3 CH 2 -。
Cycloalkyl groups: refers to a saturated or unsaturated cycloalkyl group; the halogen is fluorine, chlorine, bromine or iodine.
Alkoxy groups: means that the alkyl radical is bound to an oxygen atom to form a substituent, e.g. methoxy is-OCH 3 。
Halogenated phenyl group: refers to a group formed by substituting H on a phenyl group with a halogen.
Alkyl phenyl: refers to a group formed by substituting H on a phenyl group with an alkyl group.
5-to 10-membered aryl: refers to an aromatic single cyclic or multiple cyclic group composed of C atoms and containing no hetero atom.
The 5-to 10-membered aromatic heterocyclic group means a single cyclic group or a plurality of cyclic groups having aromaticity composed of 5 to 10 atoms of C, O, S, N, etc.
Heterocyclic group: is a saturated or unsaturated monocyclic or polycyclic hydrocarbon group carrying at least one atom of 3 to 8 selected from O, S, N.
It will be apparent that various other modifications, substitutions and alterations can be made in the present invention without departing from the basic technical concept of the invention as described above, according to the common technical knowledge and common practice in the field.
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: the corresponding transformation rate distribution diagram of 21 On-DNA pyrimidine compounds is obtained in the embodiment 3 of the invention.
Detailed Description
The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.
DNA-NH-in the invention 2 Is formed by single-stranded or double-stranded DNA and a linker group and has-NH 2 DNA constructs for linkers, e.g. DNA-NH of "compound1" in WO2005058479 2 And (5) structure. Also for example the following DNA structure:
Figure BDA0002710536470000071
wherein A is adenine, T is thymine, C is cytosine and G is guanine.
Example 1 Synthesis of On-DNA pyrimidine Compounds
Step 1, synthesis of On-DNA alpha, beta-unsaturated carbonyl compound
Figure BDA0002710536470000072
On-DNA arylethanones (1) were dissolved in 250mM boric acid buffer pH =9.4 to prepare a 1mM concentration solution (20. Mu.L, 20 nmol), benzaldehyde (4000nmol, 200 equivalents, 200mM DMSO), and sodium hydroxide (10000nmol, 500 equivalents, 500mM double distilled water) were sequentially added to the solution, and the mixture was mixed well and reacted at 30 ℃ for 2 hours.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the reacted solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the total volume, after uniformly oscillating, placing the reaction in dry ice for freezing for 0.5 hour, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain a solution of the On-DNA alpha, beta-unsaturated carbonyl compound (2), and after the quantification is carried out by an enzyme labeling instrument OD, sending LCMS to confirm that the conversion rate of the reaction is 90%. Step 2, synthesis of On-DNA pyrimidine compound
Figure BDA0002710536470000073
The On-DNA α, β -unsaturated carbonyl compound (2) was dissolved in a 250mM boric acid buffer solution having a pH of 9.4 to prepare a 1mM concentration solution (20. Mu.L, 20 nmol), ethyl amidine (8000nmol, 400 equiv, 400mM DMSO), naOH (16000nmol, 800 equiv, 1000mM double distilled water) was added to the solution and mixed uniformly, and after reacting at 30 ℃ for 4 hours, elemental iodine (2000nmol, 100 equiv, 200mM DMSO) was added to the system and mixed uniformly, and the reaction was carried out at 30 ℃ for 2 hours.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the reacted solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the total volume, after uniformly oscillating, placing the reaction in dry ice for freezing for 0.5 hour, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out the supernatant, dissolving the rest precipitate with deionized water to obtain a solution of an On-DNA product, and after quantifying by an enzyme labeling instrument OD, sending LCMS to confirm that the conversion rate of the reaction is 80%.
Example 2 Synthesis of On-DNA pyrimidine Compounds
Step 1, synthesis of On-DNA alpha, beta-unsaturated carbonyl compound
Figure BDA0002710536470000081
On-DNA arylethanones (1) were dissolved in 250mM boric acid buffer pH =9.4 to prepare a 1mM concentration solution (20. Mu.L, 20 nmol), benzaldehyde (4000nmol, 200 equivalents, 200mM DMSO), and sodium hydroxide (10000nmol, 500 equivalents, 500mM double distilled water) were sequentially added to the solution, and the mixture was mixed well and reacted at 30 ℃ for 2 hours.
And (3) after the reaction is finished, carrying out ethanol precipitation: adding a 5M sodium chloride solution with the total volume of 10% into the solution after the reaction, then continuously adding anhydrous ethanol with the volume of 3 times of the total volume, after uniformly oscillating, placing the reaction into dry ice for freezing for 0.5 hour, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out the supernatant, dissolving the rest precipitate with deionized water to obtain a solution of the On-DNA alpha, beta-unsaturated carbonyl compound (2), quantifying by an enzyme labeling instrument OD, and sending LCMS to confirm that the conversion rate of the reaction is 90%. Step 2, synthesis of On-DNA pyrimidine compound
Figure BDA0002710536470000082
The On-DNA α, β -unsaturated carbonyl compound (2) was dissolved in a 250mM boric acid buffer solution having a pH of =9.4 to prepare a 1mM concentration solution (20. Mu.L, 20 nmol), and phenylamidine (8000nmol, 400 equivalents, 400mM DMSO), naOH (10000nmol, 500 equivalents, 1000mM double distilled water) was added to the solution to mix well, and after reacting at 80 ℃ for 2 hours, elemental iodine (2000nmol, 100 equivalents, 200mM THF) was added to the system to mix well, and reacting at 30 ℃ for 1 hour.
And (3) after the reaction is finished, carrying out ethanol precipitation: and adding a 5M sodium chloride solution accounting for 10% of the total volume of the solution after the reaction, continuously adding absolute ethyl alcohol accounting for 3 times of the total volume of the solution, uniformly oscillating, placing the reaction in dry ice for freezing for 0.5 hour, centrifuging at the rotating speed of 12000rpm for half an hour, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain a solution of an On-DNA product, quantifying by an enzyme labeling instrument OD, and sending to LCMS to confirm that the reaction conversion rate is 80%.
Example 3 Synthesis of On-DNA pyrimidine Compounds
Figure BDA0002710536470000091
21 On-DNA α, β -unsaturated carbonyl compounds were dissolved in 250mM boric acid buffer solution having a pH of =9.4, respectively, to prepare 1mM concentration solutions (20. Mu.L, 20 nmol), phenylamidine (8000nmol, 400 equivalents, 400mM DMSO), naOH (10000nmol, 500 equivalents, 1000mM double distilled water) were added to the solutions, and mixed uniformly, and after reacting at 80 ℃ for 2 hours, elemental iodine (2000nmol, 100 equivalents, 200mM THF) was added to the system, and mixed uniformly, and reacted at 30 ℃ for 1 hour.
And (3) performing ethanol precipitation after the reaction is finished: and adding a 5M sodium chloride solution accounting for 10% of the total volume of the solution after the reaction, continuously adding absolute ethyl alcohol accounting for 3 times of the total volume of the solution, uniformly oscillating, placing the reaction in dry ice for freezing for 0.5 hour, centrifuging for half an hour at the rotating speed of 12000rpm, pouring out supernatant, dissolving the rest precipitate with deionized water to obtain a solution of an On-DNA product, quantifying by an enzyme labeling instrument OD (optical density) and sending to LCMS (liquid crystal display system) to confirm the conversion rate of the reaction.
As described above, the present invention can obtain an On-DNA pyrimidine compound by reacting an On-DNA α, β -unsaturated carbonyl compound with an amidino compound in the presence of a base and an oxidizing agent while controlling the conditions such as solvent, temperature, pH and the like during the reaction. The method has wide substrate application range, can be carried out in the mixed aqueous phase of an organic solvent/aqueous phase, has simple operation, does not introduce a metal reagent, is environment-friendly, and is suitable for synthesizing a DNA coding compound library by using a porous plate.

Claims (9)

1. A kind ofA method of synthesizing an On-DNA pyrimidine compound, characterized in that: the method takes an On-DNA alpha, beta-unsaturated carbonyl compound and an amidino compound as raw materials, and obtains an On-DNA product by reacting in the presence of alkali and iodine simple substances; wherein, the structural formula of the On-DNA alpha, beta-unsaturated carbonyl compound is shown in the specification
Figure FDA0004040204460000011
The amidine compound has the structural formula of->
Figure FDA0004040204460000012
Wherein the DNA in the structural formula comprises a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and/or unmodified nucleotide monomers, and the nucleotide chain is connected with the rest part in the compound through one or more chemical bonds or groups;
R 1 selected from the group consisting of groups having a molecular weight of 1000 or less which are directly attached to the DNA and carbonyl carbon atoms or are absent;
R 2 selected from groups having a molecular weight of 1000 or less directly bonded to an alkenyl carbon atom;
R 3 selected from the group consisting of groups having a molecular weight of 1000 or less directly attached to the DNA and to the alkenyl carbon atom or are absent;
R 4 selected from groups having a molecular weight below 1000 directly attached to the carbonyl carbon atom;
R 5 selected from hydrogen or a group having a molecular weight of 1000 or less directly bonded to an alkenyl carbon atom;
R 6 selected from groups having a molecular weight of 1000 or less directly attached to an amidino carbon atom;
or R 5 Are each independently of R 1 、R 2 、R 3 Or R 4 Looping;
the method comprises the following steps: dissolving an On-DNA alpha, beta-unsaturated carbonyl compound into a boric acid buffer solution with pH =9.4, adding 10-1000 times molar equivalent of an amidino compound and 10-1000 times molar equivalent of a base into an On-DNA alpha, beta-unsaturated carbonyl compound solution with molar equivalent of 1 and molar concentration of 0.5-5mM, and finally adding 10-500 times molar equivalent of an iodine simple substance to react at 10-100 ℃ for 0.5-24 hours.
2. The method of claim 1, wherein: r is as described 1 、R 2 、R 3 、R 4 Are respectively selected from alkyl, substituted alkyl, carboxyl, 5-10-membered aryl, substituted 5-10-membered aryl, 5-10-membered aromatic heterocyclic group and substituted 5-10-membered aromatic heterocyclic group; wherein the alkyl is C 1 ～C 20 Alkyl or C 3 ～C 8 A cycloalkyl group; the number of substituents of the substituted alkyl group is one or more; the substituent of the substituted alkyl is one or more independently selected from halogen, carboxyl, nitro, alkoxy, halogenated phenyl, alkyl phenyl and heterocyclic radical; the number of the substituent for substituting the 5-to 10-membered aryl is one or more, and the substituents for substituting the 5-to 10-membered aryl are independently selected from halogen, cyano, nitro, carboxyl, alkoxy and C 1 ～C 20 One or more of alkyl and trifluoromethyl; the number of the substituent(s) for substituting the 5-to 10-membered aromatic heterocyclic group is one or more, and the substituent(s) for substituting the 5-to 10-membered aromatic heterocyclic group are independently selected from the group consisting of halogen, cyano, nitro, carboxyl, alkoxy, C 1 ～C 20 One or more of alkyl and trifluoromethyl;
the R is 5 Selected from hydrogen, C 1 ～C 20 An alkyl group;
the R is 6 Is selected from C 1 ～C 20 Alkyl or phenyl.
3. The method of claim 1, wherein: the base is selected from the group consisting of sodium borate, lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, sodium phosphate, potassium phosphate, sodium hydrogen phosphate, potassium hydrogen phosphate, N-methylmorpholine, triethylamine, diisopropylethylamine, 1, 8-diazabicycloundecen-7-ene, 4-dimethylaminopyridine, 2, 6-dimethylpyridine, N-methylimidazole.
4. The method of claim 1, wherein: the reaction is carried out in a solvent, and the solvent is a water-containing mixed solvent of any one or more of water, methanol, ethanol, acetonitrile, dimethyl sulfoxide, N-dimethylformamide, N-dimethylacetamide, an inorganic salt buffer solution, an organic acid buffer solution and an organic base buffer solution.
5. The method of claim 1, wherein: the reaction temperature of the reaction is 20 ℃,30 ℃,40 ℃,50 ℃ or 60 ℃.
6. The method of claim 1, wherein: the reaction time of the reaction is 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, or 16 hours.
7. The method of claim 1, wherein: in the method, the molar equivalent of the On-DNA alpha, beta-unsaturated carbonyl compound is 1, and the molar equivalent of the amidino compound is 50, 100, 200, 300, 400, 500, 600, 800 or 1000; the molar equivalent of the base is 50 equivalents, 100 equivalents, 200 equivalents, 300 equivalents, 400 equivalents, 500 equivalents, 600 equivalents, 800 equivalents, 1000 equivalents; the molar equivalents of the iodine are 20 equivalents, 60 equivalents, 80 equivalents, 100 equivalents, 200 equivalents and 400 equivalents.
8. The method according to any one of claims 1 to 7, wherein the method is used for multi-well plate operations in batches.
9. The method of any one of claims 1 to 7, wherein the method is used for the synthesis of libraries of DNA encoding compounds for multi-well plates.
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