CN112794874A - Synthesis method of On-DNA Petasis reaction - Google Patents
Synthesis method of On-DNA Petasis reaction Download PDFInfo
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Abstract
The invention relates to a synthetic method of an On-DNA Petasis reaction, which takes an On-DNA amino compound as a substrate to react with alpha substituted aldehyde and boric acid/boric acid ester compound to obtain an On-DNA product. The method can be carried out in the mixed water phase of the organic solvent and the water phase, and has simple post-treatment and environment-friendly effect; and alpha substituted aldehyde and boric acid/borate compounds can be introduced in large scale as synthesis modules, highly diverse DNA coding compounds can be obtained in a short time and high yield, and the method is suitable for operation by using a large-scale multi-well plate.
Description
Technical Field
The invention belongs to the technical field of coding compound libraries, and particularly relates to a synthetic method of an On-DNA Petasis reaction 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 DNA-encoded compound library technologies (WO2005058479, WO2018166532, CN103882532) combine the technologies of combinatorial chemistry and molecular biology, add a DNA tag to each compound on the molecular level, and synthesize up to hundred million levels of compound libraries in a very short time, which is a trend of the next generation compound library screening technology, and begin to be widely applied in the pharmaceutical industry, resulting in many positive effects (Accounts of Chemical Research,2014,47, 1247-.
The DNA coding compound library can rapidly generate a giant compound library through combinatorial chemistry, and can screen out a lead compound with high flux, 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.
The carboxyl alpha-amino substituted compound has an important skeleton structure of a pharmaceutical compound, and the introduction of the carboxyl alpha-amino substituted compound skeleton into a DNA coding compound library can further expand the diversity of the compound library and is beneficial to improving the probability of screening effective compounds. However, no method for constructing the On-DNA carboxyl alpha-amino substituted compound by the On-DNA amino compound is reported at present. Therefore, a new method for synthesizing the On-DNA carboxyl alpha-amino substituted compound suitable for constructing a large-batch porous-plate DNA coding compound library is hoped to be developed, alpha-substituted aldehyde and boric acid/boric acid ester compounds are introduced into the method as a synthesis module in a large scale, the diversity of the DNA coding compound library is obviously increased, and the application value of the DNA coding compound library technology is further improved.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a synthesis method of an On-DNA Petasis reaction, which has the advantages of mild reaction conditions, no use of metal catalysts, high reaction selectivity, high yield, simple post-treatment, suitability for the production of DNA coding compound libraries and capability of obviously improving the diversity of molecules of the compound libraries.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a synthetic method of On-DNA Petasis reaction, said reaction regards On-DNA amidocyanogen compound as the substrate, react with alpha substituted aldehyde, boric acid/boric acid ester compound to get On-DNA product; wherein the structural formula of the On-DNA amino compound is DNA-R1-NH-R2(ii) a The structural formula of the alpha-substituted aldehyde is R3-CHO; the boric acid/boric acid ester compound has the structural formula R4-B(OR5)(OR6) (ii) a The structural formula of the On-DNA product is as follows:
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 R in the compound through one or more chemical bonds or groups1Connecting;
wherein, the DNA and R in the structural formula1Linked by a chemical bond or multiple chemical bonds or groups; when a chemical bond is present, it means DNA and R in the structural formula1Directly connecting; when there are a plurality of chemical bonds or groups, it means DNA and R in the structural formula1Are connected with a plurality of chemical bonds at intervals, for example, DNA and R1Through a methylene group (-CH)2-) are linked, i.e. linked by two chemical bonds; or DNA and R1The 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 and R1Through a methylene carbonyl group (-CH)2CO-) is attached to the amino group of the DNA, again by three consecutive chemical bonds.
R1Selected from the group consisting of groups having a molecular weight of 1000 or less directly linked to the amino nitrogen atom and the DNA;
R2selected from hydrogen or a radical having a molecular weight of 1000 or less directly attached to the nitrogen atom of the amino group, or R1、R2Connecting to form a ring;
R3selected from groups having a molecular weight of 1000 or less directly bonded to an aldehyde carbon atom;
R4selected from groups having a molecular weight of 1000 or less which are directly attached to the boron atom in the boronic acid/boronic ester;
R5、R6each independently selected from hydrogen or a group with the molecular weight of below 1000 and directly connected with an oxygen atom in the borate; or R5、R6Are connected to form a ring.
Preferably, the method comprises the following steps: said R1、R2Each independently selected from C1～C10Alkyl, substituted C1～C10Alkyl, substituted C1～C10The number of substituents of the alkyl group being one or more, substituted C1～C10The substituents of the alkyl are mutually independent and are selected from 5-10-membered aryl, hydroxyl or C1～C6An alkoxyphenyl group.
Further, R1、R2Each independently selected from C1～C6Alkyl, substituted C1～C6Alkyl radical ofC1～C6The alkyl is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl and hexyl; substituted C1～C6The substituent of the alkyl group is selected from phenyl or naphthyl;
preferably, the method comprises the following steps: said R1、R2Are connected to form a 3-8 membered saturated heterocyclic ring; or R1、R2Are connected to form 5-12 membered saturated spiroheterocyclic ring and substituted 5-12 membered saturated spiroheterocyclic ring; the number of the substituents for substituting the 5-to 12-membered saturated spiroheterocycle is one or more, and the substituents for substituting the 5-to 12-membered saturated spiroheterocycle are oxygen atoms.
Preferably, the On-DNA amino compound is specifically selected from the following structures:
said R3Selected from the group consisting of carboxyl, -C (O) R7OR-C (O) OR7Said R is7Is selected from C1～C10A linear or branched alkyl group, preferably, said R7Is selected from C1～C6The alkyl group is specifically selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl and hexyl.
Preferably, the alpha substituted aldehyde is selected from glyoxylic acid.
Preferably, R is4Selected from 5-10 membered aromatic heterocyclic group, substituted 5-10 membered aromatic heterocyclic group, 5-10 membered aryl group, substituted 5-10 membered aryl group, C2～C10Alkenyl radicals, substituted C2～C10An alkenyl group;
the number of the substituents for substituting the 5-to 10-membered aromatic heterocyclic group is one or more, and the substituents for substituting the 5-to 10-membered aromatic heterocyclic group are independently selected from C1～C10Alkyl, 3-8 membered saturated heterocyclyl substituted C1～C10Alkyl, oxo 3-8 membered saturated heterocyclyl substituted C1～C10Alkyl radical, C1～C6Alkoxy-substituted C1～C10Alkyl, 3-8 membered saturated heteroCyclic group, 3-to 8-membered saturated cycloalkyl group, oxygen atom, C1～C6One or more of alkoxy and phenyl;
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 mutually independent and selected from C1～C6Alkyl radical, C1～C6An alkoxy group; said C is1～C6Alkoxy is selected from methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, hexoxy;
substituted C2～C10The number of substituents of the alkenyl group being one or more, substituted C2～C10The substituents of the alkenyl are independently selected from phenyl and C1～C6Alkylphenyl, halogen, C1～C6Alkyl or C1～C6An alkoxy group;
said R5、R6Each independently selected from hydrogen or C1～C10A linear or branched alkyl group; further, R5、R6Each independently selected from C1～C6Straight-chain or branched alkyl, in particular selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl; or R5、R6Are connected to form a ring.
Preferably, the boronic acid compound is specifically selected from the following structures:
an On-DNA Petasis reaction method, comprising the following steps: (1) adding 5-500 times of molar equivalent of alpha substituted aldehyde into an On-DNA amino compound solution with the molar equivalent of 1 and the molar concentration of 0.5-5mM, reacting at 20-100 ℃ for 0.5-24 hours, adding 5-100 times of molar equivalent of boric acid/boric acid ester compound, and reacting at 20-100 ℃ for 0.5-24 hours until the reaction is finished.
Preferably, the reaction time is 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours or 12 hours.
Preferably, the reaction is carried out in a solvent, wherein the solvent is one or a mixture of water in acetonitrile, methanol, ethanol, DMF, DMA, DMSO, THF, water, inorganic salt buffer solution, organic acid buffer solution and organic base buffer solution; further, the solvent was a borate buffer containing DMA, and the pH of the buffer was 9.4.
Preferably, the reaction temperature is 20 ℃, 30 ℃, 40 ℃, 50 ℃,60 ℃, 70 ℃, 80 ℃ or 90 ℃.
Preferably, the boric acid/borate compound has a molar equivalent of 10 equivalents, 30 equivalents, 50 equivalents, 70 equivalents, 90 equivalents, or 100 equivalents, and the alpha-substituted aldehyde has a molar equivalent of 100 equivalents, 200 equivalents, 300 equivalents, 400 equivalents, or 500 equivalents.
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 construction of the On-DNA carboxyl alpha-amino substituted compound in a DNA coding compound library through a Petasis reaction, and can introduce alpha substituted aldehyde and boric acid/boric acid ester compounds as synthesis modules in a large scale. The method has the advantages of high yield, single product, simple post-treatment, environmental protection, suitability for large-scale multi-plate operation and provision of a new framework for a DNA coding compound library.
In the preferred embodiment of the invention, by controlling the solvent and temperature of the reaction, the generation of byproducts can be inhibited, and the accuracy of the method applied to library construction of the DNA coding compounds is improved.
Definitions of terms used in connection with 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 given to them by a person skilled in the art are to be given in light of the disclosure and the context.
"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, C1～C20The alkyl group is a straight-chain or branched alkyl group having 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-CH3ethyl-CH2CH3(ii) a The alkyl group may also be part of another group, such as a C1-C6 alkoxy group.
Alkoxy groups: means that the alkyl radical is bound to an oxygen atom to form a substituent, e.g. methoxy is-OCH3。
3-to 8-membered saturated cycloalkyl: refers to saturated cyclic groups having multiple carbon atoms and no ring heteroatoms and having a single ring or multiple rings (including fused, bridged, and spiro ring systems).
3-8 membered saturated heterocycle/group: is a monocyclic or polycyclic or monocyclic or polycyclic hydrocarbon group of 3 to 8 atoms carrying at least one atom selected from O, S, N, and no double bond is present in any position.
R1、R2Linked to form a 3-8 membered saturated heterocyclic ring: is denoted by R1、R2Are connected with N atoms at the ortho positions to form a 3-8 membered saturated heterocyclic ring.
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 is a single cyclic group or a plurality of cyclic groups having aromaticity and comprising 5 to 10 atoms of C, O, S, N and the like.
5-12 membered saturated spiroheterocycle: it means a spiro ring comprising 5 to 12 atoms such as C, O, S, N, and no double bond is present at any position.
Alkenyl: including straight or branched alkenyl groups.
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: the conversion rates of the 26 On-DNA nitrogen heterocyclic compounds obtained in the embodiment 3 of the invention are corresponding.
Detailed Description
The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.
The embodiment of the invention is abbreviated as follows: DMA: dimethylacetamide (Dimethylacetamide); DMF: dimethylformamide; DMSO, DMSO: dimethyl sulfoxide; THF: tetrahydrofuran; HATU: 2- (7-azabenzotriazole) -N, N' -tetramethyluronium hexafluorophosphate; DIPEA: n, N-diisopropylethylamine.
In the invention, the term "room temperature" means 20-25 ℃.
DNA-NH in the present invention2Is formed by single-stranded or double-stranded DNA and a linker group and has-NH2DNA constructs for linkers, e.g. DNA-NH of "compound 1" in WO20050584792And (5) structure.
Example 1 Synthesis of On-DNA Compound substituted by amino group at carboxyl position
Carboxylic acid-containing amine compound 1b (containing Fmoc protecting group) (40 equivalents, 200mM in DMA solution), HATU (100 equivalents, 200mM in DMA solution) and DIPEA (100 equivalents, 200mM in DMA solution) were activated at-20 ℃ for 5 minutes, respectively, mixed together in an equivalent ratio and then activated at 5 ℃ for 5 minutes, and DNA-NH was added2Dissolved in borate buffer (0.25M, pH 9.4) to prepare a 1mM solution, which is added to the activated mixtureMixing the mixture evenly, and reacting for 2 hours at room temperature;
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, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out a supernatant, and freeze-drying to obtain an On-DNA amino compound (containing Fmoc protecting group) compound 1c which is directly used for the next reaction.
Dissolving a compound containing an On-DNA amino compound 1c (containing Fmoc protecting group) in deionized water to prepare a 1mM concentration solution (10 muL, 10nmol), adding piperidine with the volume of 10 percent (1 muL) of the solution, uniformly mixing, and reacting at room temperature for 1 hour;
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, then continuously adding anhydrous ethanol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out a supernatant, dissolving the rest precipitate with deionized water to obtain a solution of an On-DNA amino compound, and sending LCMS to confirm that the conversion rate of the reaction is 95%.
On-DNA amino compound 1d was dissolved in borate buffer (0.25M, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), glyoxylic acid (100 equivalents, 400mM DMA solution) was added to the solution, and the mixture was reacted at 60 ℃ for 30 minutes, followed by adding phenylboronic acid (100 equivalents, 600mM DMA solution), mixing the mixture uniformly, and reacting the mixture at 90 ℃ for 6 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, then continuously adding anhydrous ethanol with the total volume of 3 times of the solution, after uniform oscillation, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out a supernatant, dissolving the rest precipitate with deionized water to obtain a solution of an On-DNA product 1e, and sending LCMS to confirm that the conversion rate of the reaction is 65%.
Example 2 Synthesis of On-DNA carboxy alpha-amino substituted Compound
Carboxylic acid-containing amine compound 2b (containing Fmoc protecting group) (40 equivalents, 200mM in DMA solution), HATU (100 equivalents, 200mM in DMA solution) and DIPEA (100 equivalents, 200mM in DMA solution) were activated at-20 ℃ for 5 minutes, respectively, mixed together in an equivalent ratio and then activated at 5 ℃ for 5 minutes, and DNA-NH was added2Dissolved in borate buffer (0.25M, pH 9.4) to prepare a 1mM concentration solution, added to the activated mixture, mixed well and reacted at room temperature 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, then continuously adding absolute ethyl alcohol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out the supernatant, and freeze-drying to obtain an On-DNA amino compound (containing Fmoc protecting group) compound which is directly used for the next reaction.
Dissolving a compound containing an On-DNA amino compound 2c (containing Fmoc protecting group) in deionized water to prepare a 1mM concentration solution (10 muL, 10nmol), adding piperidine with the volume of 10 percent (1 muL) of the solution, uniformly mixing, and reacting at room temperature for 1 hour;
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, then continuously adding anhydrous ethanol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out a supernatant, dissolving the rest precipitate with deionized water to obtain a solution of an On-DNA amino compound, and sending LCMS to confirm that the conversion rate of the reaction is 95%.
Dissolving the On-DNA amino compound 2d in borate buffer (0.25M, pH 9.4) to prepare a 1mM concentration solution (10 μ L, 10nmol), adding glyoxylic acid (X equivalent, 400mM DMA solution) and NaOH (Y equivalent, 1M aqueous solution) to the solution in this order, mixing well, reacting at 60 ℃ for 1 hour, adding boric acid compound 2e (100 equivalent, 600mM DMA solution), mixing well, and reacting at 90 ℃ for 16 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, then continuously adding anhydrous ethanol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out a supernatant, dissolving the rest precipitate with deionized water to obtain a solution of an On-DNA product, and sending to LCMS to confirm that the conversion rate of the reaction is the same.
| Condition | X (equivalent) | Y (equivalent) | Conversion rate |
| 1 | 40 | 0 | 14％ |
| 2 | 80 | 0 | 46％ |
| 3 | 120 | 0 | 79％ |
| 4 | 140 | 0 | 60％ |
| 5 | 160 | 0 | 0 |
| 6 | 120 | 20 | 36％ |
Example 3 Synthesis of On-DNA carboxy alpha-amino substituted Compound
26 On-DNA amino compounds were prepared as described in example 1.
26 On-DNA amino compounds were dissolved in borate buffer (0.25M, pH 9.4) to prepare 1mM concentrated solutions (10 μ L, 10nmol), glyoxylic acid (120 equivalents, 400mM DMA solution) was added to the solutions, the mixture was mixed well, and reacted at 60 ℃ for 1 hour, followed by adding boric acid compound (100 equivalents, 600mM DMA solution) and mixing well, and reacted at 90 ℃ for 16 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, then continuously adding anhydrous ethanol with the total volume of 3 times of the solution, after uniformly oscillating, placing the reaction in dry ice for freezing for 2 hours, then centrifuging for half an hour at the rotating speed of 12000rpm, pouring out a supernatant, dissolving the rest precipitate with deionized water to obtain 26 solutions of On-DNA products, and sending the solutions to LCMS to confirm the conversion rate of the reaction.
In conclusion, the method can obtain the On-DNA carboxyl alpha-amino substituted compound by controlling the conditions of solvent, temperature, pH and the like during reaction and using the On-DNA amino compound, glyoxylic acid and aryl boric acid/boric acid ester compound without a metal catalyst. The method can be carried out in the mixed water phase of the organic solvent and the water phase, and has simple post-treatment and environment-friendly effect; and the alpha-substituted aldehyde and the boric acid/borate compound can be introduced as synthesis modules in a large scale, a highly diverse DNA coding compound library can be obtained in a short time and at a high yield, and the method is suitable for operation using a large number of multi-well plates.
Claims (10)
1. A synthetic method of On-DNA Petasis reaction is characterized in that: the reaction takes an On-DNA amino compound as a substrate, and the On-DNA amino compound reacts with alpha substituted aldehyde and boric acid/boric acid ester compound to obtain an On-DNA product; wherein the structural formula of the On-DNA amino compound is DNA-R1-NH-R2(ii) a The structural formula of the alpha-substituted aldehyde is R3-CHO; the boric acid/boric acid ester compound has the structural formula R4-B(OR5)(OR6) (ii) a The structural formula of the On-DNA product is as follows:
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 R in the compound through one or more chemical bonds or groups1Connecting;
R1selected from the group consisting of groups having a molecular weight of 1000 or less directly linked to the amino nitrogen atom and the DNA;
R2selected from hydrogen or a radical having a molecular weight of 1000 or less directly attached to the nitrogen atom of the amino group, or R1、R2Connecting to form a ring;
R3selected from groups having a molecular weight of 1000 or less directly bonded to an aldehyde carbon atom;
R4selected from groups having a molecular weight of 1000 or less which are directly attached to the boron atom in the boronic acid/boronic ester;
R5、R6each independently selected from hydrogen or a group with the molecular weight of below 1000 and directly connected with an oxygen atom in the borate; or R5、R6Are connected to form a ring.
2. The method of claim 1, wherein: r1、R2Each independently selected from C1～C10Alkyl, substituted C1～C10Alkyl, substituted C1～C10The number of substituents of the alkyl group being one or more, substituted C1～C10The substituents of the alkyl are mutually independent and are selected from 5-10-membered aryl, hydroxyl or C1～C6An alkoxyphenyl group;
or R1、R2Are connected to form a 3-8 membered saturated heterocyclic ring; or R1、R2Are connected to form 5-12 membered saturated spiroheterocyclic ring and substituted 5-12 membered saturated spiroheterocyclic ring; the number of the substituent for substituting the 5-12 membered saturated spiroheterocycle is one or more, and the substituent for substituting the 5-12 membered saturated spiroheterocycle is an oxygen atom;
R3selected from the group consisting of carboxyl, -C (O) R7OR-C (O) OR7Said R is7Is selected from C1～C10A linear or branched alkyl group;
R4selected from 5-10 membered aromatic heterocyclic group, substituted 5-10 membered aromatic heterocyclic group, 5-10 membered aryl group, substituted 5-10 membered aryl group, C2～C10Alkenyl, substituted C2～C10An alkenyl group;
the number of the substituent group for substituting the 5-to 10-membered aromatic heterocyclic group is one or more, and the substituent groups for substituting the 5-to 10-membered aromatic heterocyclic group are mutually independent and selected from C1～C10Alkyl, 3-8 membered saturated heterocyclyl substituted C1～C10Alkyl, oxo 3-8 membered saturated heterocyclyl substituted C1～C10Alkyl radical, C1～C6Alkoxy-substituted C1～C10Alkyl, 3-8 membered saturated heterocyclic group, 3-8 membered saturated cycloalkyl group, oxygen atom, C1～C6One or more of alkoxy and phenyl;
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 mutually independent and selected from C1～C6Alkyl radical, C1～C6An alkoxy group;
said substitution C2～C10The number of substituents of the alkenyl group being one or more, substituted C2～C10The substituents of the alkenyl are independently selected from phenyl and C1～C6Alkylphenyl, halogen, C1～C6Alkyl or C1～C6An alkoxy group;
R5、R6each independently selected from C1～C10A linear or branched alkyl group; or R5、R6Are connected to form a ring.
3. The method of claim 1, wherein: the method comprises the following steps: (1) adding 5-500 times of molar equivalent of alpha substituted aldehyde into an On-DNA amino compound solution with the molar equivalent of 1 and the molar concentration of 0.5-5mM, reacting at 20-100 ℃ for 0.5-24 hours, adding 5-100 times of molar equivalent of boric acid/boric acid ester compound, and reacting at 20-100 ℃ for 0.5-24 hours until the reaction is finished.
4. The method of claim 3, wherein: the reaction time is 0.5 hour, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, or 12 hours.
5. The method of claim 3, wherein: the reaction is carried out in a solvent, wherein the solvent is one or a mixture of water in acetonitrile, methanol, ethanol, DMF, DMA, DMSO, THF, water, inorganic salt buffer solution, organic acid buffer solution and organic base buffer solution.
6. The method of claim 5, wherein: the solvent was a borate buffer containing DMA, the pH of which was 9.4.
7. The method of claim 3, wherein: the reaction temperature is 20 ℃, 30 ℃, 40 ℃, 50 ℃,60 ℃, 70 ℃, 80 ℃ or 90 ℃.
8. The method of claim 3, wherein: in the reaction, the molar equivalent of the On-DNA amino compound is 1, the molar equivalent of the boric acid/borate compound is 10 equivalents, 30 equivalents, 50 equivalents, 70 equivalents, 90 equivalents or 100 equivalents, and the molar equivalent of the alpha-substituted aldehyde is 100 equivalents, 200 equivalents, 300 equivalents, 400 equivalents or 500 equivalents.
9. The method according to any one of claims 1 to 8, wherein: the method is used for batch multi-well plate operations.
10. The method according to any one of claims 1 to 8, wherein: the method is used for the synthesis of libraries of DNA-encoding compounds for multi-well plates.
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