CN112941637A - On-DNA Aldol reaction method - Google Patents

On-DNA Aldol reaction method Download PDF

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CN112941637A
CN112941637A CN202011426217.0A CN202011426217A CN112941637A CN 112941637 A CN112941637 A CN 112941637A CN 202011426217 A CN202011426217 A CN 202011426217A CN 112941637 A CN112941637 A CN 112941637A
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equivalents
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李进
成坤朋
陈柳
蔡品文
刘观赛
马慧勇
万金桥
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Hitgen Inc
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/08—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
    • C40B50/10—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support involving encoding steps
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/10—Methods of screening libraries by measuring physical properties, e.g. mass
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries

Abstract

The invention relates to an On-DNA Aldol reaction method, which takes an On-DNA aldehyde compound and an alpha hydrogen aldehyde group or ketone group compound as raw materials to obtain an On-DNA product in the presence of a catalyst. The method can be carried out in the mixed aqueous phase of an organic solvent and an aqueous phase, is simple to operate, does not introduce a metal reagent, is environment-friendly, and is suitable for synthesizing a DNA coding compound library by using a porous plate.

Description

On-DNA Aldol reaction method
Technical Field
The invention belongs to the technical field of coding compound libraries, and particularly relates to an On-DNA Aldol reaction method 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 aldol or hydroxyketone compound is an important medicine compound framework or intermediate structure, and the introduction of the aldol or hydroxyketone compound framework 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 On-DNA aldol or hydroxyketone compounds from On-DNA aldehyde compounds has been reported. Therefore, it is desired to develop a new method for synthesizing On-DNA aldol or hydroxyketone compounds suitable for large-scale multi-well plate operation, increase the diversity of DNA coding compound libraries, and further improve the application value of the DNA coding compound library technology.
Disclosure of Invention
The invention provides an On-DNA Aldol reaction method, which has mild reaction conditions and simple post-treatment, is suitable for the production of a DNA coding compound library and can obviously improve the molecular diversity of the compound library.
The invention provides a method for On-DNA Aldol reaction, which takes an On-DNA aldehyde compound and an alpha hydrogen aldehyde group or ketone group compound as raw materials to obtain an On-DNA product in the presence of a catalyst; wherein the structural formula of the On-DNA aldehyde compound is DNA-R3-CHO, the structural formula of the alpha hydrogen aldehyde group or ketone group compound is
Figure BDA0002825395720000021
The structural formula of the On-DNA product is shown in the specification
Figure BDA0002825395720000022
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 through one or more chemical bonds or groups3Connecting; the length of the DNA is 10-200 bases.
Wherein, the DNA and R in the structural formula3Linked by a chemical bond or multiple chemical bonds or groups; when a chemical bond is present, it means DNA and R in the structural formula3Directly connecting; when there are a plurality of chemical bonds or groups, it means DNA and R in the structural formula3Are connected with a plurality of chemical bonds at intervals, for example, DNA and R3Through a methylene group (-CH)2-) are connected, i.e. by twoA plurality of chemical bonds; or DNA and R3The 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 R3Through a methylene carbonyl group (-CH)2CO-) is attached to the amino group of the DNA, i.e., by three consecutive chemical bonds.
R1Selected from hydrogen or a group with molecular weight below 1000 directly connected with carbon atom at alpha position of carbonyl;
R2selected from hydrogen or a group having a molecular weight of 1000 or less directly bonded to the carbonyl carbon atom;
R3selected from the group consisting of groups having a molecular weight of 1000 or less directly linked to DNA and an aldehyde carbon atom or being absent;
R4selected from hydrogen or a group with molecular weight below 1000 directly connected with carbon atom at alpha position of carbonyl;
or R1And R4Looping; or R2Are each independently of R1Or R4Looping;
preferably, R is1、R2、R3、R4Each independently selected from alkyl, substituted alkyl, hydroxy, C1～C20Alkoxy, 5-10-membered aryl, substituted 5-10-membered aryl, 5-10-membered aromatic heterocyclic group, substituted 5-10-membered aromatic heterocyclic group; wherein the alkyl is C1～C20Alkyl or C3～C8A cycloalkyl group; the number of substituents of the substituted alkyl group is one or more; the substituents of the substituted alkyl are independently selected from hydroxyl, halogen, carboxyl, nitro and C1～C20Alkoxy, halophenyl, phenyl, alkylphenyl, C1～C20One or more of alkoxyphenyl, 5-10 membered aromatic heterocyclic group, and carboxyl-substituted 5-10 membered aromatic heterocyclic group; the number of the substituent for substituting the 5-to 10-membered aryl is one or more, and the substituent for substituting the 5-to 10-membered aryl is independently selected from hydroxyl, halogen, cyano, nitro, carboxyl and C1～C20Alkoxy radical, C1～C20One or more of alkyl and trifluoromethyl; the number of the substituents for substituting the 5-to 10-membered aromatic heterocyclic group is one or more, and the 5-to 10-membered aromatic heterocyclic group is substitutedThe substituents are independently selected from halogen, cyano, nitro, carboxyl and C1～C20Alkoxy radical, C1～C20One or more of alkyl and trifluoromethyl.
More specifically, R is3Selected from phenyl, substituted phenyl, pyridyl, furyl; the substituent of the substituted phenyl is selected from C1～C6Alkoxy, halogen substituted C1～C6Alkoxy, halogen; said C is1～C6Alkoxy is selected from methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, hexoxy.
The On-DNA aldehyde compound is specifically selected from
Figure BDA0002825395720000031
Figure BDA0002825395720000032
The alpha-hydrogen aldehyde or ketone compound is specifically selected from
Figure BDA0002825395720000033
Figure BDA0002825395720000034
Another object of the present invention is to provide a method of On-DNA Aldol reaction comprising the steps of: adding 1-50 times of molar equivalent of catalyst into an On-DNA aldehyde compound solution with the molar equivalent of 1 and the molar concentration of 0.5-5mM, then adding 10-1000 times of molar equivalent of alpha hydrogen aldehyde or ketone compound, and reacting for 0.5-24 hours at 10-100 ℃.
Further, the catalyst is proline methyl ester, proline, pyrrolidine or piperidine; preferably, the catalyst is proline.
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, an inorganic salt buffer solution, an organic acid buffer solution and an organic base buffer solution; preferably, the reaction solvent contains a boric acid buffer.
Furthermore, the pH value of the boric acid buffer solution is 8-10; preferably, the pH is 9.4.
Further, in the reaction method, the molar equivalent of the On-DNA aldehyde-based compound is 1, the molar equivalent of the alpha hydrogen aldehyde-based or ketone-based compound is 20 equivalents, 50 equivalents, 100 equivalents, 200 equivalents, 400 equivalents, 800 equivalents, and the molar equivalent of the catalyst is 2 equivalents, 5 equivalents, 10 equivalents, 20 equivalents, 30 equivalents, 40 equivalents.
Further, the reaction temperature of the reaction is 20 ℃, 30 ℃, 40 ℃, 50 ℃ or 60 ℃.
Further, the reaction time of the reaction is 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, or 8 hours.
Furthermore, the feeding sequence of the reaction is that the On-DNA aldehyde group compound is added firstly, then the catalyst is added, and finally the alpha hydrogen aldehyde group or ketone group compound is added.
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 Aldol or hydroxyketone compound in the DNA coding compound library through Aldol reaction and is suitable for various aldehyde and ketone substrates. The method can be carried out in the mixed aqueous phase of an organic solvent and an aqueous phase, is simple to operate, does not introduce a metal reagent, is environment-friendly, and is suitable for synthesizing a DNA coding compound library by using a porous plate.
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～C12The alkyl group is a straight-chain or branched alkyl group having 1 to 12 carbon atoms.
Alkyl means a straight or branched hydrocarbon radical in an alkane molecule, e.g. methyl-CH3ethyl-CH2CH3methylene-CH2-; the alkyl group may also be part of another group, such as C1～C6Alkoxy radical, C1～C6An alkylamino group.
Cycloalkyl refers to a saturated or partially saturated cyclic group having multiple carbon atoms and no ring heteroatoms, and having a single ring or multiple rings (including fused, bridged, and spiro ring systems).
The halogen is fluorine, chlorine, bromine or iodine.
Alkoxy means that the alkyl radical is linked to an oxygen atom to form a substituent, e.g. methoxy is-OCH3。
The halophenyl group means a group in which H on a phenyl group is substituted with halogen.
Alkylphenyl refers to a group formed by substituting H on a phenyl group with an alkyl group.
Aryl means an aromatic monocyclic or multicyclic group consisting of C atoms, free of heteroatoms.
The arylheterocyclyl group is a single cyclic group or a plurality of cyclic groups having aromaticity composed of 5 to 10 atoms such as C, O, S, N.
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.
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FIG. 1: LC-MS spectrum and MS spectrum of compound 24 in example 13.
Detailed Description
The technical scheme of the invention is clearly and completely described in the following by combining specific embodiments. It is to be understood that the embodiments described are only a few embodiments of the present 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.
DNA-NH in the present invention2Or
Figure BDA0002825395720000051
Is 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. Also for example the following DNA structure:
Figure BDA0002825395720000052
wherein A is adenine, T is thymine, C is cytosine, and G is guanine.
DMA: and (3) dimethylacetamide. HATU: 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate. DIPEA: n, N-diisopropylethylamine. BBS buffer: a borate buffered solution. DMSO, DMSO: dimethyl sulfoxide (DMSO). The room temperature is 20-30 ℃.
Example 1 Synthesis of On-DNA aldol Compound 3
Figure BDA0002825395720000061
Dissolving a DNA raw material 1 into a 250mM boric acid buffer solution with the pH value of 9.4 to prepare a 1mM concentration solution (20 mu L, 20nmol), acid aldehyde (1 mu mol, 50 equivalent, 200mM DMA solution), HATU (1 mu mol, 50 equivalent, 400mM DMA solution), DIPEA (1 mu mol, 50 equivalent, 400mM DMA solution) which are respectively placed in a refrigerator with the temperature of-20 ℃ for 5 minutes and then mixed, fully mixing the mixture by vortex oscillation, and then placing the mixture in a refrigerator with the temperature of 4 ℃ for 5 minutes; the mixture was added to the solution of DNA Material 1, mixed well and left overnight at room temperature.
Ethanol precipitation standard procedure: adding 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 at 12000rpm for half an hour, and pouring out the supernatant. Directly used for the next reaction.
On-DNA aldehyde compound 2 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10nmol), proline (100nmol, 10 equiv., 200mM DMSO solution) and cyclobutylformaldehyde (1 μmol, 100 equiv., 200mM DMSO solution) were sequentially added to the solution, and the mixture was mixed well and reacted at room temperature for 2 hours.
After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5436.1, the actual measured molecular weight is 5435.3, and the conversion rate is 98%.
Example 2 Synthesis of On-DNA aldol Compound 5
Figure BDA0002825395720000062
DNA material 1 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (20 μ L, 20nmol), acid aldehyde (1 μmol, 50 equivalents, 200mM DMA solution), HATU (1 μmol, 50 equivalents, 400mM DMA solution), DIPEA (1 μmol, 50 equivalents, 400mM DMA solution) were placed in a refrigerator at-20 ℃ for 5 minutes, mixed, and the mixture was mixed by vortexing and stored in the refrigerator at 4 ℃ for 5 minutes. The mixture was added to the solution of DNA Material 1, mixed well and left overnight at room temperature. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Directly used for the next reaction.
On-DNA aldehyde compound 4 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10nmol), proline (100nmol, 10 equiv., 200mM DMSO solution) and cyclobutylformaldehyde (1 μmol, 100 equiv., 200mM DMSO solution) were sequentially added to the solution, and the mixture was mixed well and reacted at room temperature for 2 hours. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5418.1, the actual measured molecular weight is 5417.9, and the conversion rate is 86%.
Example 3 Synthesis of On-DNA aldol Compound 7
Figure BDA0002825395720000071
DNA material 1 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (20 μ L, 20nmol), acid aldehyde (1 μmol, 50 equivalents, 200mM DMA solution), HATU (1 μmol, 50 equivalents, 400mM DMA solution), DIPEA (1 μmol, 50 equivalents, 400mM DMA solution) were placed in a refrigerator at-20 ℃ for 5 minutes, mixed, and the mixture was mixed by vortexing and stored in the refrigerator at 4 ℃ for 5 minutes. The mixture was added to the solution of DNA Material 1, mixed well and left overnight at room temperature. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Directly used for the next reaction.
The On-DNA aldehyde compound 6 was dissolved in 250mM boric acid buffer solution at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10nmol), proline (100nmol, 10 equivalents, 200mM DMSO solution) and cyclobutylformaldehyde (1 μmol, 100 equivalents, 200mM DMSO solution) were sequentially added to the solution, and the mixture was mixed well and reacted at room temperature for 2 hours. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5418.1, the actual measured molecular weight is 5417.5, and the conversion rate is 88%.
Example 4 Synthesis of On-DNA aldol Compound 9
Figure BDA0002825395720000072
DNA material 1 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (20 μ L, 20nmol), acid aldehyde (1 μmol, 50 equivalents, 200mM DMA solution), HATU (1 μmol, 50 equivalents, 400mM DMA solution), DIPEA (1 μmol, 50 equivalents, 400mM DMA solution) were placed in a refrigerator at-20 ℃ for 5 minutes, mixed, and the mixture was mixed by vortexing and stored in the refrigerator at 4 ℃ for 5 minutes. The mixture was added to the solution of DNA Material 1, mixed well and left overnight at room temperature. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Directly used for the next reaction.
The On-DNA aldehyde compound 8 was dissolved in 250mM boric acid buffer solution at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10nmol), proline (100nmol, 10 equivalents, 200mM DMSO solution) and cyclobutylformaldehyde (1 μmol, 100 equivalents, 200mM DMSO solution) were sequentially added to the solution, and the mixture was mixed well and reacted at room temperature for 2 hours. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5390.1, the actual measured molecular weight is 5389.5, and the conversion rate is 92%.
Example 5 Synthesis of On-DNA aldol Compound 11
Figure BDA0002825395720000081
DNA material 1 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (20 μ L, 20nmol), acid aldehyde (1 μmol, 50 equivalents, 200mM DMA solution), HATU (1 μmol, 50 equivalents, 400mM DMA solution), DIPEA (1 μmol, 50 equivalents, 400mM DMA solution) were placed in a refrigerator at-20 ℃ for 5 minutes, mixed, and the mixture was mixed by vortexing and stored in the refrigerator at 4 ℃ for 5 minutes. The mixture was added to the solution of DNA Material 1, mixed well and left overnight at room temperature. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Directly used for the next reaction.
The On-DNA aldehyde compound 10 was dissolved in 250mM boric acid buffer solution at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10nmol), proline (100nmol, 10 equivalents, 200mM DMSO solution) and cyclobutylformaldehyde (1 μmol, 100 equivalents, 200mM DMSO solution) were sequentially added to the solution, and the mixture was mixed well and reacted at room temperature for 2 hours. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5418.1, the actual measured molecular weight is 5418.0, and the conversion rate is 84%.
Example 6 Synthesis of On-DNA aldol Compound 13
Figure BDA0002825395720000091
DNA material 1 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (20 μ L, 20nmol), acid aldehyde (1 μmol, 50 equivalents, 200mM DMA solution), HATU (1 μmol, 50 equivalents, 400mM DMA solution), DIPEA (1 μmol, 50 equivalents, 400mM DMA solution) were placed in a refrigerator at-20 ℃ for 5 minutes, mixed, and the mixture was mixed by vortexing and stored in the refrigerator at 4 ℃ for 5 minutes. The mixture was added to the solution of DNA Material 1, mixed well and left overnight at room temperature. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Directly used for the next reaction.
The On-DNA aldehyde compound 12 was dissolved in 250mM boric acid buffer solution at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10nmol), proline (100nmol, 10 equivalents, 200mM DMSO solution) and cyclobutylformaldehyde (1 μmol, 100 equivalents, 200mM DMSO solution) were sequentially added to the solution, and the mixture was mixed well and reacted at room temperature for 2 hours. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5466.1, the actual measured molecular weight is 5466.0, and the conversion rate is 77%.
Example 7 Synthesis of On-DNA aldol Compound 15
Figure BDA0002825395720000092
DNA material 1 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (20 μ L, 20nmol), and acid aldehyde (1 μmol, 50 equivalents, 200mM DMA solution), HATU (1 μmol, 50 equivalents, 400mM DMA solution), DIPEA (1 μmol, 50 equivalents, 400mM DMA solution) were placed in a refrigerator at-20 ℃ for 5 minutes, mixed, and the mixture was mixed by vortexing and stored in the refrigerator at 4 ℃ for 5 minutes. The mixture was added to the solution of DNA Material 1, mixed well and left overnight at room temperature. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Directly used for the next reaction.
The On-DNA aldehyde compound 14 was dissolved in 250mM boric acid buffer solution at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10nmol), proline (100nmol, 10 equivalents, 200mM DMSO solution) and cyclobutylformaldehyde (1 μmol, 100 equivalents, 200mM DMSO solution) were sequentially added to the solution, and the mixture was mixed well and reacted at room temperature for 2 hours. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5436.1, the actual measured molecular weight is 5434.8, and the conversion rate is 73%.
Example 8 Synthesis of On-DNA aldol Compound 17
Figure BDA0002825395720000101
DNA material 1 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (20 μ L, 20nmol), and acid aldehyde (1 μmol, 50 equivalents, 200mM DMA solution), HATU (1 μmol, 50 equivalents, 400mM DMA solution), DIPEA (1 μmol, 50 equivalents, 400mM DMA solution) were placed in a refrigerator at-20 ℃ for 5 minutes, mixed, and the mixture was mixed by vortexing and stored in the refrigerator at 4 ℃ for 5 minutes. The mixture was added to the solution of DNA Material 1, mixed well and left overnight at room temperature. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Directly used for the next reaction.
The On-DNA aldehyde compound 16 was dissolved in 250mM boric acid buffer solution at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10nmol), proline (100nmol, 10 equivalents, 200mM DMSO solution) and cyclobutylformaldehyde (1 μmol, 100 equivalents, 200mM DMSO solution) were sequentially added to the solution, and the mixture was mixed well and reacted at room temperature for 2 hours. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5436.1, the actual measured molecular weight is 5434.9, and the conversion rate is 61%.
Example 9 Synthesis of On-DNA aldol Compound 19
Figure BDA0002825395720000102
DNA material 1 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (20 μ L, 20nmol), and acid aldehyde (1 μmol, 50 equivalents, 200mM DMA solution), HATU (1 μmol, 50 equivalents, 400mM DMA solution), DIPEA (1 μmol, 50 equivalents, 400mM DMA solution) were placed in a refrigerator at-20 ℃ for 5 minutes, mixed, and the mixture was mixed by vortexing and stored in the refrigerator at 4 ℃ for 5 minutes. The mixture was added to the solution of DNA Material 1, mixed well and left overnight at room temperature. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Directly used for the next reaction.
The On-DNA aldehyde compound 18 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10nmol), proline (100nmol, 10 equiv, 200mM DMSO solution) and cyclobutylformaldehyde (1 μmol, 100 equiv, 200mM DMSO solution) were sequentially added to the solution, and the mixture was mixed well and reacted at room temperature for 2 hours. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5400.1, the actual measured molecular weight is 5399.6, and the conversion rate is 55%.
Example 10 Synthesis of On-DNA aldol Compound 21
Figure BDA0002825395720000111
DNA material 1 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (20 μ L, 20nmol), and acid aldehyde (1 μmol, 50 equivalents, 200mM DMA solution), HATU (1 μmol, 50 equivalents, 400mM DMA solution), DIPEA (1 μmol, 50 equivalents, 400mM DMA solution) were placed in a refrigerator at-20 ℃ for 5 minutes, mixed, and the mixture was mixed by vortexing and stored in the refrigerator at 4 ℃ for 5 minutes. The mixture was added to the solution of DNA Material 1, mixed well and left overnight at room temperature. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Directly used for the next reaction.
The On-DNA aldehyde compound 20 was dissolved in 250mM boric acid buffer solution at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10nmol), proline (100nmol, 10 equivalents, 200mM DMSO solution) and cyclobutylformaldehyde (1 μmol, 100 equivalents, 200mM DMSO solution) were sequentially added to the solution, and the mixture was mixed well and reacted at room temperature for 2 hours. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5401.1, the actual measured molecular weight is 5400.5, and the conversion rate is 80%.
Example 11 Synthesis of On-DNA aldol Compound 22
Figure BDA0002825395720000121
On-DNA aldehyde compound 4 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10nmol), proline (100nmol, 10 equivalents, 200mM DMSO solution) and aldehyde (1 μmol, 100 equivalents, 200mM DMSO solution) were sequentially added to the solution, and the mixture was mixed well and reacted at room temperature for 2 hours. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5526.3, the actual measured molecular weight is 5525.6, and the conversion rate is 90%.
Example 12 Synthesis of On-DNA aldol Compound 23
Figure BDA0002825395720000122
On-DNA aldehyde compound 4 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10nmol), proline (100nmol, 10 equivalents, 200mM DMSO solution) and ketone (1 μmol, 100 equivalents, 200mM DMSO solution) were sequentially added to the solution, and the mixture was mixed well and reacted at room temperature for 2 hours. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5484.1, the actual measured molecular weight is 5483.6, and the conversion rate is 89%.
Example 13 Synthesis of On-DNA aldol Compound 24
Figure BDA0002825395720000123
On-DNA aldehyde compound 4 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10nmol), proline (100nmol, 10 equivalents, 200mM DMSO solution) and ketone (1 μmol, 100 equivalents, 200mM DMSO solution) were sequentially added to the solution, and the mixture was mixed well and reacted at room temperature for 2 hours. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5529.2, the actual measured molecular weight is 5528.4, and the conversion rate is 89%.
Example 14 Synthesis of On-DNA aldol Compound 25
Figure BDA0002825395720000131
On-DNA aldehyde compound 4 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10nmol), proline (100nmol, 10 equivalents, 200mM DMSO solution) and ketone (1 μmol, 100 equivalents, 200mM DMSO solution) were sequentially added to the solution, and the mixture was mixed well and reacted at room temperature for 2 hours. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5514.2, the actual measured molecular weight is 5513.6, and the conversion rate is 85%.
Example 15 Synthesis of On-DNA aldol Compound 26
Figure BDA0002825395720000132
On-DNA aldehyde compound 4 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10 nmol). Proline (100nmol, 10 equivalents, 200mM DMSO solution) and aldehyde (1. mu. mol, 100 equivalents, 200mM DMSO solution) were added to the solution in this order, mixed well, and reacted at room temperature for 2 hours. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5494.2, the actual measured molecular weight is 5494.1, and the conversion rate is 84%.
Example 16 Synthesis of On-DNA aldol Compound 27
Figure BDA0002825395720000141
On-DNA aldehyde compound 4 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10nmol), proline (100nmol, 10 equivalents, 200mM DMSO solution) and aldehyde (1 μmol, 100 equivalents, 200mM DMSO solution) were sequentially added to the solution, and the mixture was mixed well and reacted at room temperature for 2 hours. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5436.1, the actual measured molecular weight is 5436.2, and the conversion rate is 76%.
Example 17 Synthesis of On-DNA aldol Compound 28
Figure BDA0002825395720000142
On-DNA aldehyde compound 4 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10nmol), proline (100nmol, 10 equivalents, 200mM DMSO solution) and ketone (1 μmol, 100 equivalents, 200mM DMSO solution) were sequentially added to the solution, and the mixture was mixed well and reacted at room temperature for 2 hours. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5528.2, the actual measured molecular weight is 5528.0, and the conversion rate is 64%.
Example 18 Synthesis of On-DNA aldol Compound 29
Figure BDA0002825395720000143
On-DNA aldehyde compound 4 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10nmol), proline (100nmol, 10 equivalents, 200mM DMSO solution) and ketone (1 μmol, 100 equivalents, 200mM DMSO solution) were sequentially added to the solution, and the mixture was mixed well and reacted at room temperature for 2 hours. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5490.2, the actual measured molecular weight is 5490.2, and the conversion rate is 61%.
Example 19 Synthesis of On-DNA aldol Compound 30
Figure BDA0002825395720000151
On-DNA aldehyde compound 4 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10nmol), proline (100nmol, 10 equivalents, 200mM DMSO solution) and aldehyde (1 μmol, 100 equivalents, 200mM DMSO solution) were sequentially added to the solution, and the mixture was mixed well and reacted at room temperature for 2 hours. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5496.2, the actual measured molecular weight is 5496.2, and the conversion rate is 59%.
Example 20 Synthesis of On-DNA aldol Compound 31
Figure BDA0002825395720000152
On-DNA aldehyde compound 4 was dissolved in 250mM boric acid buffer at pH 9.4 to prepare a 1mM concentration solution (10 μ L, 10nmol), proline (100nmol, 10 equivalents, 200mM DMSO solution) and aldehyde (1 μmol, 100 equivalents, 200mM DMSO solution) were sequentially added to the solution, and the mixture was mixed well and reacted at room temperature for 2 hours. After the reaction is finished, performing standard operation post-treatment on ethanol precipitation. Dissolving the rest precipitate with deionized water to obtain solution of On-DNA product, quantifying by enzyme labeling instrument OD, and monitoring by LCMS, wherein the theoretical molecular weight of the compound is 5482.2, the actual measured molecular weight is 5482.3, and the conversion rate is 58%.
In conclusion, the On-DNA aldol compound can be obtained by controlling the conditions of solvent, temperature, pH and the like of the reaction and reacting the On-DNA aldehyde compound with the alpha hydrogen aldehyde or ketone compound in the presence of the catalyst. The method can be carried out in the mixed aqueous phase of an organic solvent and an aqueous phase, is simple to operate, does not introduce a metal reagent, is environment-friendly, and is suitable for synthesizing a DNA coding compound library by using a porous plate. The above embodiments and drawings are only preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (10)

1. A method for On-DNA Aldol reaction is characterized in that: the reaction takes an On-DNA aldehyde compound and an alpha hydrogen aldehyde or ketone compound as raw materials to obtain an On-DNA product in the presence of a catalyst; wherein the structural formula of the On-DNA aldehyde compound is DNA-R3-CHO, the structural formula of the alpha hydrogen aldehyde group or ketone group compound is
Figure FDA0002825395710000011
The structural formula of the On-DNA product is shown in the specification
Figure FDA0002825395710000012
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 through one or more chemical bonds or groups3Connecting;
R1selected from hydrogen or a group with molecular weight below 1000 directly connected with carbon atom at alpha position of carbonyl;
R2selected from hydrogen or a group having a molecular weight of 1000 or less directly bonded to the carbonyl carbon atom;
R3selected from the group consisting of groups having a molecular weight of 1000 or less directly linked to DNA and an aldehyde carbon atom or being absent;
R4selected from hydrogen or a group with molecular weight below 1000 directly connected with carbon atom at alpha position of carbonyl;
or R1And R4Looping; or R2Are each independently of R1Or R4Looping.
2. The method of claim 1, wherein: said R1、R2、R3、R4Each independently selected from alkyl, substituted alkyl, hydroxy, C1～C20Alkoxy, 5-10-membered aryl, substituted 5-10-membered aryl, 5-10-membered aromatic heterocyclic group, substituted 5-10-membered aromatic heterocyclic group; wherein the alkyl is C1～C20Alkyl or C3～C8A cycloalkyl group; the number of substituents of the substituted alkyl group is one or more; the substituents of the substituted alkyl are independently selected from hydroxyl, halogen, carboxyl, nitro and C1～C20Alkoxy, halophenyl, phenyl, alkylphenyl, C1～C20One or more of alkoxyphenyl, 5-10 membered aromatic heterocyclic group, and carboxyl-substituted 5-10 membered aromatic heterocyclic group; the number of the substituent for substituting the 5-to 10-membered aryl is one or more, and the substituent for substituting the 5-to 10-membered aryl is independently selected from hydroxyl, halogen, cyano, nitro, carboxyl and C1～C20Alkoxy radical, C1～C20One 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, C1～C20Alkoxy radical, C1～C20One or more of alkyl and trifluoromethyl.
3. The method of claim 1, wherein: the reaction comprises the following reaction steps: adding 1-50 times of molar equivalent of catalyst into an On-DNA aldehyde compound solution with the molar equivalent of 1 and the molar concentration of 0.5-5mM, then adding 10-1000 times of molar equivalent of alpha hydrogen aldehyde or ketone compound, and reacting for 0.5-24 hours at 10-100 ℃.
4. The method of claim 3, wherein: the catalyst is proline, proline methyl ester, tetrahydropyrrole or piperidine.
5. The method of claim 3, wherein: the reaction is carried out in a solvent, and the solvent is one or a mixture of water, methanol, ethanol, acetonitrile, dimethyl sulfoxide, an inorganic salt buffer solution, an organic acid buffer solution and an organic base buffer solution.
6. The method of claim 3, wherein: in the reaction method, the molar equivalent of the On-DNA aldehyde group compound is 1, the molar equivalent of the alpha hydrogen aldehyde group or ketone group compound is 20 equivalents, 50 equivalents, 100 equivalents, 200 equivalents, 400 equivalents and 800 equivalents, and the molar equivalent of the catalyst is 2 equivalents, 5 equivalents, 10 equivalents, 20 equivalents, 30 equivalents and 40 equivalents.
7. The method of claim 3, wherein: the reaction temperature of the reaction is 20 ℃, 30 ℃, 40 ℃, 50 ℃ or 60 ℃.
8. The method of claim 3, wherein: the reaction time of the reaction is 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, or 8 hours.
9. The method according to any one of claims 1 to 8, wherein the method is used for a batch multi-well plate operation.
10. The method of 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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