阅读说明：本技术 DNA编码化合物库构建中通过氧化酰胺化制备On-DNA酰胺化合物的方法 (Method for preparing On-DNA amide compound through oxidative amidation in construction of DNA coding compound library ) 是由 安玉龙 曲毅 吴阿亮 陈雯婷 苏文姬 蒯乐天 杨洪芳 彭宣嘉 于 2020-05-25 设计创作，主要内容包括：本发明公开了一种DNA编码化合物库构建中通过氧化酰胺化制备On-DNA酰胺化合物的方法,将On-DNA醛基化合物与小分子伯胺、铜盐、氧化剂在一定温度下反应一段时间后得到On-DNA酰胺化合物。本发明反应普适性好、条件温和、成本低、操作方便、收率高,适合于多孔板进行的DNA编码化合物库的合成。(The invention discloses a method for preparing an On-DNA amide compound by oxidative amidation in the construction of a DNA coding compound library, which comprises the step of reacting an On-DNA aldehyde compound with micromolecular primary amine, copper salt and an oxidant at a certain temperature for a period of time to obtain the On-DNA amide compound. The method has the advantages of good reaction universality, mild conditions, low cost, convenient operation and high yield, and is suitable for synthesizing the DNA coding compound library by using a porous plate.)

1. A method for preparing On-DNA amide compounds through oxidative amidation in the construction of a DNA coding compound library is characterized in that an On-DNA aldehyde compound with the molar concentration of 0.1-2.0 mM is mixed with 10-500 molar equivalent of small molecular primary amine, 10-200 molar equivalent of copper salt and 10-500 molar equivalent of oxidant, and the mixture is reacted at 20-100 ℃ for 1-24 hours until the reaction is finished to prepare the On-DNA amide compounds;

wherein the structural formula of the On-DNA aldehyde compound is as follows: DNA-CHO, the DNA in the structural formula is connected with the CHO through one or more chemical bonds; the micromolecular primary amine is primary amine with the molecular weight less than or equal to 1000, and the structural formula is as follows: R-NH₂(ii) a The structural formula of the On-DNA amide compound is DNA-CO-NH-R;

wherein, the DNA in the On-DNA aldehyde compound and the On-DNA amide compound is a single-chain or double-chain nucleotide chain obtained by polymerizing artificially modified and/or unmodified nucleotide monomers;

wherein the reaction solvent of the reaction is a water-containing mixed solvent containing any one or more of acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, methanol, ethanol, tert-butyl alcohol, isopropanol, tetrahydrofuran, an inorganic salt buffer solution, an organic acid buffer solution and an organic base buffer solution;

wherein the copper salt is selected from one or more of copper simple substance, cuprous chloride, cuprous bromide, cuprous iodide, cuprous oxide, cuprous acetate, copper trifluoroacetate, copper acetate and copper sulfate;

wherein the oxidant is selected from one or more of iodine simple substance, hydrogen peroxide, tert-butyl peroxide and tetramethylpiperidine oxide.

2. The method of claim 1, wherein the molar concentration of the On-DNA aldehyde compound after dissolution in an aqueous solution is 0.1 to 2.0 mM; preferably, the molar concentration of the On-DNA aldehyde compound aqueous solution is 1.0 mM.

3. The method according to claim 1, wherein the reaction solution of the reaction is an aqueous mixed solvent containing any one or more of acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide, methanol, ethanol, t-butanol, isopropanol, tetrahydrofuran, an inorganic salt buffer, an organic acid buffer, and an organic base buffer.

4. The method of claim 1, wherein the molar equivalents of the small molecule amine are 50-400 equivalents; preferably, the molar equivalent of the small molecule amine is 100-300 equivalents; preferably, the molar equivalent of the small molecule amine is 200 equivalents.

5. The method of claim 1, wherein the copper salt is cuprous chloride, cuprous bromide, cuprous iodide; preferably, the copper salt is cuprous iodide.

6. The method of claim 1, wherein the molar equivalent of the copper salt is 50 to 10 equivalents; preferably, the molar equivalent of the small molecule amine is 80 equivalents.

7. The method of claim 1, wherein the oxidizing agent is hydrogen peroxide, t-butanol peroxide; preferably, the oxidizing agent is t-butanol peroxide.

8. The method of claim 1, wherein the molar equivalent of the oxidizing agent is 10 to 500 equivalents; preferably, the molar equivalent of the oxidant is 20-400 equivalents; preferably, the molar equivalent of the oxidant is 40-200 equivalents; preferably, the molar equivalent of the oxidant is 50-100 equivalents; preferably, the molar equivalent of the oxidizing agent is 100 equivalents.

9. The method of claim 1, wherein the reaction temperature of the reaction is 20 to 100 ℃; preferably, the reaction temperature of the reaction is 20-50 ℃; preferably, the reaction temperature of the reaction is 20-35 ℃; preferably, the reaction temperature of the reaction is 25 ℃.

10. The method of claim 1, wherein the reaction time is 1 to 24 hours; preferably, the reaction time of the reaction is 4-20 hours; preferably, the reaction time of the reaction is 8-16 hours; preferably, the reaction time of the reaction is 16 hours.

11. The method of claim 1, wherein the method is used for a batch multi-well plate operation; preferably, the method is used for the synthesis of libraries of DNA-encoding compounds for multiwell plates.

Technical Field

The invention belongs to the technical field of DNA coding compound libraries, and particularly relates to a method for preparing an On-DNA amide compound from an On-DNA aldehyde compound and small molecular amine through oxidative amidation.

Background

The concept of libraries of DNA-Encoded Library (DEL) was proposed in 1992 by the teaching of Sydney Brenner and Richard Lerner of the Scripps institute of America (reference: Proc. Natl.Acad.Sci.,1992,89,5381) by linking an organic small molecule reagent to a unique sequence of DNA at the molecular level, rapidly constructing a large Library of compounds each consisting of different organic small molecule reagent residues and identified by DNA of a corresponding unique base sequence using a combinatorial chemistry "combinatorial-resolution" strategy over two to many cycles, affinity-screening a small Library of DNA-Encoded compounds to a target, washing away Library molecules that are not adsorbed to the target, leaving Library molecules adsorbed to the target and then washing away, where the resulting Library molecule concentrations are low and difficult to analyze and identify by conventional means, polymerase chain Reaction (polymerase chain Reaction, PCR for short) can be carried out to copy and amplify the DNA part in the obtained library molecule adsorbed with the target until the obtained DNA quantity can be identified by a DNA sequencer, the sequenced data is decoded by a relation table between small molecule reagents and DNA base sequences established when a DNA coding compound library is constructed, then the small molecule reagents corresponding to specific compounds corresponding to potential active molecules are found, then the small molecule reagents are combined together by a traditional organic synthesis method to obtain the screened target molecules, and the biological activity of the target molecules on the target is detected and confirmed.

The method for constructing DNA coding compound Library mainly includes three kinds, the first kind is DNA-guided Chemical Library Synthesis (DTCL) mainly obtained by using DNA template technology from Ensemble corporation in America, the second kind is DNA-Recorded Chemical Library (DRCL) mainly obtained by using DNA marking technology from GSK corporation in America, X-Chem corporation and domestic leaders, the third kind is coding Self-assembly molecule Library (ESAC) mainly obtained by Fragment-based drug design (Fragment-based drug discovery, FBDD) technology from Philogen corporation in Switzerland, the method for constructing DNA coding compound Library which is industrially applied in large quantity at present is mainly the second kind, the method is simple to operate and lower in cost, and can quickly obtain a DNA coding compound library containing massive compounds by using a combinatorial chemistry method.

Besides the DNA starting fragment (see the invention patents of the present company: CN108070009A, CN109868268A), a large number of DNA tags and small organic molecule reagents which can be reacted in a certain order are required. The DNA tag code can be obtained by a computer program (see the present invention: CN107958139A), and a primer of a specific DNA base sequence can be obtained by a DNA synthesizer. The small organic molecule reagent can be obtained by screening the obtained reagent list by using a certain computer program (see the invention patent of the company: CN 108959855A).

One of the major tasks in the DEL library field at present is the development of chemical reactions On DNA, called On-DNA chemistry for short. Because DNA must be kept stable in a certain aqueous phase, pH, temperature, metal ion concentration and inorganic salt concentration, the On-DNA chemical reaction which has small DNA damage, better recovery rate and wide substrate adaptability is required for the synthesis of a DNA coding compound library in a large scale. The On-DNA chemical reactions reported at present are nearly 120 in types, including aqueous phase, solid phase and DNA template reactions (see in detail On-line database DEL ChemFinder, https:// delopen. org/reactions), each reaction condition is one less, more than ten, so to say, under the same other conditions, the more the On-DNA chemical reactions are, the more the conditions are, the better the universality is, the more the selectivity is in the design of the DNA coding compound library, the higher the synthesis success rate of the final DNA coding compound library is, and the more the diversity of the obtained DNA coding compound library is.

In the construction of libraries of DNA-encoding compounds, amide bond is one of the most common and important bonding means, and at present, amide bond formation of On-DNA is mainly performed by small-molecule carboxylic acid and amine group On DNA, and amide compound formation of On-DNA by reverse synthesis method using small-molecule organic amine and carboxyl group On DNA is also reported (references: nat. chem.biol.,2009,5,9,647 654, CN109456368A, angelw. chem.int. ed. engl.,2019,58(28),9570-9574, org. lett.,2019,21,7,2194-2199, ACS comb.sci.,2019,21,2,75-82), but the reaction universality is not good, the use of small-molecule organic amine is limited, and particularly the reaction success rate of ortho-heterocyclic arylamine is low.

In order to obtain a more universal method for reverse synthesis of On-DNA amide compounds, we studied and developed the method of the present invention.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for preparing an On-DNA amide compound by oxidative amidation in the construction of a DNA coding compound library,

in order to solve the technical problems, the invention provides the following technical scheme:

a method for preparing an On-DNA amide compound through oxidative amidation in the construction of a DNA coding compound library comprises the steps of mixing an On-DNA aldehyde compound with the molar concentration of 0.1-2.0 mM with 10-500 molar equivalents of small molecular primary amine, 10-200 molar equivalents of copper salt and 10-500 molar equivalents of oxidant, and reacting at 20-100 ℃ for 1-24 hours until the reaction is finished to prepare the On-DNA amide compound; the reaction equation is as follows:

wherein the structural formula of the On-DNA aldehyde compound is as follows: DNA-CHO, the DNA in the structural formula is connected with the CHO through one or more chemical bonds; the micromolecular primary amine is primary amine with the molecular weight less than or equal to 1000, and the structural formula is as follows: R-NH₂(ii) a The structural formula of the On-DNA amide compound is DNA-CO-NH-R;

wherein, the DNA in the On-DNA aldehyde compound and the On-DNA amide compound is a single-chain or double-chain nucleotide chain obtained by polymerizing artificially modified and/or unmodified nucleotide monomers;

wherein the reaction solvent of the reaction is a water-containing mixed solvent containing any one or more of acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, methanol, ethanol, tert-butyl alcohol, isopropanol, tetrahydrofuran, an inorganic salt buffer solution, an organic acid buffer solution and an organic base buffer solution;

wherein the copper salt is selected from one or more of copper simple substance, cuprous chloride, cuprous bromide, cuprous iodide, cuprous oxide, cuprous acetate, copper trifluoroacetate, copper acetate and copper sulfate;

wherein the oxidant is selected from one or more of iodine simple substance, hydrogen peroxide, tert-butyl peroxide and tetramethylpiperidine oxide.

In particular, the structural formula of the micromolecule primary amine R-NH₂In the above process, R is optionally selected so as not to directly react with aldehyde (CHO) and primary amino (NH) groups under the reaction conditions of the above process₂) A group that undergoes a chemical reaction.

In a preferred embodiment, the molar concentration of the On-DNA aldehyde compound is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mM. Preferably, the molar concentration of the On-DNA aldehyde compound is 0.5-1.5 mM; preferably, the molar concentration of the On-DNA aldehyde compound is 0.8-1.2 mM; preferably, the molar concentration of the On-DNA aldehyde compound is 1.0 mM.

The molar concentration of the On-DNA aldehyde compound dissolved in the aqueous solution is 0.1-2.0 mM; preferably, the molar concentration of the On-DNA aldehyde compound aqueous solution is 1.0 mM.

In a preferred embodiment, the molar equivalent of the small molecule amine is 50 to 400 equivalents; preferably, the molar equivalent of the small molecule amine is 100-300 equivalents; preferably, the molar equivalent of the small molecule amine is 200 equivalents.

In a preferred embodiment, the copper salt is selected from at least one of cuprous chloride, cuprous bromide, cuprous iodide, cuprous oxide, and cuprous acetate; preferably, the copper salt is selected from at least one of cuprous chloride, cuprous bromide and cuprous iodide; preferably, the copper salt is selected from cuprous iodide.

In a preferred embodiment, the molar equivalent of the copper salt is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 equivalents. Preferably, the molar equivalent of the small molecule amine is 50-100 equivalents; preferably, the molar equivalent of the small molecule amine is 80 equivalents.

In a preferred embodiment, the oxidizing agent is selected from hydrogen peroxide or tert-butanol peroxide; preferably, the oxidizing agent is selected from tert-butanol peroxide.

In a preferred embodiment, the molar equivalent of the oxidizing agent is 10 to 500 equivalents; preferably, the molar equivalent of the oxidant is 20-400 equivalents; preferably, the molar equivalent of the oxidant is 40-200 equivalents; preferably, the molar equivalent of the oxidant is 50-100 equivalents; preferably, the molar equivalent of the oxidizing agent is 100 equivalents.

In a preferred embodiment, the reaction temperature of the reaction is 20-100 ℃; preferably, the reaction temperature of the reaction is 20-50 ℃; preferably, the reaction temperature of the reaction is 20-35 ℃; preferably, the reaction temperature of the reaction is 25 ℃.

In a preferred embodiment, the reaction time of the reaction is 1 to 24 hours; preferably, the reaction time of the reaction is 4-20 hours; preferably, the reaction time of the reaction is 8-16 hours; preferably, the reaction time of the reaction is 16 hours.

In a preferred embodiment, the method is used for a batch multi-well plate operation; preferably, the method is used for the synthesis of libraries of DNA-encoding compounds for multiwell plates.

The invention provides a method for obtaining an On-DNA amide compound by an oxidative amidation method in the construction of a DNA coding compound library, which enlarges the diversity of the DNA coding compound library of the company and ensures that the library molecules of the obtained DNA coding compound library can better meet the market demands.

The method has the advantages of good reaction universality, mild conditions, low cost, convenient operation and high yield, and is suitable for synthesizing the DNA coding compound library by using a porous plate.

Drawings

FIG. 1 is a representative structural formula of micromolecule organic amine which is prepared by carrying out oxidative amidation On an On-DNA aryl aldehyde group compound 3 and arylamine to obtain a corresponding On-DNA amide compound.

FIG. 2 is a representative structural formula of the small molecular organic amine which is prepared by carrying out oxidative amidation On an On-DNA alkyl aldehyde group compound 9 and arylamine to obtain a corresponding On-DNA amide compound.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. 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.